Cells of the indicated genotypes that carried or not the DRT5T dimeric tRNA construct ( schematized on top ) were grown either in the presence of the optimum concentration of adenine ( left ) or in the presence of a limiting concentration of adenine ( right ) . Two independent clones of the same genotype ( # 1 and # 2 ) were used . See text for details . 3F refers to the 3 Flag epitope tag at the C - terminus of Rpc37 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O

Cells of the indicated genotypes that carried or not the DRT5T dimeric tRNA construct (schematized on top) were grown either in the presence of the optimum concentration of adenine (left) or in the presence of a limiting concentration of adenine (right). Two independent clones of the same genotype (#1 and #2) were used. See text for details. 3F refers to the 3 Flag epitope tag at the C-terminus of Rpc37.


Strand - specific RT - qPCR was used to quantify the levels of read - through transcripts ( see Methods ) . The mean ± standard deviation from 4 biological replicates is represented here . P - values were obtained using the Wilcoxon - Mann Whitney statistical test .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Strand-specific RT-qPCR was used to quantify the levels of read-through transcripts (see Methods). The mean ± standard deviation from 4 biological replicates is represented here. P-values were obtained using the Wilcoxon-Mann Whitney statistical test.


Northern blot analysis of the tRNA SPATRNAPRO . 02 using an intron - specific probe ( TCTAAACTCAGCATACAAGTGGGG ) . U5 snRNA was used as a loading control .

O O O O O O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O O O O O O O O O O O O O O O O O O O

Northern blot analysis of the tRNA SPATRNAPRO.02 using an intron-specific probe (TCTAAACTCAGCATACAAGTGGGG). U5 snRNA was used as a loading control.


ChIP - qPCR analysis of Rpc37 around SPCTRNAARG . 10 gene in the strong terminator mutant ( mean ± standard deviation from 4 biological replicates ) .

O O O O O B-MEASURED_VAR O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O O O O O O O O O O O O O O O O O

ChIP-qPCR analysis of Rpc37 around SPCTRNAARG.10 gene in the strong terminator mutant (mean ± standard deviation from 4 biological replicates).


Strand specific RT - qPCR was used to quantify read - through transcripts at SPCTRNAARG . 10 ( arg10 ) and SPCTRNASER . 09 ( ser09 ) in the strong terminator mutant ( mean ± standard deviation from 4 biological replicates ; p - value obtained using the Wilcoxon - Mann Whitney statistical test ) .

O O O O O O O O O O O O O O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O B-MEASURED_VAR O O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Strand specific RT-qPCR was used to quantify read-through transcripts at SPCTRNAARG.10 (arg10) and SPCTRNASER.09 (ser09) in the strong terminator mutant (mean ± standard deviation from 4 biological replicates; p-value obtained using the Wilcoxon-Mann Whitney statistical test).


C . MA plot highlighting genes differentially expressed between state independent of strain ( glm state term , false discovery rate - adjusted P value ( FDR ) < 0 . 05 and log2FC > 1 ) . Core pluripotent TFs highlighted for each state ( maroon = naive ESCs , teal = formative EpiLCs ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

C. MA plot highlighting genes differentially expressed between state independent of strain (glm state term, false discovery rate-adjusted P value (FDR) < 0.05 and log2FC >1). Core pluripotent TFs highlighted for each state (maroon = naive ESCs, teal = formative EpiLCs).


H . Heatmap of motif deviations from 3 biological replicates of B6 and D2 ESCs for TFs highlighted in G .

O O O O O O O O O O O O O O O O O O O O O

H. Heatmap of motif deviations from 3 biological replicates of B6 and D2 ESCs for TFs highlighted in G.


A - D . Example expression patterns for select genes identified by applying a general linear model ( glm ) including state , genotype , and interaction terms . Dots represent individual biological replicates ( N = 3 ) ; lines highlight changes in mean values for replicates between each state ( log2FC > 1 and FDR < 0 . 05 ) . A . State dependent expression is exemplified by Nanog which showed no difference between strains . B . Expression of Zfp277 exemplifies strain - dependence being consistently higher in B6 . C & D . A significant genotype x state ( GXS ) interaction was identified for Nr5a2 ( C , a marker for pluripotency ) and Sox1 ( D , a marker for neuronal differentiation ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O

A-D. Example expression patterns for select genes identified by applying a general linear model (glm) including state, genotype, and interaction terms. Dots represent individual biological replicates (N = 3); lines highlight changes in mean values for replicates between each state (log2FC >1 and FDR < 0.05). A. State dependent expression is exemplified by Nanog which showed no difference between strains. B. Expression of Zfp277 exemplifies strain-dependence being consistently higher in B6. C & D. A significant genotype x state (GXS) interaction was identified for Nr5a2 (C, a marker for pluripotency) and Sox1 (D, a marker for neuronal differentiation).


D . Feature plot of expression gradients for indicated gene overlaid on UMAP from a . Three lineage markers that help to distinguish cellular identity are shown for five cell clusters .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

D. Feature plot of expression gradients for indicated gene overlaid on UMAP from a. Three lineage markers that help to distinguish cellular identity are shown for five cell clusters.


E . Pseudocolor plots of FACS analysis on cells from EBs for both B6 and D2 strains . Cells were labeled with anti - EpCAM ( top ) or biological replicates with anti - SOX1 antibody ( bottom ) . Gated population indicates percentages of EpCAM + ( Q3 ) or SOX1 + cells .

O O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O B-MEASURED_VAR O O O O O B-MEASURED_VAR O O O

E. Pseudocolor plots of FACS analysis on cells from EBs for both B6 and D2 strains. Cells were labeled with anti-EpCAM (top) or biological replicates with anti-SOX1 antibody (bottom). Gated population indicates percentages of EpCAM+ (Q3) or SOX1+ cells.


B . Scatterplot of genomic locations of individual chromatin accessibility ( ca ) QTL ( x - axis , LOD > 5 ) versus location of ATAC peak being regulated ( y - axis ) for ESCs . Genetic effects that act locally ( n = 3 , 973 ) , i . e . in cis , fall on the diagonal line , while distal acting QTL ( n = 13 , 767 ) lie off the diagonal . Several prominent distal QTL hotspots appear as vertical lines . C . Similar to B , plotting expression ( e ) QTL position versus gene location ( LOD > 5 , n = 701 local - eQTL , n = 357 distal - eQTL ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

B. Scatterplot of genomic locations of individual chromatin accessibility (ca)QTL (x-axis, LOD > 5) versus location of ATAC peak being regulated (y-axis) for ESCs. Genetic effects that act locally (n = 3,973), i.e. in cis, fall on the diagonal line, while distal acting QTL (n = 13,767) lie off the diagonal. Several prominent distal QTL hotspots appear as vertical lines. C. Similar to B, plotting expression (e)QTL position versus gene location (LOD > 5, n = 701 local-eQTL, n = 357 distal-eQTL). D


G . Coverage profile for Gstp2 locus on Chr 19 showing H3K4me3 , ATAC , and RNA read depth from merged replicates . Locations of Chr 7 trans caQTL targets are indicated with black bars and LOD scores listed below .

O O O O O B-MEASURED_VAR O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

G. Coverage profile for Gstp2 locus on Chr 19 showing H3K4me3, ATAC, and RNA read depth from merged replicates. Locations of Chr 7 trans caQTL targets are indicated with black bars and LOD scores listed below.


F . Left - Coverage profiles ( averaged biological replicates , N = 3 ) for ChIP factor occupancy at Cd59a on Chr 2 under trans - regulation by the Chr 13d QTL . Distal ca - QTL target region used for quantification is indicated by a black box with LOD score below . Right - Scatterplots for ChIP factors comparing quantitative level of modification / binding for independent replicates between B6 and D2 ESCs .

O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

F. Left - Coverage profiles (averaged biological replicates, N=3) for ChIP factor occupancy at Cd59a on Chr 2 under trans-regulation by the Chr 13d QTL. Distal ca-QTL target region used for quantification is indicated by a black box with LOD score below. Right - Scatterplots for ChIP factors comparing quantitative level of modification/binding for independent replicates between B6 and D2 ESCs.


G . The difference in occupancy of P300 in the parental strains is correlated to difference in chromatin accessibility in the 33 BXDs based on the genotype of the BXD at the QTL for Chr 13 caQTL targets ( LOD > 7 , Pearson ' s r = - 0 . 61 ) . H . TRIM28 occupancy at the same targets of Chr 13d caQTL in F were negatively correlated with ATAC signal ( Pearson ' s r = - 0 . 64 ) .

O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

G. The difference in occupancy of P300 in the parental strains is correlated to difference in chromatin accessibility in the 33 BXDs based on the genotype of the BXD at the QTL for Chr 13 caQTL targets (LOD > 7, Pearson's r = -0.61). H. TRIM28 occupancy at the same targets of Chr 13d caQTL in F were negatively correlated with ATAC signal (Pearson's r = -0.64). I


I . Similar to F , coverage profiles for ChIP factor occupancy for a putative regulatory element on Chr 5 near Zfp932 , targeted by Chr 12 trans caQTL , showed greater occupancy of POU5F1 and increased open chromatin compared to D2 . Distal ca - QTL target region used for quantification is indicated by a black box with LOD score below .

O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O

I. Similar to F, coverage profiles for ChIP factor occupancy for a putative regulatory element on Chr 5 near Zfp932, targeted by Chr 12 trans caQTL, showed greater occupancy of POU5F1 and increased open chromatin compared to D2. Distal ca-QTL target region used for quantification is indicated by a black box with LOD score below.


J . The difference in occupancy of POU5F1 in the parental strains is correlated to difference in chromatin accessibility in the 33 BXDs based on the genotype of the BXD at the QTL for Chr 12 caQTL targets ( LOD > 7 , Pearson ' s r = - 0 . 66 ) . K . Similar to J showing negative correlation between TRIM28 occupancy and chromatin accessibility ( Pearson ' s r = - 0 . 51 ) .

O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O

J. The difference in occupancy of POU5F1 in the parental strains is correlated to difference in chromatin accessibility in the 33 BXDs based on the genotype of the BXD at the QTL for Chr 12 caQTL targets (LOD > 7, Pearson's r = -0.66). K. Similar to J showing negative correlation between TRIM28 occupancy and chromatin accessibility (Pearson's r = -0.51). D


A . Coverage profile for H3K4me3 , ATAC , P300 , and TRIM28 ChIP at a target locus for the QTL on Chr 4 for both chromatin accessibility ( black boxes ) and gene expression ( Riok3 and Rmc1 ) . LOD scores are listed under each target locus .

O O O O O B-MEASURED_VAR O O O B-MEASURED_VAR O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O B-MEASURED_VAR O O O O O O O O O O O

A. Coverage profile for H3K4me3, ATAC, P300, and TRIM28 ChIP at a target locus for the QTL on Chr 4 for both chromatin accessibility (black boxes) and gene expression (Riok3 and Rmc1). LOD scores are listed under each target locus.


E . Box and whisker plot indicating change in H3K9me3 levels between WT and Chr 4 KO mESCs grouped by whether chromatin accessibility is higher when the QTL is B6 ( left ) or D2 ( right ) . Dots represent mean values from biological duplicate experiments and lines connect means . Orange indicates loci for which H3K9me3 signal decreases in the KO ( p - value indicated on top , two - sided paired Wilcoxon test ) .

O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O

E. Box and whisker plot indicating change in H3K9me3 levels between WT and Chr 4 KO mESCs grouped by whether chromatin accessibility is higher when the QTL is B6 (left) or D2 (right). Dots represent mean values from biological duplicate experiments and lines connect means. Orange indicates loci for which H3K9me3 signal decreases in the KO (p-value indicated on top, two-sided paired Wilcoxon test).


H . MA - plot similar to D showing change in gene expression between WT and Chr 4 KO mESCs ( n = 2 ) . Chr 4 QTL targets are indicated in magenta and increase expression in the KO .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

H. MA-plot similar to D showing change in gene expression between WT and Chr 4 KO mESCs (n = 2). Chr 4 QTL targets are indicated in magenta and increase expression in the KO.


Flag - INVS interacted with HA - Akt ( lanes 1 - 3 ) , but not with HA - PDK1 ( lanes 4 - 6 ) or HA - PrKA ( lanes 7 - 9 ) in HEK293T cells . Expression levels of HA - Akt , PDK1 , PrKA , and Flag - INVS were similar , as determined by immunoblotting ( HA : anti - HA antibody ; F : anti - Flag antibody ; C : control antibody ) . The results presented ( A - E ) are representative of at least two independent experiments .

O O B-MEASURED_VAR O O O O B-MEASURED_VAR O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O B-MEASURED_VAR O B-MEASURED_VAR O B-MEASURED_VAR O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Flag-INVS interacted with HA-Akt (lanes 1-3), but not with HA-PDK1 (lanes 4-6) or HA-PrKA (lanes 7-9) in HEK293T cells. Expression levels of HA-Akt, PDK1, PrKA, and Flag-INVS were similar, as determined by immunoblotting (HA: anti-HA antibody; F: anti-Flag antibody; C: control antibody). The results presented (A-E) are representative of at least two independent experiments.


B . Flag - tagged INVS interacted with HA - tagged Akt1 ( lane 1 - 3 ) , Akt2 ( lane 4 - 6 ) , and Akt3 ( lane 7 - 9 ) in co - immunoprecipitationassays . Expression of each Akt isoform and INVS were similar .

O O O O O B-MEASURED_VAR O O O O O B-MEASURED_VAR O O O O O O O B-MEASURED_VAR O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O B-MEASURED_VAR O O B-MEASURED_VAR O O O

B. Flag-tagged INVS interacted with HA-tagged Akt1 (lane 1-3), Akt2 (lane 4-6), and Akt3 (lane 7-9) in co-immunoprecipitationassays. Expression of each Akt isoform and INVS were similar.


C . INVS was co - immunoprecipitated with endogenous Akt from HEK293 cells , in which INVS is also endogenously expressed ( lane 2 ) .

O O B-MEASURED_VAR O O O O O O B-MEASURED_VAR O O O O O O B-MEASURED_VAR O O O O O O O O O

C. INVS was co-immunoprecipitated with endogenous Akt from HEK293 cells, in which INVS is also endogenously expressed (lane 2).


D . To determine the Akt domain responsible for interacting with INVS , Akt subfragments were generated . The C - terminus of Akt is the primary domain for INVS interaction ( lanes 7 - 9 ) . Similar levels of each Akt fragment were immunoprecipitated using anti - HA antibody .

O O O O O B-MEASURED_VAR O O O O O B-MEASURED_VAR O B-MEASURED_VAR O O O O O O O O O B-CONTROLLED_VAR O O O O O B-MEASURED_VAR O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O

D. To determine the Akt domain responsible for interacting with INVS, Akt subfragments were generated. The C-terminus of Akt is the primary domain for INVS interaction (lanes 7-9). Similar levels of each Akt fragment were immunoprecipitated using anti-HA antibody.


E . Schematic showing the structure and the functional domains of Flag - tagged full - length and fragmented INVS in mammalian expression vectors ( N - Term : 1 - 421 , Intermediate domain : 421 - 678 , and C - Term : 678 - 1065 ) used in the current study . Intermediate ( lanes 6 - 9 ) and C - terminal ( lanes 10 - 12 ) fragments of INVS were important for interaction with Akt .

O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O B-MEASURED_VAR O

E. Schematic showing the structure and the functional domains of Flag-tagged full-length and fragmented INVS in mammalian expression vectors (N-Term: 1-421, Intermediate domain: 421-678, and C-Term: 678-1065) used in the current study. Intermediate (lanes 6-9) and C-terminal (lanes 10-12) fragments of INVS were important for interaction with Akt.


A . The indicated recombinant INVS proteins ( lanes 1 - 4 ) were used for IVK . Akt phosphorylates WT ( lane5 ) , 1 - 970 ( lane 6 ) , and 1 - 898 ( lane 7 ) INVS , but failed to phosphorylate 1 - 670 ( lane 8 ) INVS .

O O O O O B-MEASURED_VAR O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O B-CONTROLLED_VAR O

A. The indicated recombinant INVS proteins (lanes 1-4) were used for IVK. Akt phosphorylates WT (lane5), 1-970 (lane 6), and 1-898 (lane 7) INVS, but failed to phosphorylate 1-670 (lane 8) INVS.


The indicated recombinant INVS proteins ( lanes 1 - 10 ) were used for IVK . Akt phosphorylates WT ( lane 6 ) and 1 - 898 ( lane7 ) INVS , but failed to phosphorylate other subfragments of INVS ( 1 - 822 , 1 - 746 , and 1 - 670 , in lane 8 , 9 , and 10 , respectively ) , as detected using a phospho - Akt substrate antibody . .

O O O B-MEASURED_VAR O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O

The indicated recombinant INVS proteins (lanes 1-10) were used for IVK. Akt phosphorylates WT (lane 6) and 1-898 (lane7) INVS, but failed to phosphorylate other subfragments of INVS (1-822, 1-746, and 1-670, in lane 8, 9, and 10, respectively), as detected using a phospho-Akt substrate antibody..


C . Using the indicated subfragments of INVS for IVK , we confirmed that Akt phosphorylates 675 - 1065 ( lane 4 ) , but not 675 - 822 ( lane 5 ) , or 675 - 746 ( lane 6 ) INVS .

O O O O O O O B-MEASURED_VAR O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O

C. Using the indicated subfragments of INVS for IVK, we confirmed that Akt phosphorylates 675-1065 (lane 4), but not 675-822 (lane 5), or 675-746 (lane 6) INVS.


E . Purified recombinant WT or 3A INVS were generated ( top panel , lanes 1 - 4 ) . In IVK , Akt phosphorylated the WT fragment ( 824 - 898 , middle panel , lane 3 ) , but failed to phosphorylate 3A INVS ( middle panel , lane 4 ) , as detected by using a phospho - Akt substrate antibody .

O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O B-MEASURED_VAR O O O

E. Purified recombinant WT or 3A INVS were generated (top panel, lanes 1-4). In IVK, Akt phosphorylated the WT fragment (824-898, middle panel, lane 3), but failed to phosphorylate 3A INVS (middle panel, lane 4), as detected by using a phospho-Akt substrate antibody.


F . Akt phosphorylated WT ( lane 10 ) , 1 - 898 ( lane 12 ) and 675 - 1065 ( lane 15 ) INVS , but failed to phosphorylate both full - length and 675 - 1065 , ∆ 727 - 896 , 1 - 857 , or 1 - 822 , 3A INVS ( lanes 16 , 11 , 14 , and 15 , respectively ) , all of which lacked the T864 / S865 / T866 target amino acid .

O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O

F. Akt phosphorylated WT (lane 10), 1-898 (lane 12) and 675-1065 (lane 15) INVS, but failed to phosphorylate both full-length and 675-1065, ∆727-896, 1-857, or 1-822, 3A INVS (lanes 16, 11, 14, and 15, respectively), all of which lacked the T864/S865/T866 target amino acid.


A . Confocal microscopy shows that active Akt ( phospho - S473Akt ) is localized in the basal body of primary cilia ( right side panels show higher magnification ) . B . INVS is localized in the proximal region of the INVS compartment in primary cilia ( INVS compartment , right side panels show higher magnification ) . C . INVS co - localizes with γ - tubulin , a centrosome marker . D . Anti - phospho - Akt substrates stain positively at the basal region of the primary cilia ( arrows ) , as determined by anti - acetylated tubulin ( right side panels show higher magnification ) .

O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O B-MEASURED_VAR O O O O O B-MEASURED_VAR O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

A. Confocal microscopy shows that active Akt (phospho-S473Akt) is localized in the basal body of primary cilia (right side panels show higher magnification).B. INVS is localized in the proximal region of the INVS compartment in primary cilia (INVS compartment, right side panels show higher magnification).C. INVS co-localizes with γ-tubulin, a centrosome marker.D. Anti-phospho-Akt substrates stain positively at the basal region of the primary cilia (arrows), as determined by anti-acetylated tubulin (right side panels show higher magnification).


E . Silver - intensified immunogold electron microscopy shows that both phospho - Akt ( left side panels ) and INVS ( right side panels ) were localized to the ciliary pocket of primary cilia in the presence of PDGF - AA ( white arrow ) .

O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O O O O O

E. Silver-intensified immunogold electron microscopy shows that both phospho-Akt (left side panels) and INVS (right side panels) were localized to the ciliary pocket of primary cilia in the presence of PDGF-AA (white arrow).


PDGF - AA stimulation enhances localization of active Akt ( phospho - S473Akt ) at the basal body of primary cilia ( right side panels show higher magnification ) .

B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O

PDGF-AA stimulation enhances localization of active Akt (phospho-S473Akt) at the basal body of primary cilia (right side panels show higher magnification).


Quantification of co - localization of phosphorylated Akt ( green ) and INVS ( red ) at the indicated time points after PDGF - AA stimulation revealed a time - dependent increase . Colocalization was measured by counting yellow pixels ( colocalized area ) using Imaris software ( Bitplane AG ) . Results presented are means ± SE ( n = 31 at time 0 , n = 35 at 1 min . , and n = 33 at 3 min , respectively ) . Three independent experiments were analyzed with similar results . Statistical significance was determined by student ' s t test .

O O O O O O O B-MEASURED_VAR O O O O B-MEASURED_VAR O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Quantification of co-localization of phosphorylated Akt (green) and INVS (red) at the indicated time points after PDGF-AA stimulation revealed a time-dependent increase. Colocalization was measured by counting yellow pixels (colocalized area) using Imaris software (Bitplane AG). Results presented are means ±SE (n=31 at time 0, n=35 at 1 min., and n=33 at 3 min, respectively). Three independent experiments were analyzed with similar results. Statistical significance was determined by student's t test.


Co - immunoprecipitation assays using HEK293T cells show that INVS preferentially binds to WT - Akt ( second panel , lane1 ) or phospho - mimetic Akt ( T308D / S473D , lane2 ) , but fails to interact with phosphorylation - defective forms of Akt ( T308A / S473A , lane 3 ) . PDGF - AA stimulation of HEK293T cells results in augmented interaction of endogenous INVS with Akt ( top panel , comparison between lane 1 and lane 2 , untreated and PDGF - AA - stimulated conditions , respectively ) .

O O O O O O O O O B-MEASURED_VAR O O O O O B-MEASURED_VAR O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O B-MEASURED_VAR O B-MEASURED_VAR O O O O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O

Co-immunoprecipitation assays using HEK293T cells show that INVS preferentially binds to WT-Akt (second panel, lane1) or phospho-mimetic Akt (T308D/S473D, lane2), but fails to interact with phosphorylation-defective forms of Akt (T308A/S473A, lane 3).PDGF-AA stimulation of HEK293T cells results in augmented interaction of endogenous INVS with Akt (top panel, comparison between lane 1 and lane 2, untreated and PDGF-AA-stimulated conditions, respectively).


Both WT and 3A - INVS , which are localized at the INVS compartment of primary cilia under serum - starved condition ( left side panels , lower panels show higher magnification ) , translocate to the basal body of the cilium after PDGF - AA stimulation ( right side panels , lower panels show higher magnification ) . Results presented are means ± SE of yellow pixels demonstrating colocalization of EGFP - INVS ( green ) with γ - tubulin ( red ) in hTERT - RPE1 cells ( n = 28 ) . Three independent experiments showed similar results . Statistical significance was determined by student ' s t test . Fluorescence intensities of INVS ( green ) , acetylated - α - tubulin ( blue ) , and γ - tubulin ( red ) along the line ( a - b ) are plotted underneath .

O O O O O B-CONTROLLED_VAR O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Both WT and 3A-INVS, which are localized at the INVS compartment of primary cilia under serum-starved condition (left side panels, lower panels show higher magnification), translocate to the basal body of the cilium after PDGF-AA stimulation (right side panels, lower panels show higher magnification). Results presented are means ± SE of yellow pixels demonstrating colocalization of EGFP-INVS (green) with γ-tubulin (red) in hTERT-RPE1 cells (n=28). Three independent experiments showed similar results. Statistical significance was determined by student's t test. Fluorescence intensities of INVS (green), acetylated-α-tubulin (blue), and γ-tubulin (red) along the line (a-b) are plotted underneath.


A . PDGF - AA stimulation resulted in the phosphorylation of WT INVS ( top panel , lane 2 ) , but not 3A INVS ( top panel , lane 6 ) . PDGF - AA - stimulated INVS phosphorylation is inhibited by Akt inhibitors ( top panel , LY294002 , MK2206 , and GSK690693 , lanes 3 , 4 , and 5 , respectively ) .

O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O B-MEASURED_VAR O O O O B-MEASURED_VAR O O O O O B-CONTROLLED_VAR O B-CONTROLLED_VAR O O B-CONTROLLED_VAR O O O O O O O O O O O O

A. PDGF-AA stimulation resulted in the phosphorylation of WT INVS (top panel, lane 2), but not 3A INVS (top panel, lane 6). PDGF-AA-stimulated INVS phosphorylation is inhibited by Akt inhibitors (top panel, LY294002, MK2206, and GSK690693, lanes 3, 4, and 5, respectively).


B . Myr - Akt , a constitutively active form of Akt , was expressed together with WT or 3A INVS in HEK293 cells and INVS phosphorylation was quantified . WT , but not 3A INVS , can be phosphorylated in the presence of Myr - Akt .

O O O O B-CONTROLLED_VAR O O O O O O B-MEASURED_VAR O O O O O O O O B-CONTROLLED_VAR O O O O B-MEASURED_VAR O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O B-MEASURED_VAR O

B. Myr-Akt, a constitutively active form of Akt, was expressed together with WT or 3A INVS in HEK293 cells and INVS phosphorylation was quantified. WT, but not 3A INVS, can be phosphorylated in the presence of Myr-Akt.


C . 3A INVS exhibited a weaker interaction with Akt as compared to WT INVS ( top panel , compare lanes 1 and 2 , WT and phospho - defective mutant , respectively ) . Expression of Akt and INVS are shown underneath . D . 3A INVS exhibited weaker dimerization as compared to WT INVS ( top panel , compare lanes 1 and 2 , WT and 3A , respectively ) . Expression of Akt and INVS are shown below each .

O O O B-CONTROLLED_VAR O O O O O B-MEASURED_VAR O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O B-MEASURED_VAR O O O O O O O B-CONTROLLED_VAR O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O B-MEASURED_VAR O O O O O

C. 3A INVS exhibited a weaker interaction with Akt as compared to WT INVS (top panel, compare lanes 1 and 2, WT and phospho-defective mutant, respectively). Expression of Akt and INVS are shown underneath.D. 3A INVS exhibited weaker dimerization as compared to WT INVS (top panel, compare lanes 1 and 2, WT and 3A, respectively). Expression of Akt and INVS are shown below each.


E . Length of primary cilia in confluent hTERT - RPE cells was analyzed after siRNA - mediated knock down of Akt1 / 2 . Ciliary length decreased in cells expressing siRNA . Results presented are means ± SE of the longitudinal length of acetylated - tubulin ( red ) , a marker of primary cilia , in siRNA - transfected ( green ) hTERT - RPE1 cells ( n = 175 for control siRNA and Akt siRNA , and n = 195 for Akt siRNA plus siRNA resistant Akt , respectively ) . Three independent experiments showed similar results . Statistical significance was analyzed by student ' s t test . Note that re - introduction of siRNA - resistant Akt ( Matsuda - Lennikov et al . , 2014 ) in Akt knockdown cells rescued the effect on ciliary length .

O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O B-CONTROLLED_VAR O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O

E. Length of primary cilia in confluent hTERT-RPE cells was analyzed after siRNA-mediated knock down of Akt1/2. Ciliary length decreased in cells expressing siRNA. Results presented are means ±SE of the longitudinal length of acetylated-tubulin (red), a marker of primary cilia, in siRNA-transfected (green) hTERT-RPE1 cells (n=175 for control siRNA and Akt siRNA, and n=195 for Akt siRNA plus siRNA resistant Akt, respectively). Three independent experiments showed similar results. Statistical significance was analyzed by student's t test. Note that re-introduction of siRNA-resistant Akt (Matsuda-Lennikov et al., 2014) in Akt knockdown cells rescued the effect on ciliary length.


F . Expression of 3A INVS ( bottom panels ) but not WT INVS ( middle panels ) inhibited the development of primary cilia as compared to control cells ( top panels ) .

O O O O O B-MEASURED_VAR O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O

F. Expression of 3A INVS (bottom panels) but not WT INVS (middle panels) inhibited the development of primary cilia as compared to control cells (top panels).


Quantification of ciliary length . Results are mean ± SE of longitudinal length of acetylated - tubulin ( blue ) , a marker of primary cilia , in EGFP - INVS ( green ) transfected in hTERT - RPE1 cells ( n = 58 for EGFP vector and for EGFP - INVS WT , and n = 52 for EGFP - INVS 3A mutant , respectively ) . Three independent experiments showed similar results . Statistical significance was determined by student ' s t test .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O

Quantification of ciliary length. Results are mean ± SE of longitudinal length of acetylated-tubulin (blue), a marker of primary cilia, in EGFP-INVS (green) transfected in hTERT-RPE1 cells (n=58 for EGFP vector and for EGFP-INVS WT, and n=52 for EGFP-INVS 3A mutant, respectively). Three independent experiments showed similar results. Statistical significance was determined by student's t test.


Schematic describing the measurement of spindle angle ( θ ) ( Kikuchi et al . , 2010 ) . Z - stack images were obtained from 0 . 45 mm - thick sections of metaphase cells and the angle between the axis of metaphase spindle and that of the substrate surface , which is termed the spindle angle , was calculated . Results presented are scatter plots of the angle ( θ ) of the mitotic spindle ( n = 28 for all samples of vector , HA - WT , - 3D , - 3A , - R899X , - Q671X , and - R603X INVS stably transfected MDCK cells ) . Three independent experiments showed similar results . Statistical significance was determined by Mann - Whitney ' s non - parametric median test .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Schematic describing the measurement of spindle angle (θ) (Kikuchi et al., 2010). Z-stack images were obtained from 0.45 mm-thick sections of metaphase cells and the angle between the axis of metaphase spindle and that of the substrate surface, which is termed the spindle angle, was calculated. Results presented are scatter plots of the angle (θ) of the mitotic spindle (n=28 for all samples of vector, HA-WT, -3D, -3A, -R899X, -Q671X, and -R603X INVS stably transfected MDCK cells). Three independent experiments showed similar results. Statistical significance was determined by Mann-Whitney's non-parametric median test.


Spindle angles were measured in MDCK cells transfected with the indicated INVS plasmids ( + ) . The spindle axis was misaligned in cells expressing mutant forms of INVS as compared to cells expressing WT INVS or sham - transduced cells . Expression of 3A INVS and of clinically relevant truncated forms of INVS but not WT INVS , resulted in disorientation of the spindle axis during metaphase . Similar expression of all INVS constructs was verified by immunoblotting ( Appendix figure S4A ) .

O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O B-CONTROLLED_VAR O O O O O O O O O B-CONTROLLED_VAR O O O O O O O B-CONTROLLED_VAR O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O

Spindle angles were measured in MDCK cells transfected with the indicated INVS plasmids (+). The spindle axis was misaligned in cells expressing mutant forms of INVS as compared to cells expressing WT INVS or sham-transduced cells. Expression of 3A INVS and of clinically relevant truncated forms of INVS but not WT INVS, resulted in disorientation of the spindle axis during metaphase. Similar expression of all INVS constructs was verified by immunoblotting (Appendix figure S4A).


WT INVS robustly inhibited the transactivation of INVS in a luciferase reporter assay , which could be reverted by C - terminal truncated INVS ( R899X , Q671X , or Q603X ) , but not by a phospho - mimetic mutant or 3A INVS . Results presented are mean ± SE of the ratio of triplicates between sequentially measured firefly TCF / LEF signal and Renilla luciferase signal in HA - INVS transfected 293T cells . Dual - Luciferase reporter ® Assay System ( Promega ) was used and experiments were performed in triplicates . Two independent experiments showed similar results . Statistical significance was determined by student ' s t test .

O B-MEASURED_VAR O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

WT INVS robustly inhibited the transactivation of INVS in a luciferase reporter assay, which could be reverted by C-terminal truncated INVS (R899X, Q671X, or Q603X), but not by a phospho-mimetic mutant or 3A INVS. Results presented are mean ± SE of the ratio of triplicates between sequentially measured firefly TCF/LEF signal and Renilla luciferase signal in HA-INVS transfected 293T cells. Dual-Luciferase reporter® Assay System (Promega) was used and experiments were performed in triplicates. Two independent experiments showed similar results. Statistical significance was determined by student's t test.


The expression of HA - Dvl ( upper panels ) and the indicated forms of Flag - INVS ( lower panels ) are shown by immunoblots ; α - tubulin was used as an internal control .

O O O O O B-MEASURED_VAR O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O

The expression of HA-Dvl (upper panels) and the indicated forms of Flag-INVS (lower panels) are shown by immunoblots; α-tubulin was used as an internal control.


WT INVS - transduced MDCK cells in 3D cultures form uniform round tubules . However , expression of 3A or NPHP2 - related truncated INVS mutants , which lack Akt phosphorylation site ( s ) , disrupts this uniformity . Results presented are representative of three independent experiments and show mean ± SE of percent normal acini in vector - or WT INVS - transduced MDCK cells ( n = 422 for Vector , n = 398 for HA - WT , n = 401 for - 3D , n = 365 for - 3A , n = 344 for - R899X , n = 331 for - Q671X , and n = 325 for - R603X INVS stably transfected MDCK cells , respectively ) . Three independent experiments showed similar results . Statistical significance was determined by student ' s t test .

O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O

WT INVS- transduced MDCK cells in 3D cultures form uniform round tubules. However, expression of 3A or NPHP2-related truncated INVS mutants, which lack Akt phosphorylation site (s), disrupts this uniformity. Results presented are representative of three independent experiments and show mean ± SE of percent normal acini in vector- or WT INVS-transduced MDCK cells (n=422 for Vector, n=398 for HA-WT, n=401 for -3D, n=365 for -3A, n= 344 for -R899X, n=331 for -Q671X, and n=325 for -R603X INVS stably transfected MDCK cells, respectively). Three independent experiments showed similar results. Statistical significance was determined by student's t test.


WT transduced MDCK cells form a relatively smaller lumen ; however , expression of 3A INVS or the indicated NPHP2 - related truncation mutants result in relatively larger acini . Both truncation mutants ( Q671X or R603X ) lack the Akt phosphorylation site and exhibit a larger lumen size . Three independent experiments showed similar results .

O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

WT transduced MDCK cells form a relatively smaller lumen; however, expression of 3A INVS or the indicated NPHP2-related truncation mutants result in relatively larger acini. Both truncation mutants (Q671X or R603X) lack the Akt phosphorylation site and exhibit a larger lumen size. Three independent experiments showed similar results.


C . Quantification of normal lumen in cells transduced with WT or mutant INVS . Results presented are mean ± SE of percent normal acini in vector - or WT INVS - transduced MDCK cells ( n = 166 for Vector , n = 204 for HA - WT , n = 233 for - 3D , n = 150 for - 3A , n = 164 for - R899X , n = 128 for - Q671X , and n = 137 for - R603X INVS stably transfected MDCK cells , respectively ) . Three independent experiments showed similar results . Statistical significance was determined by student ' s t test .

O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O

C. Quantification of normal lumen in cells transduced with WT or mutant INVS. Results presented are mean ±SE of percent normal acini in vector- or WT INVS-transduced MDCK cells (n=166 for Vector, n=204 for HA-WT, n=233 for -3D, n=150 for -3A, n=164 for -R899X, n=128 for -Q671X, and n=137 for -R603X INVS stably transfected MDCK cells, respectively). Three independent experiments showed similar results. Statistical significance was determined by student's t test.


D . Size of lumen in cells transduced with WT or mutant INVS is shown . Results presented are scatter plots of acini size ( n = 205 for Vector , n = 143 for HA - WT , n = 131 for - 3D , n = 167 for - 3A , n = 144 for - R899X , n = 128 for - Q671X , and n = 144 for - R603X INVS stably transfected MDCK cells , respectively ) . Two independent experiments showed similar results . Statistical significance was determined by Mann - Whitney ' s non - parametric median test .

O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

D. Size of lumen in cells transduced with WT or mutant INVS is shown. Results presented are scatter plots of acini size (n=205 for Vector, n=143 for HA-WT, n=131 for -3D, n=167 for -3A, n=144 for -R899X, n=128 for -Q671X, and n=144 for -R603X INVS stably transfected MDCK cells, respectively). Two independent experiments showed similar results. Statistical significance was determined by Mann-Whitney's non-parametric median test.


B ) Electrophoretic mobility shift assay ( EMSA ) examination of the binding affinity of CENPC - CT and CM peptide to a CENP - A nucleosome . Binding to a canonical H3 nucleosome was also examined using EMSA .

O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O

B) Electrophoretic mobility shift assay (EMSA) examination of the binding affinity of CENPC-CT and CM peptide to a CENP-A nucleosome. Binding to a canonical H3 nucleosome was also examined using EMSA.


C ) EMSA results of the binding affinities of CENPC - CT WT , CENPC - CT 3A679 - 681 , and CENPC - CT ∆ 678 - 683 to the CENP - A nucleosome .

O O O O O O O O O B-MEASURED_VAR O O O O B-MEASURED_VAR O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O

C) EMSA results of the binding affinities of CENPC-CT WT, CENPC-CT 3A679-681, and CENPC-CT ∆678-683 to the CENP-A nucleosome.


D ) Stable expression of GFP - fused CENPC - CT and its mutants ( shown in ( B ) ) in CENP - C knockout chicken DT40 cells . α - Tubulin ( Tub ) was probed as a loading control . Parental CENP - C knockout cells ( parental ) were also examined .

O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O

D) Stable expression of GFP-fused CENPC-CT and its mutants (shown in (B)) in CENP-C knockout chicken DT40 cells. α-Tubulin (Tub) was probed as a loading control. Parental CENP-C knockout cells (parental) were also examined.


E ) Localization analysis of GFP - fused CENPC - CT WT , CENPC - CT 3A679 - 681 , and CENPC - CT ∆ 678 - 683 ( green ) on the mitotic chromosomes in chicken DT40 cells . CENP - T ( red ) was used as a centromere marker . DNA was stained using 4 ' , 6 - diamidino - 2 - phenylindole ( DAPI ; blue ) . Scale bar indicates 10 μm .

O O O O O O O O B-MEASURED_VAR O O O O B-MEASURED_VAR O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

E) Localization analysis of GFP-fused CENPC-CT WT, CENPC-CT 3A679-681, and CENPC-CT ∆678-683 (green) on the mitotic chromosomes in chicken DT40 cells. CENP-T (red) was used as a centromere marker. DNA was stained using 4',6-diamidino-2-phenylindole (DAPI; blue). Scale bar indicates 10 μm.


F ) Localization of CENP - C in CENP - A knockout DT40 cells stably expressing GFP - fused gCENP - A WT , RG - AA , or ∆ RG . Scale bar indicates 10 μm . CENP - C was stained by an anti - CENP - C antibody ( red ) and DNA was stained by DAPI ( blue ) . CENP - C signals on kinetochores in mitotic cells were quantified in each cell . Bar graph indicates mean ± SD ( n = 7 ; * * * * , P < 0 . 0001 , unpaired t test , two tailed ) .

O O O O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O B-MEASURED_VAR I-MEASURED_VAR O O O O O O O O O O O O O O O O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O O O O O O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O O O O O O O O O O O O O O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

F) Localization of CENP-C in CENP-A knockout DT40 cells stably expressing GFP-fused gCENP-A WT, RG-AA, or ∆RG. Scale bar indicates 10 μm. CENP-C was stained by an anti-CENP-C antibody (red) and DNA was stained by DAPI (blue). CENP-C signals on kinetochores in mitotic cells were quantified in each cell. Bar graph indicates mean ± SD (n = 7; ****, P < 0.0001, unpaired t test, two tailed).


C ) EMSA results of the binding affinities of CENP - C fragments indicated in ( B ) to the CENP - A nucleosome . All fragments were phosphorylated by CDK1 .

O O O O O O O O O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O

C) EMSA results of the binding affinities of CENP-C fragments indicated in (B) to the CENP-A nucleosome. All fragments were phosphorylated by CDK1.


D ) Stable expression of GFP - fused CENPC - CT and CENPC643 - 864 ( shown in ( B ) ) in CENP - C knockout chicken DT40 cells . α - Tubulin ( Tub ) was probed as a loading control .

O O O O O O O O B-MEASURED_VAR O O O B-MEASURED_VAR O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O

D) Stable expression of GFP-fused CENPC-CT and CENPC643-864 (shown in (B)) in CENP-C knockout chicken DT40 cells. α-Tubulin (Tub) was probed as a loading control.


E ) Localization analysis of GFP - fused CENPC - CT and CENPC643 - 864 lacking the CM upstream region ( green ) on mitotic chromosomes in CENP - C knockout DT40 cells . CENP - T ( red ) was used as a centromere marker . DNA was stained using DAPI ( blue ) . Scale bar indicates 10 μm .

O O O O O O O O B-MEASURED_VAR O O O B-MEASURED_VAR O O O O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O

E) Localization analysis of GFP-fused CENPC-CT and CENPC643-864 lacking the CM upstream region (green) on mitotic chromosomes in CENP-C knockout DT40 cells. CENP-T (red) was used as a centromere marker. DNA was stained using DAPI (blue). Scale bar indicates 10 μm.


A ) The position of a CDK1 phosphorylation site , T651CENP - C , is indicated in the diagram of chicken CENPC - CT . EMSA was performed to examine the binding affinities of phosphorylated or non - phosphorylated CENPC - CT ( left panel ) and CM peptide ( right panel ) to the CENP - A nucleosome .

O O O O O O O O O O O B-MEASURED_VAR I-MEASURED_VAR O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O

A) The position of a CDK1 phosphorylation site, T651CENP-C, is indicated in the diagram of chicken CENPC-CT. EMSA was performed to examine the binding affinities of phosphorylated or non-phosphorylated CENPC-CT (left panel) and CM peptide (right panel) to the CENP-A nucleosome.


D ) All constructs analyzed in ( E ) were stably expressed in CENP - C knockout cells .

O O O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O

D) All constructs analyzed in (E) were stably expressed in CENP-C knockout cells.


E ) Localization analysis of GFP - fused CENPC - CT , CENPC - CTR656E , and CENPC - CTR655E _ R656E ( green ) on mitotic chromosomes in chicken DT40 cells . DNA was stained using DAPI ( blue ) . Scale bar indicates 10 μm .

O O O O O O O O B-MEASURED_VAR O O O B-MEASURED_VAR O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

E) Localization analysis of GFP-fused CENPC-CT, CENPC-CTR656E, and CENPC-CTR655E_R656E (green) on mitotic chromosomes in chicken DT40 cells. DNA was stained using DAPI (blue). Scale bar indicates 10 μm.


A ) Competitive pull - down assays for the CENP - A nucleosome binding of CENPC - CT and CENPN - NT . The left panel displays a complex of MBP - CENPN - NT ( MBP - CN - NT ) with the CENP - A nucleosome ( CA - nuc ) incubated in the absence or presence of phosphorylated CENPC - CT ( CC - CT ) and pulled down by MBP affinity . The right panel displays a complex of phosphorylated MBP - CENPC - CT ( MBP - CC - CT ) with the CENP - A nucleosome ( CA - nuc ) incubated in the absence or presence of CENPN - NT ( CN - NT ) and pulled down by MBP affinity . The signal intensities of all histones in CENP - A nucleosomes , which were precipitated with MBP proteins in indicated lanes ( * ) , were quantified . The signal intensities were normalized to MBP - CN - NT or MBP - CC - CT signals . Bar graph indicates mean with SD ( n = 3 ) .

O O O O O O O O O O O O O O O B-MEASURED_VAR O O O B-MEASURED_VAR O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

A) Competitive pull-down assays for the CENP-A nucleosome binding of CENPC-CT and CENPN-NT. The left panel displays a complex of MBP-CENPN-NT (MBP-CN-NT) with the CENP-A nucleosome (CA-nuc) incubated in the absence or presence of phosphorylated CENPC-CT (CC-CT) and pulled down by MBP affinity. The right panel displays a complex of phosphorylated MBP-CENPC-CT (MBP-CC-CT) with the CENP-A nucleosome (CA-nuc) incubated in the absence or presence of CENPN-NT (CN-NT) and pulled down by MBP affinity. The signal intensities of all histones in CENP-A nucleosomes, which were precipitated with MBP proteins in indicated lanes (*), were quantified. The signal intensities were normalized to MBP-CN-NT or MBP-CC-CT signals. Bar graph indicates mean with SD (n = 3).


Immunoprecipitation of SATB2 from mouse neonatal cortical lysates . LEMD2 was detected by Western blotting in the SATB2 - immunoprecipitate from control but not SATB2 - deficient cortical lysate . The equal input of total protein from control and SATB2 - deficient cortical lysates was controlled by ERK2 detection . Representative images of the immunoblots are shown ; I ( Input ) , F ( Flow - through ) , B ( Beads ) .

O O B-MEASURED_VAR O O O O O O B-MEASURED_VAR O O O O O O O B-MEASURED_VAR O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Immunoprecipitation of SATB2 from mouse neonatal cortical lysates. LEMD2 was detected by Western blotting in the SATB2-immunoprecipitate from control but not SATB2-deficient cortical lysate. The equal input of total protein from control and SATB2-deficient cortical lysates was controlled by ERK2 detection. Representative images of the immunoblots are shown; I (Input), F (Flow-through), B (Beads).


Reverse immunoprecipitation with LEMD2 antibody and control IgG antibody , followed by WB detection of LEMD2 and SATB2 . Lysates from primary cortical culture lysates were used .

O O O B-MEASURED_VAR O O O O O O O O O O O B-MEASURED_VAR O B-MEASURED_VAR O O O O O O O O O O

Reverse immunoprecipitation with LEMD2 antibody and control IgG antibody, followed by WB detection of LEMD2 and SATB2. Lysates from primary cortical culture lysates were used.


GST pull - down assays . Representative images of n = 3 independent experiments are shown . GST pull - down assay of transiently overexpressed V5 - tagged LEMD2 ( i ) , SATB2 ( ii ) , and LEMD2ΔLEM ( iii ) from HeLa cell lysates using GST , GST - SATB2 , and GST - LEMD2 ( 413 - 503 ) hybrid proteins .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O B-MEASURED_VAR O O O O O B-MEASURED_VAR O O O O O O O O O O O O B-CONTROLLED_VAR O O O O B-CONTROLLED_VAR O O O O O O O O

GST pull-down assays. Representative images of n = 3 independent experiments are shown. GST pull-down assay of transiently overexpressed V5-tagged LEMD2 (i), SATB2 (ii), and LEMD2ΔLEM (iii) from HeLa cell lysates using GST, GST-SATB2, and GST-LEMD2(413-503) hybrid proteins.


Immunoprecipitations using anti - V5 - tag antibody from lysates of HeLa cells transfected with expression plasmids encoding V5 - tagged full - length SATB2 ( Satb21 - 733 ) and EGFP as control . The equal input of total protein was controlled by GAPDH detection . Representative images of the immunoblots are shown ; I ( Input ) , F ( Flow ‐ through ) , B ( Beads ) , M ( Molecular weight marker ) .

O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Immunoprecipitations using anti-V5-tag antibody from lysates of HeLa cells transfected with expression plasmids encoding V5-tagged full-length SATB2 (Satb21-733) and EGFP as control. The equal input of total protein was controlled by GAPDH detection. Representative images of the immunoblots are shown; I (Input), F (Flow‐through), B (Beads), M (Molecular weight marker).


The CUT - like domain of SATB2 is required for the interaction with LEMD2 . Immunoprecipitations using anti - V5 - tag antibody from lysates of HeLa cells transfected with V5 - tagged Satb21 - 247 and Satb21 - 157 deletion mutants . LEMD2 and BAF were detected only in immunoprecipitates from HeLa cells transfected with the deletion mutant containing the CUT - like domain ( Satb21 - 247 ) . The equal input of total protein was controlled by GAPDH detection . Representative images of the immunoblots are shown ; I ( Input ) , F ( Flow ‐ through ) , B ( Beads ) , M ( Molecular weight marker ) .

O O O O O O B-MEASURED_VAR O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

The CUT-like domain of SATB2 is required for the interaction with LEMD2. Immunoprecipitations using anti-V5-tag antibody from lysates of HeLa cells transfected with V5-tagged Satb21-247 and Satb21-157 deletion mutants. LEMD2 and BAF were detected only in immunoprecipitates from HeLa cells transfected with the deletion mutant containing the CUT-like domain (Satb21-247). The equal input of total protein was controlled by GAPDH detection. Representative images of the immunoblots are shown; I (Input), F (Flow‐through), B (Beads), M (Molecular weight marker).


CUT1 and CUT2 domains are required for the interaction with LEMD2 . Immunoprecipitations using anti - V5 - tag antibody from HeLa cells transfected with V5 - tagged Satb2346 - 733 and Satb2616 - 733 deletion mutants . LEMD2 and BAF were detected only in immunoprecipitates from HeLa cells expressing the CUT1 and CUT2 containing deletion mutant ( Satb2346 - 733 ) .

O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O

CUT1 and CUT2 domains are required for the interaction with LEMD2. Immunoprecipitations using anti-V5-tag antibody from HeLa cells transfected with V5-tagged Satb2346-733 and Satb2616-733 deletion mutants. LEMD2 and BAF were detected only in immunoprecipitates from HeLa cells expressing the CUT1 and CUT2 containing deletion mutant (Satb2346-733).


Representative images of z - axis projected stacks of confocal images of hippocampal neuronal nuclei immunostained for Lamin B2 . Examples of spherical and infolded nuclei are shown . Scale bars : 3 μm .

O O O O O O O O O O O O O O O O O B-MEASURED_VAR I-MEASURED_VAR O O O O O O O O O O O O O O O O

Representative images of z-axis projected stacks of confocal images of hippocampal neuronal nuclei immunostained for Lamin B2. Examples of spherical and infolded nuclei are shown. Scale bars: 3 μm.


Activity - induced formation of nuclear infoldings is abolished in SATB2 - defficient hippocampal neurons . Bic - induced AP bursting for 1 h caused a significant increase in the percentage of infolded nuclei in DIV10 hippocampal cultures derived from Satb2flx / flx mice but not from Satb2flx / flx : : Nes - Cre mice ( Satb2NesCre ) ( n = 6 independent primary cultures , two - way ANOVA , F1 , 20 = 9 . 51 , significant interaction p = 0 . 0058 , simple main effects analysis , Satb2flx / flx cultures , Bic - treated vs untreated p = 0 . 0000043 , Satb2NesCre cultures , Bic - treated vs untreated p > 0 . 05 , adjustment for multiple comparisons : Bonferroni , number of analyzed nuclei : Satb2flx / flx cultures , untreated - 680 , Satb2flx / flx cultures , Bic - treated - 711 , Satb2NesCre , untreated - 755 , Satb2NesCre , Bic - treated - 720 ) . Data are presented as mean ± SEM , * * * p < 0 . 001 .

O O O O O O O O O O B-CONTROLLED_VAR O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O B-CONTROLLED_VAR O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O B-CONTROLLED_VAR O O O O O O O O O O B-CONTROLLED_VAR O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O B-CONTROLLED_VAR O O O O B-CONTROLLED_VAR O O O O O B-CONTROLLED_VAR O O O O O B-CONTROLLED_VAR O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O

Activity-induced formation of nuclear infoldings is abolished in SATB2-defficient hippocampal neurons. Bic-induced AP bursting for 1 h caused a significant increase in the percentage of infolded nuclei in DIV10 hippocampal cultures derived from Satb2flx/flx mice but not from Satb2flx/flx::Nes-Cre mice (Satb2NesCre) (n = 6 independent primary cultures, two-way ANOVA, F1,20 = 9.51, significant interaction p = 0.0058, simple main effects analysis, Satb2flx/flx cultures, Bic-treated vs untreated p = 0.0000043, Satb2NesCre cultures, Bic-treated vs untreated p > 0.05, adjustment for multiple comparisons: Bonferroni, number of analyzed nuclei: Satb2flx/flx cultures, untreated - 680, Satb2flx/flx cultures, Bic-treated - 711, Satb2NesCre, untreated - 755, Satb2NesCre, Bic-treated - 720). Data are presented as mean ± SEM, ***p < 0.001.


Activity - induced formation of nuclear infoldings is impaired in SATB2 - deficient cortical neurons . DIV14 cortical cultures form Satb2flx / flx and Satb2NesCre mice were silenced with NBQX for 1 h followed by stimulation with Bic for 1 h . The percentage of infolded nuclei following AP bursting was increased in control Satb2flx / flx cultures but not in SATB2 - deficient cultures ( n = 3 - 4 independent primary cultures , two - way ANOVA , F1 , 10 = 39 . 124 , significant interaction p = 0 . 000094 , simple main effects analysis , Satb2flx / flx cultures : Bic - treated vs NBQX - treated p = 7 . 66E - 07 , Satb2NesCre cultures : Bic - treated vs NBQX - treated p > 0 . 05 , adjustment for multiple comparisons : Bonferroni , number of analyzed nuclei : Satb2flx / flx cultures , NBQX - 606 , Satb2flx / flx cultures , Bic - 910 ; Satb2NesCre cultures , NBQX - 604 , Satb2NesCre cultures , Bic - 835 ) . Data are presented as mean ± SEM , * * * p < 0 . 001 .

O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O B-CONTROLLED_VAR O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O B-CONTROLLED_VAR O O O O O O O O B-CONTROLLED_VAR O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O

Activity-induced formation of nuclear infoldings is impaired in SATB2-deficient cortical neurons. DIV14 cortical cultures form Satb2flx/flx and Satb2NesCre mice were silenced with NBQX for 1 h followed by stimulation with Bic for 1 h. The percentage of infolded nuclei following AP bursting was increased in control Satb2flx/flx cultures but not in SATB2-deficient cultures (n = 3-4 independent primary cultures, two-way ANOVA, F1,10 = 39.124, significant interaction p = 0.000094, simple main effects analysis, Satb2flx/flx cultures: Bic-treated vs NBQX-treated p = 7.66E-07, Satb2NesCre cultures: Bic-treated vs NBQX-treated p > 0.05, adjustment for multiple comparisons: Bonferroni, number of analyzed nuclei: Satb2flx/flx cultures, NBQX - 606, Satb2flx/flx cultures, Bic - 910; Satb2NesCre cultures, NBQX -604, Satb2NesCre cultures, Bic - 835). Data are presented as mean ± SEM, ***p < 0.001.


Confocal images ( z - axis - projected stacks ) of Lamin B2 stained neuronal nuclei from the CA1 pyramidal cell layer of adult Satb2flx / flx and Satb2flx / flx : : CamK2a - Cre ( Satb2CamkCre ) mice . Scale bars : 10 μm .

O O O O O O O O O O O B-MEASURED_VAR I-MEASURED_VAR O O O O O O O O O O O B-CONTROLLED_VAR O O O B-CONTROLLED_VAR O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O

Confocal images (z-axis-projected stacks) of Lamin B2 stained neuronal nuclei from the CA1 pyramidal cell layer of adult Satb2flx/flx and Satb2flx/flx::CamK2a-Cre (Satb2CamkCre) mice. Scale bars: 10 μm.


Analysis of the percentage of infolded nuclei in the hippocampus of Satb2flx / flx mice vs Satb2CamkCre mice . In Satb2flx / flx mice , the number of infolded nuclei was significantly higher in the CA1 area compared to the CA3 area ( p = 0 . 000001 ) and DG ( p = 1 . 8 E - 12 ) . In Satb2CamkCre mice , the number of infolded nuclei in the CA1 area was significantly reduced compared to the corresponding number in littermate controls ( p = 1 . 9 E - 09 ) . No significant differences were observed in this number in the CA3 and DG between Satb2CamkCre mice and floxed controls ( n = 4 mice , two - way ANOVA , significant interaction , F2 , 18 = 47 . 81 , p = 0 . 00001 , simple main effects analysis , CA1 area , Satb2flx / flx vs CKO mice p = 1 . 9 E - 09 , CA3 area , Satb2flx / flx vs CKO mice p = 0 . 909 , DG , Satb2flx / flx vs CKO mice p = 0 . 262 , adjustment for multiple comparisons : Bonferroni ) . Number of analyzed nuclei : Satb2flx / flx mice : 469 ( CA1 ) , 225 ( CA3 ) , 665 ( DG ) ; Satb2CamkCre : 508 ( CA1 ) , 224 ( CA3 ) , 575 ( DG ) . Data are presented as mean ± SEM , * * * p < 0 . 001 .

O O O O O O O O O O O B-CONTROLLED_VAR O O O O B-CONTROLLED_VAR O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Analysis of the percentage of infolded nuclei in the hippocampus of Satb2flx/flx mice vs Satb2CamkCre mice. In Satb2flx/flx mice, the number of infolded nuclei was significantly higher in the CA1 area compared to the CA3 area (p = 0.000001) and DG (p = 1.8 E-12). In Satb2CamkCre mice, the number of infolded nuclei in the CA1 area was significantly reduced compared to the corresponding number in littermate controls (p = 1.9 E-09). No significant differences were observed in this number in the CA3 and DG between Satb2CamkCre mice and floxed controls (n = 4 mice, two-way ANOVA, significant interaction, F2,18 = 47.81, p = 0.00001, simple main effects analysis, CA1 area, Satb2flx/flx vs CKO mice p = 1.9 E-09, CA3 area, Satb2flx/flx vs CKO mice p = 0.909, DG, Satb2flx/flx vs CKO mice p = 0.262, adjustment for multiple comparisons: Bonferroni). Number of analyzed nuclei: Satb2flx/flx mice: 469 (CA1), 225 (CA3), 665 (DG); Satb2CamkCre: 508 (CA1), 224 (CA3), 575 (DG). Data are presented as mean ± SEM, ***p < 0.001.


Analysis of the percentage of infolded nuclei in the CA1 pyramidal cell layer of Satb2CamkCre mice vs . littermate controls after stereotaxic injections of AAV - SATB2 - V5 or AAV8 - EGFP . Viral delivery of SATB2 resulted in restoration of the number of infolded nuclei in the CA1 pyramidal cell layer of Satb2CamkCre mice ( n = 3 - 5 mice , one - way ANOVA followed by Hochberg post hoc test , F2 , 9 = 33 . 1 , Satb2CamkCre : : AAV - SATB2 vs . Satb2flx / flx : : AAV - EGFP , p = 0 . 0012 ; Satb2flx / flx : : AAV - EGFP vs . Satb2CamkCre : : AAV - EGFP , p = 0 . 00008 , Satb2flx / flx : : AAV - EGFP vs . Satb2CamkCre : : AAV - SATB2 , p = 0 . 072 ) . Number of analyzed nuclei : 388 ( Satb2flx / flx : : AAV - EGFP ) , 560 ( Satb2CamkCre : : AAV - EGFP ) , 546 ( Satb2CamkCre : : AAV - SATB2 ) . Data are presented as mean ± SEM , * * * p < 0 . 001 compared to Satb2flx / flx : : AAV - EGFP , # # p < 0 . 01 , compared to Satb2CamkCre : : AAV - EGFP .

O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O B-CONTROLLED_VAR O O B-CONTROLLED_VAR O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O B-CONTROLLED_VAR O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O B-CONTROLLED_VAR O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O

Analysis of the percentage of infolded nuclei in the CA1 pyramidal cell layer of Satb2CamkCre mice vs. littermate controls after stereotaxic injections of AAV-SATB2-V5 or AAV8-EGFP. Viral delivery of SATB2 resulted in restoration of the number of infolded nuclei in the CA1 pyramidal cell layer of Satb2CamkCre mice (n = 3-5 mice, one-way ANOVA followed by Hochberg post hoc test, F2,9 = 33.1, Satb2CamkCre::AAV-SATB2 vs. Satb2flx/flx::AAV-EGFP, p = 0.0012; Satb2flx/flx::AAV-EGFP vs. Satb2CamkCre::AAV-EGFP, p = 0.00008, Satb2flx/flx::AAV-EGFP vs. Satb2CamkCre::AAV-SATB2, p = 0.072). Number of analyzed nuclei: 388 (Satb2flx/flx::AAV-EGFP), 560 (Satb2CamkCre::AAV-EGFP), 546 (Satb2CamkCre::AAV-SATB2). Data are presented as mean ± SEM, ***p < 0.001 compared to Satb2flx/flx::AAV-EGFP, ##p < 0.01, compared to Satb2CamkCre::AAV-EGFP.


Nuclear infolding triggered by SATB2 overexpression requires LEMD2 . Analysis of the percentage of infolded nuclei in DIV10 hippocampal neurons upon Satb2 overexpression and Lemd2 knockdown . AAV - SATB2 - transduced neurons ( both non - transfected and scrambled siRNA - transfected , Scr ) exhibited higher percentage of infolded nuclei compared to AAV - EGFP - transduced neurons . In contrast , LEMD2 - depleted AAV - SATB2 - transduced cultures showed similar percentage of infolded nuclei to AAV - EGFP - transduced neurons ( n = 3 - 4 independent primary cultures ; one - way ANOVA F3 , 11 = 30 . 982 , p = 0 . 000011 ; Hochberg post hoc test , AAV - EGFP vs . AAV - SATB2 p = 0 . 0001 , AAV - EGFP vs . AAV - SATB2 + Scr p = 0 . 0001 , AAV - EGFP vs . AAV - SATB2 + siLemd2 p = 0 . 9999 , AAV - SATB2 + Scr vs . AAV - SATB2 + siLemd2 p = 0 . 0001 ) . Number of nuclei analyzed : 460 ( AAV - EGFP ) , 466 ( AAV - SATB2 ) , 537 ( AAV - SATB2 + Scr ) , 597 ( AAV - SATB2 + siLem2 ) . Data are presented as mean ± SEM , * * * p < 0 . 001 compared to AAV - EGFP ; # # # p < 0 . 001 compared to AAV - SATB2 + Scr .

O O O O B-CONTROLLED_VAR O O B-CONTROLLED_VAR O O O O O O O O O O O O O B-CONTROLLED_VAR O O B-CONTROLLED_VAR O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O B-CONTROLLED_VAR O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O B-CONTROLLED_VAR O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O

Nuclear infolding triggered by SATB2 overexpression requires LEMD2. Analysis of the percentage of infolded nuclei in DIV10 hippocampal neurons upon Satb2 overexpression and Lemd2 knockdown. AAV-SATB2-transduced neurons (both non-transfected and scrambled siRNA-transfected, Scr) exhibited higher percentage of infolded nuclei compared to AAV-EGFP-transduced neurons. In contrast, LEMD2-depleted AAV-SATB2-transduced cultures showed similar percentage of infolded nuclei to AAV-EGFP-transduced neurons (n = 3-4 independent primary cultures; one-way ANOVA F3,11 = 30.982, p = 0.000011; Hochberg post hoc test, AAV-EGFP vs. AAV-SATB2 p = 0.0001, AAV-EGFP vs. AAV-SATB2 + Scr p = 0.0001, AAV-EGFP vs. AAV-SATB2 + siLemd2 p= 0.9999, AAV-SATB2 + Scr vs. AAV-SATB2 + siLemd2 p = 0.0001). Number of nuclei analyzed: 460 (AAV-EGFP), 466 (AAV-SATB2), 537 (AAV-SATB2 + Scr), 597 (AAV-SATB2+siLem2). Data are presented as mean ± SEM, ***p < 0.001 compared to AAV-EGFP; ###p < 0.001 compared to AAV-SATB2 + Scr.


Activity - induced formation of nuclear infoldings is impaired in LEMD2 - depleted cortical neurons . Bic - triggered AP bursting for 1 h caused a significant increase in the percentage of infolded nuclei in cortical neurons transduced with AAV - scrambled but not with AAV - shLemd2 virus ( n = 5 independent primary cultures , two - way ANOVA , F1 , 16 = 8 . 35 , significant interaction p = 0 . 0107 , simple main effects analysis : AAV - scrambled - transduced cultures , Bic - treated vs untreated p = 0 . 0049 , AAV - shLemd2 - transduced cultures , Bic - treated vs untreated p > 0 . 99 , adjustment for multiple comparisons : Bonferroni ) . Data are presented as mean ± SEM , * * p < 0 . 01 compared to NBQX - treated cultures .

O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Activity-induced formation of nuclear infoldings is impaired in LEMD2-depleted cortical neurons. Bic-triggered AP bursting for 1 h caused a significant increase in the percentage of infolded nuclei in cortical neurons transduced with AAV-scrambled but not with AAV-shLemd2 virus (n = 5 independent primary cultures, two-way ANOVA, F1,16 = 8.35, significant interaction p = 0.0107, simple main effects analysis: AAV-scrambled-transduced cultures, Bic-treated vs untreated p = 0.0049, AAV-shLemd2-transduced cultures, Bic-treated vs untreated p > 0.99, adjustment for multiple comparisons: Bonferroni). Data are presented as mean ± SEM, **p < 0.01 compared to NBQX-treated cultures.


LEMD2 is required for the nuclear envelope infoldings of CA1 pyramidal neurons in vivo . Top panel : representative confocal images ( z - axis - projected stack ) of Lamin B2 / DAPI - stained coronal brain sections from wild - type mice after stereotaxic injection of AAV - shRNA viruses ( AAV - shLemd2 - mCherry or AAV - scrambled - mCherry ) into the dorsal hippocampus . Merged , colocalization of the three signals . Scale bars : 50 μm . Middle panel : higher magnification images of AAV - shLemd2 - transduced CA1 pyramidal neurons . Arrows show examples of mCherry - positive nuclei that are devoid of nuclear infoldings , whereas stars denote non - transduced infolded nuclei . Lower panel : higher magnification images of AAV - scrambled siRNA - transduced CA1 pyramidal neurons . Arrows show examples of infolded , mCherry - positive nuclei ; stars denote infolded , non - transduced neurons . Scale bars ( middle and lower panels ) : 10 μm .

B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR I-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

LEMD2 is required for the nuclear envelope infoldings of CA1 pyramidal neurons in vivo. Top panel: representative confocal images (z-axis-projected stack) of Lamin B2/DAPI-stained coronal brain sections from wild-type mice after stereotaxic injection of AAV-shRNA viruses (AAV-shLemd2-mCherry or AAV-scrambled-mCherry) into the dorsal hippocampus. Merged, colocalization of the three signals. Scale bars: 50 μm. Middle panel: higher magnification images of AAV-shLemd2-transduced CA1 pyramidal neurons. Arrows show examples of mCherry-positive nuclei that are devoid of nuclear infoldings, whereas stars denote non-transduced infolded nuclei. Lower panel: higher magnification images of AAV-scrambled siRNA-transduced CA1 pyramidal neurons. Arrows show examples of infolded, mCherry-positive nuclei; stars denote infolded, non-transduced neurons. Scale bars (middle and lower panels): 10 μm.


Analysis of the percentage of infolded nuclei in DIV10 hippocampal neurons upon SATB2 overexpression and Vps4a / Vps4b knockdown . AAV - SATB2 - transduced neurons ( non - transfected or scrambled siRNA ( Scr ) - transfected ) exhibited higher percentage of infolded nuclei compared to AAV - EGFP - transduced neurons . In contrast , VPS4 - depleted AAV - SATB2 - transduced cultures showed similar number of infolded nuclei to AAV - EGFP - transduced neurons ( n = 4 independent primary cultures , one - way ANOVA followed by Tuckey post hoc test , F3 , 11 = 15 . 866 , p = 0 . 00026 , AAV - EGFP vs . AAV - SATB2 p = 0 . 0104 , AAV - EGFP vs . AAV - SATB2 + Scr p = 0 . 0028 , AAV - EGFP vs . AAV - SATB2 + siVps4 p = 0 . 605 , AAV - SATB2 + Scr vs . AAV - SATB2 + siVps4 p = 0 . 0007 ) . Number of nuclei analyzed : 594 ( AAV - EGFP ) , 657 ( AAV - SATB2 ) , 612 ( AAV - SATB2 + Scr ) , 428 ( AAV - SATB2 + siVps4 ) . Data are presented as mean ± SEM , * * p < 0 . 01 compared with AAV - EGFP ; # # # p < 0 . 001 compared with AAV - SATB2 + Scr .

O O O O O O O O O O O O B-CONTROLLED_VAR O O B-CONTROLLED_VAR O B-CONTROLLED_VAR O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O B-CONTROLLED_VAR O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O B-CONTROLLED_VAR O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O

Analysis of the percentage of infolded nuclei in DIV10 hippocampal neurons upon SATB2 overexpression and Vps4a/ Vps4b knockdown. AAV-SATB2-transduced neurons (non-transfected or scrambled siRNA (Scr)-transfected) exhibited higher percentage of infolded nuclei compared to AAV-EGFP-transduced neurons. In contrast, VPS4-depleted AAV-SATB2-transduced cultures showed similar number of infolded nuclei to AAV-EGFP-transduced neurons (n = 4 independent primary cultures, one-way ANOVA followed by Tuckey post hoc test, F3,11 =15.866, p = 0.00026, AAV-EGFP vs. AAV-SATB2 p = 0.0104, AAV-EGFP vs. AAV-SATB2 + Scr p = 0.0028, AAV-EGFP vs. AAV-SATB2 + siVps4 p = 0.605, AAV-SATB2 + Scr vs. AAV-SATB2 + siVps4 p = 0.0007). Number of nuclei analyzed: 594 (AAV-EGFP), 657 (AAV-SATB2), 612 (AAV-SATB2 + Scr), 428 (AAV-SATB2 + siVps4). Data are presented as mean ± SEM, **p < 0.01 compared with AAV-EGFP; ###p < 0.001 compared with AAV-SATB2 + Scr.


Activity - induced formation of nuclear infoldings is impaired in VPS4 - depleted cortical neurons . Upon AP bursting , the percentage of infolded nuclei was increased in non - transfected cultures and in cultures transfected with scramble siRNA ( Scr ) but not with siVps4 ( n = 3 - 4 independent primary cultures , one - way ANOVA followed by Tuckey post hoc test , F3 , 8 = 90 . 525 , p = 1 . 63E - 06 , Bic - treated vs NBQX - treated p = 0 . 000012 , Scr Bic - treated vs NBQX - treated p = 0 . 000052 , siVps4 Bic - treated vs NBQX - treated p = 0 . 396 , siVps4 Bic - treated vs Scr Bic - treated p = 0 . 000015 ) . Number of analyzed nuclei : 474 ( NBQX ) , 456 ( Bic ) , 409 ( Bic + Scr ) , 383 ( Bic + siVps4 ) . Data are presented as mean ± SEM , * * * p < 0 . 001 compared to NBQX - treated , # # # p < 0 . 001 compared to Scr Bic - treated .

O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Activity-induced formation of nuclear infoldings is impaired in VPS4-depleted cortical neurons. Upon AP bursting, the percentage of infolded nuclei was increased in non-transfected cultures and in cultures transfected with scramble siRNA (Scr) but not with siVps4 (n = 3-4 independent primary cultures, one-way ANOVA followed by Tuckey post hoc test, F3,8 = 90.525, p = 1.63E-06, Bic-treated vs NBQX-treated p = 0.000012, Scr Bic-treated vs NBQX-treated p = 0.000052, siVps4 Bic-treated vs NBQX-treated p = 0.396, siVps4 Bic-treated vs Scr Bic-treated p = 0.000015). Number of analyzed nuclei: 474 (NBQX), 456 (Bic), 409 (Bic + Scr), 383 (Bic + siVps4). Data are presented as mean ± SEM, ***p < 0.001 compared to NBQX-treated, ###p < 0.001 compared to Scr Bic-treated.


Representative confocal images ( Z - axis projection ) of CA1 pyramidal neurons of : ( top panel ) Satb2 floxed mice ( n = 3 ) maintained in their home cages ( HC ) , ( middle panel ) Satb2 floxed mice ( n = 4 ) subjected to NEE for 1 . 5 h , ( bottom panel ) Satb2CamkCre mice ( n = 5 ) exposed to NE for 1 . 5 h . Brain sections were stained for cFos and Lamin B2 . Scale bars : 10 μm . Higher magnification views of boxed areas in A ( middle panel ) . Arrows show examples of infolded , cFos - positive nuclei of the CA1 field of Satb2 floxed mice subjected to NEE . Scale bars : 5 μm .

O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O B-MEASURED_VAR I-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O

Representative confocal images (Z-axis projection) of CA1 pyramidal neurons of: (top panel) Satb2 floxed mice (n = 3) maintained in their home cages (HC), (middle panel) Satb2 floxed mice (n = 4) subjected to NEE for 1.5 h, (bottom panel) Satb2CamkCre mice (n = 5) exposed to NE for 1.5 h. Brain sections were stained for cFos and Lamin B2. Scale bars: 10 μm. Higher magnification views of boxed areas in A (middle panel). Arrows show examples of infolded, cFos-positive nuclei of the CA1 field of Satb2 floxed mice subjected to NEE. Scale bars: 5 μm.


( B ) Quantitative PCR analysis of post - natal lungs for mitochondrial DNA ( mtDNA ) . Actin was used as a normalization control . ( mean ± SD , : p < 0 . 05 ; : p < 0 . 01 ; N = 3 ) .

O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(B) Quantitative PCR analysis of post-natal lungs for mitochondrial DNA (mtDNA). Actin was used as a normalization control. (mean ± SD, : p<0.05;: p<0.01; N=3).


( C ) Quantitative analysis of oxidative stress in post - natal pups of indicated age ( in days ) using the peroxide linked oxidative stress marker DHR - 123 , as described in methods ( mean ± SD , : p < 0 . 01 ; N = 8 ) .

O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(C) Quantitative analysis of oxidative stress in post-natal pups of indicated age (in days) using the peroxide linked oxidative stress marker DHR-123, as described in methods (mean ± SD, : p<0.01; N=8).


( E ) Immunofluorescence analysis of oxidative DNA damage ( left panel ) and the DNA double - strand breaks sensor kinase marker pATM ( right panel ) in post - natal murine lungs . Here anti - 8 - oxo - 7 , 8 - dihydroguanine was used for the detection of oxidative DNA modification ( 8 - oxoG , left panel ) , and anti - pATM Serine 1981 was used for the analysis of the activation of the DDR ( pATM , right panel ) . Green cytoplasmic staining represents α - Tubulin ; blue DAPI staining represents nucleus ( scale 10μm ) . No primary antibody control served as a negative control ( P14 ) of the staining .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O O O O O O O O O O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O

(E) Immunofluorescence analysis of oxidative DNA damage (left panel) and the DNA double-strand breaks sensor kinase marker pATM (right panel) in post-natal murine lungs. Here anti-8-oxo-7, 8-dihydroguanine was used for the detection of oxidative DNA modification (8-oxoG, left panel), and anti-pATM Serine 1981 was used for the analysis of the activation of the DDR (pATM, right panel). Green cytoplasmic staining represents α-Tubulin; blue DAPI staining represents nucleus (scale 10μm). No primary antibody control served as a negative control (P14) of the staining.


( F ) Mean percentage of nuclei positive for oxidative DNA damage marker 8 - oxoG in post - natal murine lungs , as determined by immunofluorescence analysis ( mean ± SEM , : p < 0 . 01 ; : p < 0 . 001 ; N = 8 ) . ( G ) Mean percentage of nuclei positive for DNA damage marker pATM in post - natal murine lungs , as determined by immunofluorescence analysis ( mean ± SEM , : p < 0 . 01 ; N = 8 ) .

O O O O O O O O O O O O O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(F) Mean percentage of nuclei positive for oxidative DNA damage marker 8-oxoG in post-natal murine lungs, as determined by immunofluorescence analysis (mean ± SEM, : p<0.01; : p<0.001; N=8). (G) Mean percentage of nuclei positive for DNA damage marker pATM in post-natal murine lungs, as determined by immunofluorescence analysis (mean ± SEM, : p<0.01; N=8).


( H ) Quantitative analysis of the NAD + / NADH ratio in post - natal lungs after homogenization in extraction buffer ( mean ± SD , : p < 0 . 01 ; : p < 0 . 001 ) .

O O O O O O O B-MEASURED_VAR I-MEASURED_VAR O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(H) Quantitative analysis of the NAD+/NADH ratio in post-natal lungs after homogenization in extraction buffer (mean ± SD, : p<0.01; : p<0.001).


( A ) Immunofluorescence analysis of DSBs associated foci as marked by γH2AX in lung adenocarcinoma ( A549 ) cells , cultured either in low glucose ( 5 . 5mM , left panel ) or high glucose ( 30mM , right panel ) for 5 days , then treated with etoposide ( 5µM for 60 minutes ) . The resolution of DNA - DSBs foci , as marked by γH2AX was monitored over 24 hours after drug treatment , CML ( marked in green ) served as induction control ( scale 10µm ) . ( B ) Mean percentage of DSBs positive nuclei as evidenced by γH2AX positivity after pre - treatment of different reducing carbohydrates at 0 , 3 or 24 hours . More than 400 cells were analyzed for each bar ( mean ± SD , : p < 0 . 01 ; : p < 0 . 001 ) .

O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(A) Immunofluorescence analysis of DSBs associated foci as marked by γH2AX in lung adenocarcinoma (A549) cells, cultured either in low glucose (5.5mM, left panel) or high glucose (30mM, right panel) for 5 days, then treated with etoposide (5µM for 60 minutes). The resolution of DNA-DSBs foci, as marked by γH2AX was monitored over 24 hours after drug treatment, CML (marked in green) served as induction control (scale 10µm). (B) Mean percentage of DSBs positive nuclei as evidenced by γH2AX positivity after pre-treatment of different reducing carbohydrates at 0, 3 or 24 hours. More than 400 cells were analyzed for each bar (mean ± SD, : p<0.01;: p<0.001).


( C ) Still images of A549 cells showing the live recruitment of hPARP - mCherry and hXRCC4 - GFP , at the site of laser - induced DNA - DSBs from the cells pre - treated with the indicated reducing sugars as described in methods .

O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(C) Still images of A549 cells showing the live recruitment of hPARP-mCherry and hXRCC4-GFP, at the site of laser-induced DNA-DSBs from the cells pre-treated with the indicated reducing sugars as described in methods.


( D ) Quantitative analysis of the efficiency of NHEJ - repair in a reporter cell line , as described in Methods . Cells were cultured in low glucose ( 5 . 5mM ) or high glucose ( 30mM ) for 5 days , fructose ( 30mM ) , ribose ( 20mM ) , for 3 days ( graph bar 4 ) . I - SceI , untransfected cells served as a negative control . Shown is the average from three independent experiments ( mean ± SEM , : p < 0 . 01 : p < 0 . 001 ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O B-CONTROLLED_VAR O O O O O O O B-CONTROLLED_VAR O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(D) Quantitative analysis of the efficiency of NHEJ-repair in a reporter cell line, as described in Methods. Cells were cultured in low glucose (5.5mM) or high glucose (30mM) for 5 days, fructose (30mM), ribose (20mM), for 3 days (graph bar 4). I-SceI, untransfected cells served as a negative control. Shown is the average from three independent experiments (mean ± SEM, : p<0.01 : p<0.001).


( E ) Quantitative analysis of the NAD + / NADH ratio in A549 cells , cultured in either low ( 5 . 5mM , LG ) , or high glucose ( 30mM , HG ) for 5 days , fructose ( 30mM , FR ) , ribose ( 20mM , RI ) , for 3 days ( graph bar 4 ) . Quantification was performed after homogenization in extraction buffer ( Data represent mean ± SD , : p < 0 . 01 ; : p < 0 . 001 ; N = 3 ) .

O O O O O O O B-MEASURED_VAR I-MEASURED_VAR O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(E) Quantitative analysis of the NAD+/NADH ratio in A549 cells, cultured in either low (5.5mM, LG), or high glucose (30mM, HG) for 5 days, fructose (30mM, FR), ribose (20mM, RI), for 3 days (graph bar 4). Quantification was performed after homogenization in extraction buffer (Data represent mean ± SD, : p<0.01;: p<0.001; N=3).


( F ) A549 cells were cultured in either low ( 5 . 5mM ; lane 1 ) or high glucose ( 30mM ; lane 2 ) for 5 days , fructose ( 30mM ; lane 3 ) , ribose ( 20mM ; lane 4 ) , for 3 days . The cell extracts were then for immunoprecipitation by using anti - PARP , or a non - specific species control antibody . The PARP , or its interacting partner DBC1 was then detected using PARP , or a DBC1 specific antibody .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O B-MEASURED_VAR O O O O O B-MEASURED_VAR O O O O B-MEASURED_VAR O O O B-MEASURED_VAR O O O

(F) A549 cells were cultured in either low (5.5mM; lane 1) or high glucose (30mM; lane 2) for 5 days, fructose (30mM; lane 3), ribose (20mM; lane 4), for 3 days. The cell extracts were then for immunoprecipitation by using anti-PARP, or a non-specific species control antibody. The PARP, or its interacting partner DBC1 was then detected using PARP, or a DBC1 specific antibody.


( A ) Representative images of lungs for nuclei positive for the DNA - DSBs marker γH2AX , versus DAPI , in age - matched 3 - , or 6 - months control , versus STZ induced diabetic mice , as determined by immunofluorescence analysis ( scale 10μm ) . ( B ) Mean percentage of nuclei positive for the DNA - DSBs marker γH2AX in lungs of age - matched control versus 6 - month STZ induced diabetic mice , as determined by immunofluorescence analysis of lungs ( mean ± SEM , : p < 0 . 05 ; : p < 0 . 01 ; : p < 0 . 001 ; N = 6 ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(A) Representative images of lungs for nuclei positive for the DNA-DSBs marker γH2AX, versus DAPI, in age-matched 3-, or 6- months control, versus STZ induced diabetic mice, as determined by immunofluorescence analysis (scale 10μm). (B) Mean percentage of nuclei positive for the DNA-DSBs marker γH2AX in lungs of age-matched control versus 6-month STZ induced diabetic mice, as determined by immunofluorescence analysis of lungs (mean ± SEM, :p<0.05;: p<0.01; : p<0.001; N=6).


( C ) Representative images of lungs from age - matched control versus 3 or 6 - month STZ mice , stained for the peroxide linked oxidative stress marker DHR - 123 ( in red ) , as determined by mean fluorescence intensity . Blue nuclear staining represents DAPI ( Scale 10µm ) . ( D ) Quantitative analysis of respectively age - matched control versus 3 or 6 - month STZ induced diabetic lungs for persistent DNA damage signaling associated inflammatory marker IL - 6 , as determined by mean fluorescence intensity of respective group lungs ( mean ± SD ; : p < 0 . 01 : p < 0 . 001 ; N = 6 ) .

O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(C) Representative images of lungs from age-matched control versus 3 or 6-month STZ mice, stained for the peroxide linked oxidative stress marker DHR-123(in red), as determined by mean fluorescence intensity. Blue nuclear staining represents DAPI (Scale 10µm). (D) Quantitative analysis of respectively age-matched control versus 3 or 6-month STZ induced diabetic lungs for persistent DNA damage signaling associated inflammatory marker IL-6, as determined by mean fluorescence intensity of respective group lungs (mean ± SD; : p<0.01: p<0.001; N=6).


( E ) Representative images of lungs from age - matched controls , versus 3 or 6 - month STZ induced diabetic mice . Sections stained for cellular senescence - associated β - galactosidase [ β - Gal ] as described in Methods and visualized by bright field and polarized light , the senescent areas are recognized by its bluish - green staining ( Scale 40µm ) .

O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(E) Representative images of lungs from age-matched controls, versus 3 or 6-month STZ induced diabetic mice. Sections stained for cellular senescence-associated β-galactosidase [β-Gal] as described in Methods and visualized by bright field and polarized light, the senescent areas are recognized by its bluish-green staining (Scale 40µm).


( A ) Pressure - volume curves were determined using the FlexiVent system in age - matched control versus 3 - month STZ induced diabetic mice . The curves represent group averages ( mean ± SD ; : p < 0 . 05 ; N = 5 ) .

O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O

(A) Pressure-volume curves were determined using the FlexiVent system in age-matched control versus 3-month STZ induced diabetic mice. The curves represent group averages (mean ± SD; : p<0.05; N=5).


( B ) Quantitative analysis of static pulmonary compliance in age - matched control versus 3 months STZ induced diabetic mice , as described in Figure 3a . The curves represent group averages ( mean ± SD ; : P < 0 . 05 , : p < 0 . 01 ; N = 5 ) .

O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(B) Quantitative analysis of static pulmonary compliance in age-matched control versus 3 months STZ induced diabetic mice, as described in Figure 3a. The curves represent group averages (mean ± SD; : P<0.05, : p<0.01; N=5).


( C ) Representative images of lungs from age ( 3 - or 6 - month ) matched control versus STZ induced diabetic mice , stained for Masson ' s Trichrome stain , as described in Methods and visualized by bright field and polarized light , the accumulated ECM is recognized by its blue staining ( Scale 40µm ) .

O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(C) Representative images of lungs from age (3- or 6- month) matched control versus STZ induced diabetic mice, stained for Masson's Trichrome stain, as described in Methods and visualized by bright field and polarized light, the accumulated ECM is recognized by its blue staining (Scale 40µm).


( D ) Percentage total lung capacity of controls versus patients with diabetes . The dotted line represents the cut - off decided as per the guidelines of the European Respiratory Society ( ERS ) and American Thoracic Society ( ATS ) , ( mean ± SD , : p < 0 . 05 ; triplicates from each Ncontrol = 44 , Ntype - 1 = 35 , Ntype - 2 = 110 ) . ( E ) Percentage forced vital capacity of lungs in controls versus patients with diabetes . The dotted line represents the cut - off decided as per the guidelines of ERS and ATS , ( mean ± SD ; : p < 0 . 05 ; triplicates from each Ncontrol = 44 , Ntype - 1 = 35 , Ntype - 2 = 110 ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(D) Percentage total lung capacity of controls versus patients with diabetes. The dotted line represents the cut-off decided as per the guidelines of the European Respiratory Society (ERS) and American Thoracic Society (ATS), (mean ± SD, : p<0.05; triplicates from each Ncontrol=44, Ntype-1=35, Ntype-2=110). (E) Percentage forced vital capacity of lungs in controls versus patients with diabetes. The dotted line represents the cut-off decided as per the guidelines of ERS and ATS, (mean ± SD; : p<0.05; triplicates from each Ncontrol=44, Ntype-1=35, Ntype-2=110).