( G ) NEF prevents induction of F - actin remodeling in BHEL during IS . BHEL cells ( blue ) and CD4OT - II cells ( green ) were placed on PLL - coated slides and subjected to live - cell time - lapse imaging . Shown are representative still images from the indicated time points post contact formation ( see Movies EV1 and EV2 ) . Red signals indicate actin polymerization specifically in BHEL cells . Scale bars , 5µm . Filled and empty arrowheads indicate examples of accumulation of polarized actin or p - Tyr signal at B - T interface during IS formation or lack thereof , respectively . Asterisks mark pairs of interacting T and B cell . ( H ) Quantification of B cell actin polymerization as shown in G with the frequency of actin polymerization in B cells co - cultured with control T cells arbitrarily set to 100 % . Shown are mean values with SD from three independent experiments with at least 50 conjugates counted per experiment .

O O O B-GENEPROD O O O B-SUBCELLULAR I-SUBCELLULAR I-SUBCELLULAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-SUBCELLULAR B-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O B-SUBCELLULAR O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O O B-CELL_TYPE I-CELL_TYPE O O O O O O B-CELL_TYPE I-CELL_TYPE B-SUBCELLULAR O O O O O O O O O B-SUBCELLULAR B-EXP_ASSAY O B-CELL_TYPE I-CELL_TYPE O O O O O B-CELL_TYPE I-CELL_TYPE O 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) NEF prevents induction of F-actin remodeling in BHEL during IS. BHEL cells (blue) and CD4OT-II cells (green) were placed on PLL-coated slides and subjected to live-cell time-lapse imaging. Shown are representative still images from the indicated time points post contact formation (see Movies EV1 and EV2). Red signals indicate actin polymerization specifically in BHEL cells. Scale bars, 5µm. Filled and empty arrowheads indicate examples of accumulation of polarized actin or p-Tyr signal at B-T interface during IS formation or lack thereof, respectively. Asterisks mark pairs of interacting T and B cell. (H) Quantification of B cell actin polymerization as shown in G with the frequency of actin polymerization in B cells co-cultured with control T cells arbitrarily set to 100%. Shown are mean values with SD from three independent experiments with at least 50 conjugates counted per experiment.


( L ) Relative B cell proliferation ( quantified by gating on the last 3 - 4 generations ) is plotted as mean values with SD from four independent experiments . Control T cells + Ag were arbitrarily set to 1 .

O O O O B-CELL_TYPE I-CELL_TYPE B-EXP_ASSAY O 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-CELL_TYPE I-CELL_TYPE O B-SMALL_MOLECULE O O O O O O

(L) Relative B cell proliferation (quantified by gating on the last 3-4 generations) is plotted as mean values with SD from four independent experiments. Control T cells +Ag were arbitrarily set to 1.


( B ) Production of anti - HEL IgG at the indicated day post immunization with HEL - OVA / CFA following adoptive transfer of BHEL and CD4OT - II cells expressing NEF or various NEF mutants . Shown are relative levels of HEL IgG production with the median of IgG levels in mice receiving control cells on day 14 post immunization arbitrarily set to 1 . Each dot represents one animal . Shown are mean values with SD from 5 - 12 mice from 4 experiments .

O O O O O O O B-GENEPROD B-SUBCELLULAR O O O O O O O B-GENEPROD O B-GENEPROD O O O O O O O O O O O O O B-GENEPROD O O B-GENEPROD O O O O O O O B-GENEPROD B-SUBCELLULAR O O O O O B-SUBCELLULAR O O B-ORGANISM O O O O O 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-ORGANISM O O O O

(B) Production of anti-HEL IgG at the indicated day post immunization with HEL-OVA/CFA following adoptive transfer of BHEL and CD4OT-II cells expressing NEF or various NEF mutants. Shown are relative levels of HEL IgG production with the median of IgG levels in mice receiving control cells on day 14 post immunization arbitrarily set to 1. Each dot represents one animal. Shown are mean values with SD from 5-12 mice from 4 experiments.


( D ) Quantification of B cell actin polymerization as shown in C with frequency of actin polymerization in B cells co - cultured with control T cells arbitrarily set to 100 % . Shown are mean values with SD from three independent experiments with at least 30 conjugates evaluated per condition .

O O O O O B-CELL_TYPE I-CELL_TYPE B-SUBCELLULAR B-EXP_ASSAY O O O O O O O B-SUBCELLULAR B-EXP_ASSAY O B-CELL_TYPE I-CELL_TYPE O O O O O B-CELL_TYPE I-CELL_TYPE O 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) Quantification of B cell actin polymerization as shown in C with frequency of actin polymerization in B cells co-cultured with control T cells arbitrarily set to 100%. Shown are mean values with SD from three independent experiments with at least 30 conjugates evaluated per condition.


( H ) Relative surface expression of CD4 , CD28 , OX40 , ICOS , CD45 and CD54 on T cells expressing NEF WT or the indicated NEF mutants . Analysis of surface expression was performed using flow cytometry 3 - 7 d post transduction of T cells . Depicted are mean values with SD from 3 - 6 independent experiments , with control T cells arbitrarily set to 100 % as indicated by the dashed line .

O O O O O B-EXP_ASSAY O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-CELL_TYPE I-CELL_TYPE O B-GENEPROD O O O O B-GENEPROD O O O O O B-EXP_ASSAY O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O B-CELL_TYPE I-CELL_TYPE O O O O O O O O O O O O O O O O B-CELL_TYPE I-CELL_TYPE O O O O O O O O O O O O

(H) Relative surface expression of CD4, CD28, OX40, ICOS, CD45 and CD54 on T cells expressing NEF WT or the indicated NEF mutants. Analysis of surface expression was performed using flow cytometry 3-7 d post transduction of T cells. Depicted are mean values with SD from 3-6 independent experiments, with control T cells arbitrarily set to 100% as indicated by the dashed line.


( I ) Representative confocal images of the subcellular distribution of IL - 4 in CD4OT - II cells expressing the indicated proteins engaged in an IS with BHEL cells . B cells and T cells were placed on PLL - coated cover glasses for 60 - 90min , fixed and stained for intracellular IL - 4 . Filled and empty arrowheads indicate presence or absence of IL - 4 accumulation at the IS , respectively . Scale bars , 5µm . ( J ) Quantification of IL - 4 accumulation at the IS as depicted in I . Shown are mean values with SD from 3 independent experiments relative to control cells set to 100 % .

O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O O O O O O O O B-CELL_TYPE I-CELL_TYPE O B-CELL_TYPE I-CELL_TYPE O O O O O O O O O O O O O O O B-EXP_ASSAY O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O 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) Representative confocal images of the subcellular distribution of IL-4 in CD4OT-II cells expressing the indicated proteins engaged in an IS with BHEL cells. B cells and T cells were placed on PLL-coated cover glasses for 60-90min, fixed and stained for intracellular IL-4. Filled and empty arrowheads indicate presence or absence of IL-4 accumulation at the IS, respectively. Scale bars, 5µm. (J) Quantification of IL-4 accumulation at the IS as depicted in I. Shown are mean values with SD from 3 independent experiments relative to control cells set to 100%.


( A ) Representative cross - sections of draining LNs ( dLNs ) from mice 7d post immunization with HEL - OVA / CFA following adoptive transfer of BHEL and transduced CD4OT - II cells . dLN were cryosectioned and stained with fluorophore - conjugated antibodies against GL - 7 to reveal GCs ( green ) , CD21 / 35 to stain for follicular dendritic cells ( FDC , red ) and Hoechst to stain for cell nuclei ( blue ) . Scale bar , 250µm . ( B , C ) Quantification of the number of GCs relative to B cell follicles ( B ) and size of GC ( C ) on confocal micrographs as shown in A . Shown are mean values with SD from dLNs from three mice in two independent experiment . Each dot represents one LN ( 3 LNs / mouse ) in B and one GC in C .

O O O O O O O O O B-TISSUE O B-TISSUE O O B-ORGANISM O O O O B-GENEPROD O B-GENEPROD O O O O O O O O O O O O O O B-TISSUE O O O B-EXP_ASSAY O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O B-CELL_TYPE O O O O B-GENEPROD O O O B-EXP_ASSAY O B-CELL_TYPE I-CELL_TYPE I-CELL_TYPE O B-CELL_TYPE O O O O B-SMALL_MOLECULE O B-EXP_ASSAY O O B-SUBCELLULAR O O O O O O O O O O O O O O O O O O O B-CELL_TYPE O O B-CELL_TYPE I-CELL_TYPE I-CELL_TYPE O O O O O O B-CELL_TYPE O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O B-TISSUE O O B-ORGANISM O O O O O O O O O B-TISSUE O O B-TISSUE O B-ORGANISM O O O O O B-CELL_TYPE O O O

(A) Representative cross-sections of draining LNs (dLNs) from mice 7d post immunization with HEL-OVA/CFA following adoptive transfer of BHEL and transduced CD4OT-II cells. dLN were cryosectioned and stained with fluorophore-conjugated antibodies against GL-7 to reveal GCs (green), CD21/35 to stain for follicular dendritic cells (FDC, red) and Hoechst to stain for cell nuclei (blue). Scale bar, 250µm. (B, C) Quantification of the number of GCs relative to B cell follicles (B) and size of GC (C) on confocal micrographs as shown in A. Shown are mean values with SD from dLNs from three mice in two independent experiment. Each dot represents one LN (3 LNs/mouse) in B and one GC in C.


( A ) Still images of the Movies EV3 ( Control , upper panel ) and EV4 ( NEF , lower panel ) , obtained by intravital time lapse 2PM imaging of draining popliteal LNs 24hrs post immunization with HEL - OVA / CFA , following adoptive transfer of GFP BHEL cells ( green ) and mTomato CD4OT - II cells ( red ) expressing empty vector ( Control ; upper panel ) or NEF WT ( lower panel ) , respectively . High endothelial venules ( HEVs ) ( white ) were labeled by intravenous injection of Alexa660 - labeled Meca - 79 antibody and collagen ( blue ) is visualized by second harmonic generation ( SHG ) . Yellow and orange arrows indicate stable and transient ISs , respectively . Numbering of ISs corresponds to that in the respective Supplemental movies . Scale bar 20µm . ( B ) Quantification of the duration of interactions as indicated by the arrows in A . Touching T and B cells were manually quantification using virtual reality ( InViewR , Arivis ) . Each dot represents one T - B interaction pair . Red bar shows median . Data is derived from 5 to 6 movies , acquired from 4 mice in 2 independent experiments . Percentages on top indicate frequency of interactions that lasted 20min or longer .

O O O O O O O O O O O O O O O O O O B-GENEPROD O O O O O O O O O O O O O O O B-TISSUE O O O O B-GENEPROD O B-GENEPROD O O O O O O O B-GENEPROD O O O O O O B-ORGANISM O O O O O O O O O O O O O O O O O B-GENEPROD O O O O O O O O B-TISSUE I-TISSUE I-TISSUE O B-TISSUE O O O O O O O O O O B-SMALL_MOLECULE O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-CELL_TYPE I-CELL_TYPE O O O O O O O O O O O O O O O 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-ORGANISM O O O O O O O O O O O O O O O O O O

(A) Still images of the Movies EV3 (Control, upper panel) and EV4 (NEF, lower panel), obtained by intravital time lapse 2PM imaging of draining popliteal LNs 24hrs post immunization with HEL-OVA/CFA, following adoptive transfer of GFP BHEL cells (green) and mTomato CD4OT-II cells (red) expressing empty vector (Control; upper panel) or NEF WT (lower panel), respectively. High endothelial venules (HEVs) (white) were labeled by intravenous injection of Alexa660-labeled Meca-79 antibody and collagen (blue) is visualized by second harmonic generation (SHG). Yellow and orange arrows indicate stable and transient ISs, respectively. Numbering of ISs corresponds to that in the respective Supplemental movies. Scale bar 20µm. (B) Quantification of the duration of interactions as indicated by the arrows in A. Touching T and B cells were manually quantification using virtual reality (InViewR, Arivis). Each dot represents one T-B interaction pair. Red bar shows median. Data is derived from 5 to 6 movies, acquired from 4 mice in 2 independent experiments. Percentages on top indicate frequency of interactions that lasted 20min or longer.


( C ) Still images of the Movies EV5 ( Control , upper panel ) and EV6 ( NEF , lower panel ) , obtained by intravital time lapse 2PM imaging of draining popliteal LNs 8d post immunization with HEL - OVA / CFA , following adoptive transfer of GFP BHEL cells ( green ) and mTomato CD4OT - II cells ( red ) expressing empty vector ( Control ; upper panel ) or NEF WT ( lower panel ) . Follicular dendritic cells ( FDCs ) ( white ) were labeled by subcutaneous injection of Alexa647 - labeled anti - CD21 / CD35 antibody and collagen ( blue ) is visualized by SHG . Yellow and orange arrows indicate stable and transient ISs , respectively . Numbering of ISs corresponds to that in the respective Supplemental movies . Scale bar 20µm . ( D ) Quantification of the duration of interactions as indicated by the arrows in C . Touching T and B cells were manually quantification using virtual reality ( InViewR , Arivis ) . Each dot represents one T - B interaction pair . Red bar shows median . Shown is one representative dataset out of two , derived from 5 to 8 movies , acquired from 4 mice . Percentages on top indicate frequency of interactions that lasted 20min or longer .

O O O O O O O O O O O O O O O O O O B-GENEPROD O O O O O O O O O O O O O O O B-TISSUE O O O O B-GENEPROD O B-GENEPROD O O O O O O O B-GENEPROD O O O O O O B-ORGANISM O O O O O O O O O O O O O O O O O B-GENEPROD O O O O O O B-CELL_TYPE I-CELL_TYPE I-CELL_TYPE O B-CELL_TYPE O O O O O O O O O O B-SMALL_MOLECULE O O O O B-GENEPROD O B-GENEPROD O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-CELL_TYPE I-CELL_TYPE O O O O O O O O O O O O O O O O O O O O O O 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-ORGANISM O O O O O O O O O O O O O O

(C) Still images of the Movies EV5 (Control, upper panel) and EV6 (NEF, lower panel), obtained by intravital time lapse 2PM imaging of draining popliteal LNs 8d post immunization with HEL-OVA/CFA, following adoptive transfer of GFP BHEL cells (green) and mTomato CD4OT-II cells (red) expressing empty vector (Control; upper panel) or NEF WT (lower panel). Follicular dendritic cells (FDCs) (white) were labeled by subcutaneous injection of Alexa647-labeled anti-CD21/CD35 antibody and collagen (blue) is visualized by SHG. Yellow and orange arrows indicate stable and transient ISs, respectively. Numbering of ISs corresponds to that in the respective Supplemental movies. Scale bar 20µm. (D) Quantification of the duration of interactions as indicated by the arrows in C. Touching T and B cells were manually quantification using virtual reality (InViewR, Arivis). Each dot represents one T-B interaction pair. Red bar shows median. Shown is one representative dataset out of two, derived from 5 to 8 movies, acquired from 4 mice. Percentages on top indicate frequency of interactions that lasted 20min or longer.


( B ) Virus replication as assessed by quantification of RT activity in the cell culture supernatant . Shown are mean values with SD from 6 donors .

O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O O O O

(B) Virus replication as assessed by quantification of RT activity in the cell culture supernatant. Shown are mean values with SD from 6 donors.


( C ) Production of IgG antibodies against TT from infected blocks at indicated time point . Each dot represents one donor , bar indicates mean value . Tonsillar blocks were infected with indicated virus for 24h . After washing off the virus , blocks were stimulated with TT antigen for 24h . Samples were collected at different time intervals to estimate viral replication and antibody production .

O O O O O B-SUBCELLULAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-EXP_ASSAY I-EXP_ASSAY O O O O

(C) Production of IgG antibodies against TT from infected blocks at indicated time point. Each dot represents one donor, bar indicates mean value. Tonsillar blocks were infected with indicated virus for 24h. After washing off the virus, blocks were stimulated with TT antigen for 24h. Samples were collected at different time intervals to estimate viral replication and antibody production.


Distribution of RT shifts calculated with the - ΔL + ΔE method described in Appendix Figure S1 . Different transgenes in a mid - late - replicating region are compared : the IL2R reporter gene under the control of the βA - globin promoter ( βA pro ) containing an inactive ( i , N = 10 ) or an active origin ( ii , N = 8 ) flanked by two copies of FIV ( 2xFIV ) , the blasticidin resistance gene ( BsR ) under the control of the β - actin promoter ( β - act pro ) ( iii , N = 7 ) or a combination of these two transgenes ( iv , N = 8 ) . Blue and black triangles represent reactive loxP sites and recombined inactive loxP sites respectively . Rectangle edges correspond to the 0 . 25 and 0 . 75 quartiles , the thick black lines represent the median , the white triangles represent the mean and the whiskers extend to the smallest and largest - ΔL + ΔE values . Statistical analysis was performed with Wilcoxon nonparametric two - tailed tests ( ns , not significant ; * * p < 0 . 01 ; * * * p < 0 . 001 ) .

O O B-EXP_ASSAY I-EXP_ASSAY O O O O 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-GENEPROD O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O B-GENEPROD O O O O O O O O O O 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-GENEPROD I-GENEPROD I-GENEPROD O B-GENEPROD O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Distribution of RT shifts calculated with the -ΔL+ΔE method described in Appendix Figure S1. Different transgenes in a mid-late-replicating region are compared: the IL2R reporter gene under the control of the βA-globin promoter (βA pro) containing an inactive (i, N=10) or an active origin (ii, N=8) flanked by two copies of FIV( 2xFIV), the blasticidin resistance gene (BsR) under the control of the β-actin promoter (β-act pro) (iii, N=7) or a combination of these two transgenes (iv, N=8). Blue and black triangles represent reactive loxP sites and recombined inactive loxP sites respectively. Rectangle edges correspond to the 0.25 and 0.75 quartiles, the thick black lines represent the median, the white triangles represent the mean and the whiskers extend to the smallest and largest -ΔL+ΔE values. Statistical analysis was performed with Wilcoxon nonparametric two-tailed tests (ns, not significant; **p<0.01; ***p<0.001).


A . UCSC genome browser visualization of the mid - late insertion site of chromosome 1 ( genomic positions : chr1 : 71 , 000 , 000 - 74 , 100 , 000 bp , 3 . 1 Mb ; galGal5 ) . RT weighted average ( WA ) values for the wt and the βA - globin + β - actin cell lines are shown . Early - replicated regions ( E ) are represented in orange and late replicating regions ( L ) in blue . Annotated genes ( Ref Seq genes ) are represented below . The mid - late insertion site is indicated with a red arrow and dotted line .

O O O O O O O O O O O O O O B-SUBCELLULAR O O O O O B-SUBCELLULAR O O O O O O O O O O O O O O O O O O O O O O B-EXP_ASSAY O O O O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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. UCSC genome browser visualization of the mid-late insertion site of chromosome 1 (genomic positions: chr1:71,000,000-74,100,000 bp, 3.1 Mb; galGal5). RT weighted average (WA) values for the wt and the βA-globin+β-actin cell lines are shown. Early-replicated regions (E) are represented in orange and late replicating regions (L) in blue. Annotated genes (Ref Seq genes) are represented below. The mid-late insertion site is indicated with a red arrow and dotted line.


B . UCSC genome browser visualization of the mid - late insertion site of chromosome 1 ( genomic positions : chr1 : 72 , 450 , 000 - 72 , 650 , 000 bp ; 200 kb ; galGal5 ) . Tracks of nascent strands ( NS ) enrichments in the four S phase fractions were represented separately ( S1 to S4 ) for the wt and the 2x ( βA - globin + β - actin ) cell lines . NS enriched and depleted regions for each fraction are represented in purple and blue respectively . Single reads form SNS aligned and track of replication origins ( Ori peaks ) determined in ( Massip et al , 2019 ) are reported in between . The three mid - late insertion sites 1 , 2 and 3 are indicated with red arrows . Annotated genes and CpG Islands are shown below . Initiation zones ( IZ ) and termination zones ( TZ ) are reported .

O O O O O O O O O O O O O O B-SUBCELLULAR O O O O O B-SUBCELLULAR O O O O O O O O O O O O O O O O O O O O O O O O O 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-GENEPROD I-GENEPROD I-GENEPROD O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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. UCSC genome browser visualization of the mid-late insertion site of chromosome 1 (genomic positions: chr1:72,450,000-72,650,000 bp; 200 kb; galGal5). Tracks of nascent strands (NS) enrichments in the four S phase fractions were represented separately (S1 to S4) for the wt and the 2x(βA-globin+β-actin) cell lines. NS enriched and depleted regions for each fraction are represented in purple and blue respectively. Single reads form SNS aligned and track of replication origins (Ori peaks) determined in (Massip et al, 2019) are reported in between. The three mid-late insertion sites 1, 2 and 3 are indicated with red arrows. Annotated genes and CpG Islands are shown below. Initiation zones (IZ) and termination zones (TZ) are reported.


A . Distribution of - ΔL + ΔE values for clonal cell lines containing one advanced replicon ( βA - globin + β - actin ) inserted at site 1 ( i , N = 8 ) or 3 ( iii , N = 3 ) or at both sites on the same chromosome with one GFP reporter construct composed of the GFP reporter gene under the control of the βA - globin promoter / origin linked to a 1 . 6 kb fragment of human chromosome 7 ( h . K7 ) inserted at site 2 , either on the same chromosome as 1 and 3 ( 1 + 2 + 3 ) , or on the other chromosome ( 1 + 3 ) ( ii , N = 5 ) . Blue and black triangles represent reactive loxP sites and recombined inactive loxP sites respectively . Black vertical bars represent insertion sites . Rectangle edges correspond to the 0 . 25 and 0 . 75 quartiles , the thick black lines represent the median , the white triangles represent the mean and the whiskers extend to the smallest and largest - ΔL + ΔE values . Statistical analysis was performed with Wilcoxon nonparametric two - tailed tests ( * * p < 0 . 01 ) .

O O O O O O O O O O O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O B-GENEPROD I-GENEPROD I-GENEPROD O O O O 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-SUBCELLULAR O O B-GENEPROD O O O O O B-GENEPROD O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O O O O B-SUBCELLULAR O O O O O O O O O O O O O O O B-SUBCELLULAR O O O O O O O O O O O O O O O O B-SUBCELLULAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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. Distribution of -ΔL+ΔE values for clonal cell lines containing one advanced replicon (βA-globin+β-actin) inserted at site 1 (i, N=8) or 3 (iii, N=3) or at both sites on the same chromosome with one GFP reporter construct composed of the GFP reporter gene under the control of the βA-globin promoter/origin linked to a 1.6 kb fragment of human chromosome 7 (h.K7) inserted at site 2, either on the same chromosome as 1 and 3 (1+2+3), or on the other chromosome (1+3) (ii, N=5). Blue and black triangles represent reactive loxP sites and recombined inactive loxP sites respectively. Black vertical bars represent insertion sites. Rectangle edges correspond to the 0.25 and 0.75 quartiles, the thick black lines represent the median, the white triangles represent the mean and the whiskers extend to the smallest and largest -ΔL+ΔE values. Statistical analysis was performed with Wilcoxon nonparametric two-tailed tests (**p<0.01).


C . Interaction profiles of allele 1 + 3 / 2 with chr1 for combined Hi - C libraries : distance - corrected interactions , i . e . observed / expected ( OE ) , binned at 500 kb with corresponding compartment ( EV1 ) tracks . Red arrows represent the loci that show preferred interactions with allele 1 + 3 and that coincide with A compartment .

O O B-EXP_ASSAY O O O O O O O O O B-SUBCELLULAR O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O O O O 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-EXP_ASSAY O O O O O O O O O B-SUBCELLULAR I-SUBCELLULAR O

C. Interaction profiles of allele 1+3/2 with chr1 for combined Hi-C libraries: distance-corrected interactions, i.e. observed/expected (OE), binned at 500 kb with corresponding compartment ( EV1) tracks. Red arrows represent the loci that show preferred interactions with allele 1+3 and that coincide with A compartment.


D . OE interactions of allele 1 + 3 ( orange rectangles ) and allele 2 ( blue rectangles ) averaged over A and B compartments for p and q - arms at 100kb bin size . A random compartment status permutation test was made and then we calculated the one - sided p - value for the observed value of ' difference allele 1 + 3 / 2 A - B ' . Values corresponding to OE interactions of allele 1 + 3 and allele 2 averaged over shuffled A ( red circles , N = 1000 ) and B compartments ( blue circles , N = 1000 ) for p and q - arms . ns , not significant ; * * p < 0 . 01 ; * * * p < 0 . 001 .

O O O B-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O B-SUBCELLULAR I-SUBCELLULAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O B-SUBCELLULAR I-SUBCELLULAR O O O O O O O O O O O O O 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. OE interactions of allele 1+3 (orange rectangles) and allele 2 (blue rectangles) averaged over A and B compartments for p and q-arms at 100kb bin size. A random compartment status permutation test was made and then we calculated the one-sided p-value for the observed value of 'difference allele 1+3 / 2 A - B'. Values corresponding to OE interactions of allele 1+3 and allele 2 averaged over shuffled A (red circles, N=1000) and B compartments (blue circles, N=1000) for p and q-arms. ns, not significant; **p<0.01;***p<0.001.


E . Scatterplot of density of interactions between allele 1 + 3 and allele 2 with chromosomes larger than 5 , 000 , 000 bp .

O O O O O O B-EXP_ASSAY O O O O O O O O O B-SUBCELLULAR O O O O O O O O O

E. Scatterplot of density of interactions between allele 1+3 and allele 2 with chromosomes larger than 5,000,000 bp.


A . Distribution of - ΔL + ΔE values for clonal cell lines containing one β - actin construct at site 1 ( i , N = 7 ) or at site 1 and 3 ( ii , N = 5 ) , on the same chromosome . Data information : Blue and black triangles represent reactive loxP sites and recombined inactive loxP sites respectively . Black vertical bars represent insertion sites . Rectangle edges correspond to the 0 . 25 and 0 . 75 quartiles , the thick black lines represent the median , the white triangles represent the mean and the whiskers extend to the smallest and largest - ΔL + ΔE values . Statistical analysis was performed with Wilcoxon nonparametric two - tailed tests ( * * p < 0 . 01 ) .

O O O O O O O O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O 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-SUBCELLULAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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. Distribution of -ΔL+ΔE values for clonal cell lines containing one β-actin construct at site 1 (i, N=7) or at site 1 and 3 (ii, N=5), on the same chromosome. Data information: Blue and black triangles represent reactive loxP sites and recombined inactive loxP sites respectively. Black vertical bars represent insertion sites. Rectangle edges correspond to the 0.25 and 0.75 quartiles, the thick black lines represent the median, the white triangles represent the mean and the whiskers extend to the smallest and largest -ΔL+ΔE values. Statistical analysis was performed with Wilcoxon nonparametric two-tailed tests (**p<0.01).


B . Distribution of - ΔL + ΔE values for clonal cell lines containing one βA - globin construct at site 1 ( i , N = 8 ) or at site 1 and 3 ( ii , N = 6 ) , on the same chromosome . Data information : Blue and black triangles represent reactive loxP sites and recombined inactive loxP sites respectively . Black vertical bars represent insertion sites . Rectangle edges correspond to the 0 . 25 and 0 . 75 quartiles , the thick black lines represent the median , the white triangles represent the mean and the whiskers extend to the smallest and largest - ΔL + ΔE values . Statistical analysis was performed with Wilcoxon nonparametric two - tailed tests ( * * p < 0 . 01 ) .

B. Distribution of -ΔL+ΔE values for clonal cell lines containing one βA-globin construct at site 1 (i, N=8) or at site 1 and 3 (ii, N=6), on the same chromosome. Data information: Blue and black triangles represent reactive loxP sites and recombined inactive loxP sites respectively. Black vertical bars represent insertion sites. Rectangle edges correspond to the 0.25 and 0.75 quartiles, the thick black lines represent the median, the white triangles represent the mean and the whiskers extend to the smallest and largest -ΔL+ΔE values. Statistical analysis was performed with Wilcoxon nonparametric two-tailed tests (**p<0.01).


C . After 1 , 8 , 24 and 48 h of 4 - hydroxytamoxifen treatment , genomic DNA was extracted and quantified by semi - quantitative PCR ( see Appendix Figure S6 ) . Error bars correspond to the standard deviation for PCR duplicates . The percentages of cells with the large 1 + 3 and loxP _ LE ( 1 ) + βA - globin + β - actin ( 3 ) or β - actin ( 1 + 3 ) excision at each time point are shown for different cell lines .

O O O O O O O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O O O B-EXP_ASSAY O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O B-GENEPROD I-GENEPROD I-GENEPROD O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O O O O O O O O

C. After 1, 8, 24 and 48 h of 4-hydroxytamoxifen treatment, genomic DNA was extracted and quantified by semi-quantitative PCR (see Appendix Figure S6). Error bars correspond to the standard deviation for PCR duplicates. The percentages of cells with the large 1+3 and loxP_LE (1) + βA-globin+β-actin (3) or β-actin (1+3) excision at each time point are shown for different cell lines.


A , B . Clones 1 + 2 + 3 , 1 + 3 ( A ) and loxP _ LE ( 1 ) + βA - globin + β - actin ( 3 ) ( B ) were used to assess chromatin accessibility within the transgenes . The positions of the amplicons used for quantification in each shifting construct individually ( thick black lines ; 5 ' 2xFIV - 1 or - 3 , β - actin pro - 1 or - 3 , BsR gene , PuroR gene , LoxP site ) , in both shifting constructs ( βA pro 1 and 2 , IL2R gene , 3 ' 2xFIV1 - 3 ) or in the GFP reporter construct ( βA - GFP , GFP gene , h . K7 ) are shown . The insertion sites on the wt chromosome ( insertion sites 1 , 2 and 3 ) and a genomic region located 5 kb downstream from the insertion site ( insertion site + 5kb ) were used as controls for the targeted genomic region . The endogenous active MED14 promoter was analyzed as a control . Blue and black triangles represent reactive loxP sites and recombined inactive loxP sites respectively . Black vertical bars represent insertion sites . Quantifications by real - time qPCR of total chromatin ( input ) extracted from two 1 + 2 + 3 , 1 + 3 and loxP _ LE ( 1 ) + βA - globin + β - actin ( 3 ) clonal cell lines , after digestion with micrococcal nuclease ( MNAse , 2 . 5 U , 10 U , 40 U , 160 U / mL ) and size selection were shown . Error bars indicate the standard deviation for qPCR triplicates , for two independent clones . Data are presented as total chromatin input relative to the condensed genomic regions ( cond1 and cond2 ) used for the normalization .

O 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-GENEPROD I-GENEPROD I-GENEPROD O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O B-SUBCELLULAR O O O O O O O O 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-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O B-GENEPROD O O B-GENEPROD O O O O O O O O O O O O O O O O O B-GENEPROD O O O O O O O O O O O B-GENEPROD O O O O O B-GENEPROD O B-GENEPROD O O O O O O O O O O O O O O O B-SUBCELLULAR O O O O O O O O O O O O O O O O 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-GENEPROD O O O O O O O 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-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O B-SUBCELLULAR O O O O O O O O O O O O O O O O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O O O O B-SMALL_MOLECULE O O O O O O 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-EXP_ASSAY O O O O O O O O O O O O B-SUBCELLULAR O O O O O O O O O O O O O O O O O

A, B. Clones 1+2+3, 1+3 (A) and loxP_LE (1) + βA-globin+β-actin (3) (B) were used to assess chromatin accessibility within the transgenes. The positions of the amplicons used for quantification in each shifting construct individually (thick black lines; 5' 2xFIV-1 or -3, β-actin pro-1 or -3, BsR gene, PuroR gene, LoxP site), in both shifting constructs (βA pro 1 and 2, IL2R gene, 3' 2xFIV1-3) or in the GFP reporter construct (βA-GFP, GFP gene, h.K7) are shown. The insertion sites on the wt chromosome (insertion sites 1, 2 and 3) and a genomic region located 5 kb downstream from the insertion site (insertion site +5kb) were used as controls for the targeted genomic region. The endogenous active MED14 promoter was analyzed as a control. Blue and black triangles represent reactive loxP sites and recombined inactive loxP sites respectively. Black vertical bars represent insertion sites. Quantifications by real-time qPCR of total chromatin (input) extracted from two 1+2+3, 1+3 and loxP_LE (1)+ βA-globin+β-actin (3) clonal cell lines, after digestion with micrococcal nuclease (MNAse, 2.5 U, 10 U, 40 U, 160 U/mL) and size selection were shown. Error bars indicate the standard deviation for qPCR triplicates, for two independent clones. Data are presented as total chromatin input relative to the condensed genomic regions (cond1 and cond2) used for the normalization.


A , B . UCSC genome browser visualization of two parts of chromosome 1 containing the insertion sites ( genomic positions : chr1 : 69 , 000 , 000 - 74 , 200 , 000 ; 5 . 2 Mb ( A ) and chr1 : 175 , 300 , 000 - 180 , 500 , 000 ; 5 . 2 Mb ( B ) ; galGal5 ) . RT weighted average ( WA ) values obtained after cells sorting into four S - phase fractions , BrdU pulse - labeled nascent strands ( NS ) immunoprecipitation and sequencing are shown . Early - replicated regions are represented in orange and late - replicating regions in blue . Below , track of eigenvector 1 values corresponding to A / B compartments at 100 kb resolution after Hi - C analysis in wt cells , track of replication origins determined in ( Massip et al , 2019 ) ( Ori peaks ) and annotated genes are reported . The three sites chosen for insertion are indicated with red arrows and dotted lines ( mid - late , late 1 ( A ) and late 2 ( B ) insertion ) . At the bottom , an enlargement of late insertion sites ( chr1 : 69 , 500 , 000 - 71 , 500 , 000 ( A ) , chr1 : 177 , 000 , 000 - 179 , 000 , 000 ( B ) ; galGal5 ) , displaying genes , mRNAs and ESTs for the chicken and all other species together with track of replication origins determined in ( Massip et al , 2019 ) ( Ori peaks ) is shown . Tracks of RefSeq genes are represented in blue , tracks of Ensembl genes in red , tracks of AUGUSTUS ab initio gene predictions and TransMap alignements in green and tracks of mRNAs and ESTs in black . Insertion sites are indicated with red dotted lines and black dotted lines determine the borders of the enlarged regions .

O O O O O O O O O O O O B-SUBCELLULAR O O O O O O O O O B-SUBCELLULAR O O O O O O O O O O O O O O O O O O O O O B-SUBCELLULAR 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-EXP_ASSAY O O O O O O O O O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O B-EXP_ASSAY O B-EXP_ASSAY O O O O O O O O O O O O O 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-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-SUBCELLULAR O O O O O O O O O O O O O O O O B-SUBCELLULAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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, B. UCSC genome browser visualization of two parts of chromosome 1 containing the insertion sites (genomic positions: chr1:69,000,000-74,200,000; 5.2 Mb (A) and chr1: 175,300,000-180,500,000; 5.2 Mb (B); galGal5). RT weighted average (WA) values obtained after cells sorting into four S-phase fractions, BrdU pulse-labeled nascent strands (NS) immunoprecipitation and sequencing are shown. Early-replicated regions are represented in orange and late-replicating regions in blue. Below, track of eigenvector 1 values corresponding to A/B compartments at 100 kb resolution after Hi-C analysis in wt cells, track of replication origins determined in (Massip et al, 2019) (Ori peaks) and annotated genes are reported. The three sites chosen for insertion are indicated with red arrows and dotted lines (mid-late, late 1 (A) and late 2 (B) insertion). At the bottom, an enlargement of late insertion sites (chr1: 69,500,000-71,500,000 (A), chr1: 177,000,000-179,000,000 (B); galGal5), displaying genes, mRNAs and ESTs for the chicken and all other species together with track of replication origins determined in (Massip et al, 2019) (Ori peaks) is shown. Tracks of RefSeq genes are represented in blue, tracks of Ensembl genes in red, tracks of AUGUSTUS ab initio gene predictions and TransMap alignements in green and tracks of mRNAs and ESTs in black. Insertion sites are indicated with red dotted lines and black dotted lines determine the borders of the enlarged regions.


A , B . RT profiles of chromosomal alleles following the targeted integration of a βA - globin + β - actin construct into two late - replicating loci , 1 ( A ) and 2 ( B ) in two or three clonal cell lines respectively . Nascent strands ( NS ) were quantified by real - time qPCR in four S phase fractions . Specific primer pairs determine the RT profile for the modified allele ( With ) , the wt allele ( Without ) and both alleles ( Both ) . The endogenous β - globin locus was analyzed as an early - replicated control . The modified ( Mod ) and wild - type ( wt ) alleles are shown . Difference - ΔL + ΔE values calculated at the target site following transgene integration are indicated ( see Appendix Figure S1 ) . Error bars correspond to the standard deviation for qPCR duplicates . The black vertical bars represent the precise insertion position .

O O O O B-EXP_ASSAY I-EXP_ASSAY O B-SUBCELLULAR O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O 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-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-EXP_ASSAY O O O O O O O O O O O O

A, B. RT profiles of chromosomal alleles following the targeted integration of a βA-globin+β-actin construct into two late-replicating loci, 1 (A) and 2 (B) in two or three clonal cell lines respectively. Nascent strands (NS) were quantified by real-time qPCR in four S phase fractions. Specific primer pairs determine the RT profile for the modified allele (With), the wt allele (Without) and both alleles (Both). The endogenous β-globin locus was analyzed as an early-replicated control. The modified (Mod) and wild-type (wt) alleles are shown. Difference -ΔL+ΔE values calculated at the target site following transgene integration are indicated (see Appendix Figure S1). Error bars correspond to the standard deviation for qPCR duplicates. The black vertical bars represent the precise insertion position.


B , C Both mRNA and protein levels of RBM47 were elevated in SeV - infected ( B ) or IFN - α - stimulated ( C ) 293T , HFF , HUVEC , and THP - 1 cells . Cells were infected with SeV ( MOI = 1 ) or stimulated with IFN - α for 6 or 12 h , and the expression of RBM47 mRNA ( at 6 and 12 h post treatment ) and protein ( at 6 and 12 h post treatment ) were tested . The PCR results are represented as the means ± SD of n = 3 biological replicates . Data information : Data are representative of n = 3 independent experiments . NS , non - significant ; * P ≤ 0 . 05 , * * P ≤ 0 . 01 , and * * * P ≤ 0 . 001 ( Student ' s t - test ) .

O O O O O O B-EXP_ASSAY I-EXP_ASSAY O B-GENEPROD O O O B-ORGANISM O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O B-CELL_LINE O B-CELL_LINE O B-CELL_LINE O O B-CELL_LINE I-CELL_LINE I-CELL_LINE O O O O O O B-ORGANISM O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O B-EXP_ASSAY O B-GENEPROD O 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-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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, C Both mRNA and protein levels of RBM47 were elevated in SeV-infected (B) or IFN-α-stimulated (C) 293T, HFF, HUVEC, and THP-1 cells. Cells were infected with SeV (MOI = 1) or stimulated with IFN-α for 6 or 12 h, and the expression of RBM47 mRNA (at 6 and 12 h post treatment) and protein (at 6 and 12 h post treatment) were tested. The PCR results are represented as the means ± SD of n = 3 biological replicates. Data information: Data are representative of n = 3 independent experiments. NS, non-significant; * P ≤ 0.05, **P ≤ 0.01, and ***P ≤ 0.001 (Student's t-test).


C , D Levels of viral genome , protein , and progeny of VSV ( C ) or HSV - 1 ( D ) were quantified in virus - infected 293T cells with or without RBM47 ectopic expression at the indicated time points . The viral protein levels were tested 12 h post VSV infection and 24 h post HSV - 1 infection . The PCR and viral titer data are represented as the means ± SD of n = 3 biological replicates . Data information : The data shown are representative of n = 3 independent experiments . * * P ≤ 0 . 01 , and * * * P ≤ 0 . 001 ( Student ' s t - test ) .

O O O O O O O O O O O O O B-ORGANISM O O O O B-ORGANISM I-ORGANISM I-ORGANISM O O O O O O O O O B-CELL_LINE O O O O B-GENEPROD O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O B-ORGANISM O O O O O B-ORGANISM I-ORGANISM I-ORGANISM O O O B-EXP_ASSAY O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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, D Levels of viral genome, protein, and progeny of VSV (C) or HSV-1 (D) were quantified in virus-infected 293T cells with or without RBM47 ectopic expression at the indicated time points. The viral protein levels were tested 12 h post VSV infection and 24 h post HSV-1 infection.The PCR and viral titer data are represented as the means ± SD of n = 3 biological replicates. Data information: The data shown are representative of n = 3 independent experiments.**P ≤ 0.01, and ***P ≤ 0.001 (Student's t-test).


D , E mRNA expression of IFIT1 , ISG15 , and Cig5 in SeV - infected 293T cells overexpressing RBM47 ( D ) or RBM47 knocked down cells ( E ) . Cells were infected with SeV for 24 h , and gene expression was determined using qRT - PCR . The PCR results are represented as the means ± SD of n = 3 biological replicates . Data information : The data shown are representative of n = 3 independent experiments . NS , non - significant ; * P ≤ 0 . 05 , * * P ≤ 0 . 01 , and * * * P ≤ 0 . 001 ( Student ' s t - test ) .

O O O B-EXP_ASSAY I-EXP_ASSAY O B-GENEPROD O B-GENEPROD O O B-GENEPROD O B-ORGANISM O O B-CELL_LINE O O B-GENEPROD O O O O B-GENEPROD O O O O O O O O O O O B-ORGANISM O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O B-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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, E mRNA expression of IFIT1, ISG15, and Cig5 in SeV-infected 293T cells overexpressing RBM47 (D) or RBM47 knocked down cells (E). Cells were infected with SeV for 24 h, and gene expression was determined using qRT-PCR.The PCR results are represented as the means ± SD of n = 3 biological replicates. Data information: The data shown are representative of n = 3 independent experiments. NS, non-significant; * P ≤ 0.05, **P ≤ 0.01, and ***P ≤ 0.001 (Student's t-test).


F The expression of IFIT1 , ISG15 , and Cig5 in RBM47 - overexpressing or control 293T cells stimulated by IFN - α for 12 h . The PCR results are represented as the means ± SD of n = 3 biological replicates . Data information : The data shown are representative of n = 3 independent experiments . NS , non - significant ; * P ≤ 0 . 05 , * * P ≤ 0 . 01 , and * * * P ≤ 0 . 001 ( Student ' s t - test ) .

O O B-EXP_ASSAY O B-GENEPROD O B-GENEPROD O O B-GENEPROD O B-GENEPROD O O O O B-CELL_LINE O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O B-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 The expression of IFIT1, ISG15, and Cig5 in RBM47-overexpressing or control 293T cells stimulated by IFN-α for 12 h. The PCR results are represented as the means ± SD of n = 3 biological replicates. Data information: The data shown are representative of n = 3 independent experiments. NS, non-significant; * P ≤ 0.05, **P ≤ 0.01, and ***P ≤ 0.001 (Student's t-test).


G The expression of IFIT1 , ISG15 , and Cig5 in WT and RBM47 + / - peritoneal macrophages stimulated by IFN - α for 12 h . The PCR results are represented as the means ± SD of n = 3 biological replicates . Data information : The data shown are representative of n = 3 independent experiments . NS , non - significant ; * P ≤ 0 . 05 , * * P ≤ 0 . 01 , and * * * P ≤ 0 . 001 ( Student ' s t - test ) .

O O B-EXP_ASSAY O B-GENEPROD O B-GENEPROD O O B-GENEPROD O O O B-GENEPROD O O O B-CELL_TYPE I-CELL_TYPE O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O B-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 expression of IFIT1, ISG15, and Cig5 in WT and RBM47+/- peritoneal macrophages stimulated by IFN-α for 12 h. The PCR results are represented as the means ± SD of n = 3 biological replicates. Data information: The data shown are representative of n = 3 independent experiments. NS, non-significant; * P ≤ 0.05, **P ≤ 0.01, and ***P ≤ 0.001 (Student's t-test).


B IFNAR1 mRNA stability after DRB treatment . 293T cells were transfected with RBM47 - Flag or empty vectors and treated with DRB ( 50 μg / mL ) . Total RNA was harvested at 0 , 15 , 30 , 45 , and 60 min , and IFNAR1 mRNA levels were measured using qRT - PCR and normalized to β - actin . The PCR results are represented as the means ± SD of n = 3 biological replicates . Data information : Data are representative of n = 3 independent experiments . NS , non - significant ; * * P ≤ 0 . 01 , and * * * P ≤ 0 . 001 ( Student ' s t - test ) .

O B-GENEPROD O O O B-SMALL_MOLECULE O O B-CELL_LINE O O O O B-GENEPROD O B-GENEPROD O O O O O O B-SMALL_MOLECULE O 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-GENEPROD B-EXP_ASSAY I-EXP_ASSAY O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O B-GENEPROD I-GENEPROD I-GENEPROD O O B-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 IFNAR1 mRNA stability after DRB treatment. 293T cells were transfected with RBM47-Flag or empty vectors and treated with DRB (50 μg/mL). Total RNA was harvested at 0, 15, 30, 45, and 60 min, and IFNAR1 mRNA levels were measured using qRT-PCR and normalized to β-actin. The PCR results are represented as the means ± SD of n = 3 biological replicates. Data information: Data are representative of n = 3 independent experiments. NS, non-significant; **P ≤ 0.01, and ***P ≤ 0.001 (Student's t-test).


A mRNA of IFNAR1 , but not IFNAR2 or STAT1 , was stabilized by RBM47 ectopic expression in 293T cells with or without IFN - α stimulation . Cells were treated with IFN - α or PBS for 6 h , and the relative gene expression levels were determined using qRT - PCR . The PCR results are represented as the means ± SD of n = 3 biological replicates . Data information : The data shown are representative of n = 3 independent experiments . NS , non - significant ; * * P ≤ 0 . 01 , and * * * P ≤ 0 . 001 ( Student ' s t - test ) . The data shown are representative of three independent experiments .

O O O B-GENEPROD O O O B-GENEPROD O B-GENEPROD O O O O B-GENEPROD O O O B-CELL_LINE O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O B-SMALL_MOLECULE O O O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O B-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 mRNA of IFNAR1, but not IFNAR2 or STAT1, was stabilized by RBM47 ectopic expression in 293T cells with or without IFN-α stimulation. Cells were treated with IFN-α or PBS for 6 h, and the relative gene expression levels were determined using qRT-PCR.The PCR results are represented as the means ± SD of n = 3 biological replicates. Data information: The data shown are representative of n = 3 independent experiments. NS, non-significant; **P ≤ 0.01, and ***P ≤ 0.001 (Student's t-test). The data shown are representative of three independent experiments.


F , G Protein levels of IFNAR1 and phosphorylation levels of TYK2 , STAT1 , and STAT2 in RBM47 - overexpressing ( F ) or knockdown ( G ) 293T cells . Cells were treated with PBS or IFN - α for 6 h , and the relative band densities were analyzed using ImageJ software and normalized to GAPDH . Data information : The data shown are representative of n = 3 independent experiments . NS , non - significant ; * * P ≤ 0 . 01 , and * * * P ≤ 0 . 001 ( Student ' s t - test ) . The data shown are representative of three independent experiments .

O O O B-EXP_ASSAY I-EXP_ASSAY O B-GENEPROD O B-EXP_ASSAY O O B-GENEPROD O B-GENEPROD O O B-GENEPROD O B-GENEPROD O O O O O O O O O O B-CELL_LINE O O O O O O B-SMALL_MOLECULE O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O O O O O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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, G Protein levels of IFNAR1 and phosphorylation levels of TYK2, STAT1, and STAT2 in RBM47-overexpressing (F) or knockdown (G) 293T cells. Cells were treated with PBS or IFN-α for 6 h, and the relative band densities were analyzed using ImageJ software and normalized to GAPDH. Data information: The data shown are representative of n = 3 independent experiments. NS, non-significant; **P ≤ 0.01, and ***P ≤ 0.001 (Student's t-test). The data shown are representative of three independent experiments.


G RBM47 + / + and RBM47 + / - mice were infected with HSV - 1 ( 1 × 107 pfu ) via caudal vein injection . Viral genome DNA and progeny titers were tested using blood samples from infected mice . Data are represented as the means ± SD of n = 6 biological replicates . H qRT - PCR analysis of IFNAR1 and Cig5 mRNA in blood samples from HSV - 1 - infected mice . The PCR results are represented as the means ± SD of n = 6 biological replicates . Data information : The data shown are representative of n = 3 independent experiments . NS , non - significant ; * P ≤ 0 . 05 , * * P ≤ 0 . 01 , and * * * P ≤ 0 . 001 ( Student ' s t - test ) .

O B-GENEPROD O O O O B-GENEPROD O O O B-ORGANISM O O O B-ORGANISM I-ORGANISM I-ORGANISM O O O O O O O B-TISSUE I-TISSUE O O B-SUBCELLULAR I-SUBCELLULAR I-SUBCELLULAR O B-EXP_ASSAY I-EXP_ASSAY O O O B-TISSUE O O O B-ORGANISM O O O O O O O O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O B-GENEPROD O B-GENEPROD O O B-TISSUE O O B-ORGANISM I-ORGANISM I-ORGANISM O O B-ORGANISM O O B-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 RBM47+/+ and RBM47+/- mice were infected with HSV-1 (1× 107 pfu) via caudal vein injection. Viral genome DNA and progeny titers were tested using blood samples from infected mice. Data are represented as the means ± SD of n = 6 biological replicates. H qRT-PCR analysis of IFNAR1 and Cig5 mRNA in blood samples from HSV-1-infected mice. The PCR results are represented as the means ± SD of n = 6 biological replicates. Data information: The data shown are representative of n = 3 independent experiments. NS, non-significant; * P ≤ 0.05, **P ≤ 0.01, and ***P ≤ 0.001 (Student's t-test).


D The mRNA levels of VSV and RBM47 in blood samples of IFNAR1 deficient ( IFNAR1 - / - ) mice ( n = 6 per group ) infected with VSV . Mice were infected with shmRBM47 or control lentivirus for 7 days and then challenged with VSV for another 24 h . Each dot represents one mouse . The PCR results are represented as the means ± SD of n = 6 biological replicates . Data information : The data shown are representative of n = 3 independent experiments . NS , non - significant ; * * P ≤ 0 . 01 , and * * * P ≤ 0 . 001 ( Student ' s t - test ) .

O O B-EXP_ASSAY I-EXP_ASSAY O B-ORGANISM O B-GENEPROD O B-TISSUE O O B-GENEPROD O O B-GENEPROD O O O O B-ORGANISM O O O O O O O O O B-ORGANISM O B-ORGANISM O O O O O O B-ORGANISM O O O O O O O B-ORGANISM O O O O O O O O O B-ORGANISM O O B-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 The mRNA levels of VSV and RBM47 in blood samples of IFNAR1 deficient (IFNAR1-/-) mice (n = 6 per group) infected with VSV. Mice were infected with shmRBM47 or control lentivirus for 7 days and then challenged with VSV for another 24 h. Each dot represents one mouse. The PCR results are represented as the means ± SD of n = 6 biological replicates. Data information: The data shown are representative of n = 3 independent experiments. NS, non-significant; **P ≤ 0.01, and ***P ≤ 0.001 (Student's t-test).


A ) Upper panels : Representative single z - planes from 3D - SIM image stacks of the DAPI - stained nuclei of AC16 cells kept at 37 ° C or exposed to different cold temperatures for 24h . Lower panels : DAPI signal segmented into chromatin and interchromatin regions according to relative intensity . Scale bar : 5 μm . Arrows mark chromatin concentrated at the nuclear periphery ( P ) and around nucleoli ( N ) . B ) Boxplots of the ratio of chromatin volume to nuclear volume for cells exposed to different temperature conditions . Median ( central line ) , interquartile range ( IQR , box edges ) , most extreme data point no more than 1 . 5x IQR from box edges ( whiskers ) . Number of nuclei : 37 ° C ( 31 ) , 28 ° C 24h ( 26 ) , 18 ° C 5h ( 19 ) , 18 ° C 24h ( 22 ) , 18 ° C 24h then 37 ° C 2h ( 25 ) , 8 ° C 24h ( 23 ) . C ) Left and central panels : representative single z - planes from 3D - SIM image stacks of the DAPI - stained nuclei of cells kept at 37 ° C or exposed to 18 ° C for 24h showing the spatial distribution of the immunofluorescence ( IF ) signal of hnRNPC ( green ) and nuclear pore complexes ( NPCs , red ) . Right panel : DAPI signal overlaid with dots indicating IF signal centroid coordinates for centroids located in the z - plane shown . Scale bar whole nuclei : 5 μm . Scale bar enlarged section : 1 μm . D ) Bar graph of the mean NPC centroid to nearest hnRNPC centroid distance for each temperature condition . Number of distances : 37 ° C ( 112511 ) , 28 ° C 24h ( 94521 ) , 18 ° C 5h ( 72340 ) , 18 ° C 24h ( 74998 ) , 18 ° C 24h then 37 ° C 2h ( 92896 ) , 8 ° C 24h ( 80570 ) .

O O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O B-SMALL_MOLECULE O B-EXP_ASSAY B-SUBCELLULAR O B-CELL_LINE O O O O O O O O O O O O O O O O O O B-SMALL_MOLECULE O O O B-SUBCELLULAR O B-SUBCELLULAR I-SUBCELLULAR O O O B-EXP_ASSAY O O O O O O O O O B-SUBCELLULAR O O O B-SUBCELLULAR I-SUBCELLULAR O O O O O B-SUBCELLULAR O O O O O O O O O O O B-SUBCELLULAR B-EXP_ASSAY O B-SUBCELLULAR B-EXP_ASSAY O O O O O O O O O O O O O O O O O O O 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-SUBCELLULAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O B-SMALL_MOLECULE O B-EXP_ASSAY B-SUBCELLULAR O O O O O O O O O O O O O O O O O O O O O B-EXP_ASSAY O B-EXP_ASSAY O O O B-GENEPROD O O O O B-SUBCELLULAR I-SUBCELLULAR I-SUBCELLULAR O B-SUBCELLULAR O O O O O O O B-SMALL_MOLECULE O O O O O B-EXP_ASSAY O O O O O O O O O O O O O O O O B-SUBCELLULAR O O O O O O O O O O O O O O O O O O O B-SUBCELLULAR O O O B-GENEPROD O B-EXP_ASSAY O O O O O O O B-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Upper panels: Representative single z-planes from 3D-SIM image stacks of the DAPI-stained nuclei of AC16 cells kept at 37°C or exposed to different cold temperatures for 24h. Lower panels: DAPI signal segmented into chromatin and interchromatin regions according to relative intensity. Scale bar: 5 μm. Arrows mark chromatin concentrated at the nuclear periphery (P) and around nucleoli (N). B) Boxplots of the ratio of chromatin volume to nuclear volume for cells exposed to different temperature conditions. Median (central line), interquartile range (IQR, box edges), most extreme data point no more than 1.5x IQR from box edges (whiskers). Number of nuclei: 37°C (31), 28°C 24h (26), 18°C 5h (19), 18°C 24h (22), 18°C 24h then 37°C 2h (25), 8°C 24h (23). C) Left and central panels: representative single z-planes from 3D-SIM image stacks of the DAPI-stained nuclei of cells kept at 37°C or exposed to 18°C for 24h showing the spatial distribution of the immunofluorescence (IF) signal of hnRNPC (green) and nuclear pore complexes (NPCs, red). Right panel: DAPI signal overlaid with dots indicating IF signal centroid coordinates for centroids located in the z-plane shown. Scale bar whole nuclei: 5 μm. Scale bar enlarged section: 1 μm. D) Bar graph of the mean NPC centroid to nearest hnRNPC centroid distance for each temperature condition. Number of distances: 37°C (112511), 28°C 24h (94521), 18°C 5h (72340), 18°C 24h (74998), 18°C 24h then 37°C 2h (92896), 8°C 24h (80570).


A ) Number of genes showing significant ( adjusted p < 0 . 05 , Wald test ) up or downregulation in nuclear or cytoplasmic RNA levels upon transfer of AC16 cells from 37 ° C to different cold temperatures for 24h .

O O O O O O O O O O O O O O O O O O O O O O B-SUBCELLULAR O B-SUBCELLULAR B-EXP_ASSAY I-EXP_ASSAY O O O B-CELL_LINE O O O O O O O O O O O O

A) Number of genes showing significant (adjusted p < 0.05, Wald test) up or downregulation in nuclear or cytoplasmic RNA levels upon transfer of AC16 cells from 37°C to different cold temperatures for 24h.


B ) Relationship between the log2 fold change in RNA level in the nucleus and that in the cytoplasm for all genes upon transfer of AC16 cells from 37 ° C to different cold temperatures for 24h . Points are color - coded from low to high density ( black < blue < red < yellow ) .

O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O O B-SUBCELLULAR O O O O B-SUBCELLULAR O O O O O O B-CELL_LINE O O O O O O O O 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) Relationship between the log2 fold change in RNA level in the nucleus and that in the cytoplasm for all genes upon transfer of AC16 cells from 37°C to different cold temperatures for 24h. Points are color-coded from low to high density (black < blue < red < yellow).


C ) Relationship between the log2 fold change in RNA level for all genes upon transfer of AC16 cells from 37 ° C to 28 ° C or 18 ° C ( top ) , 28 ° C or 8 ° C ( middle ) and 18 ° C or 8 ° C ( bottom ) for 24h in both the nucleus and cytoplasm . Points are shaded from low to high density ( grey < black ) . Genes that show significant differential expression at a specific temperature or at both temperatures are highlighted with colored circles or triangles , respectively .

O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O B-CELL_LINE O O O O O O O O O O O O O O O O O O O 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-SUBCELLULAR O B-SUBCELLULAR O O O O O O O O O O O O O O O O O O O O B-EXP_ASSAY O O O O O O O O O O O O O O O O O O

C) Relationship between the log2 fold change in RNA level for all genes upon transfer of AC16 cells from 37°C to 28°C or 18°C (top), 28°C or 8°C (middle) and 18°C or 8°C (bottom) for 24h in both the nucleus and cytoplasm. Points are shaded from low to high density (grey < black). Genes that show significant differential expression at a specific temperature or at both temperatures are highlighted with colored circles or triangles, respectively.


A ) Boxplots of standardized nuclear and cytoplasmic RNA levels at time points during the transfer of AC16 cells from 37 ° C to 18 ° C for 24h and then back to 37 ° C for 24h for the group of 1285 genes showing significant upregulation in nuclear RNA levels at the 18 ° C for 24h time point . Range ( whiskers ) , interquartile range ( IQR , box edges ) , median + / - IQR / [ square root ( number of genes ) ] ( notch ) and median ( central line ) .

O O O O O B-SUBCELLULAR O B-SUBCELLULAR B-EXP_ASSAY I-EXP_ASSAY O O O O O O O B-CELL_LINE O O O O O O 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-SUBCELLULAR B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O O O O 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) Boxplots of standardized nuclear and cytoplasmic RNA levels at time points during the transfer of AC16 cells from 37°C to 18°C for 24h and then back to 37°C for 24h for the group of 1285 genes showing significant upregulation in nuclear RNA levels at the 18°C for 24h time point. Range (whiskers), interquartile range (IQR, box edges), median +/-IQR/[square root(number of genes)] (notch) and median (central line).


B ) Log2 fold change in cytoplasmic ( black line , left axis ) and nuclear ( red line , right axis ) RNA level of core circadian clock negative limb genes at time points during the transfer of AC16 cells from 37 ° C to 18 ° C for 24h and then back to 37 ° C for 24h relative to cells kept at 37 ° C . Error bars show the standard error .

O O O O O O B-SUBCELLULAR O O O O O O O O B-SUBCELLULAR O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O O B-CELL_LINE O O O O O O O O O O O O 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) Log2 fold change in cytoplasmic (black line, left axis) and nuclear (red line, right axis) RNA level of core circadian clock negative limb genes at time points during the transfer of AC16 cells from 37°C to 18°C for 24h and then back to 37°C for 24h relative to cells kept at 37°C. Error bars show the standard error.


C ) Nuclear and cytoplasmic full - length RNA - Seq data from AC16 cells kept at 37 ° C or exposed to 18 ° C for 24h for REV - ERBα and CRY2 . Data for each track have been normalized to show counts per million aligned counts ( CPM ) for each nucleotide . Number of 3 ' end RNA - seq sample replicates : 37 ° C ( C : 6 , N : 7 ) , 18 ° C 5h ( 4 ) , 10h ( 4 ) , 24h ( 6 ) , 18 ° C 24h then 37 ° C 2h ( 4 ) , 5h ( 2 ) , 10h ( 2 ) , 24h ( 2 ) . Number of cytoplasmic ( C ) and nuclear ( N ) samples are the same for each condition except 37 °

O O B-SUBCELLULAR O B-SUBCELLULAR O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O B-CELL_LINE O O O O O O O O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O B-GENEPROD O O O O O O O O O O O O O O O O O O O O B-SMALL_MOLECULE O O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-SUBCELLULAR O O O O B-SUBCELLULAR O O O O O O O O O O O O O

C) Nuclear and cytoplasmic full-length RNA-Seq data from AC16 cells kept at 37°C or exposed to 18°C for 24h for REV-ERBα and CRY2. Data for each track have been normalized to show counts per million aligned counts (CPM) for each nucleotide. Number of 3' end RNA-seq sample replicates: 37°C (C: 6, N: 7), 18°C 5h (4), 10h (4), 24h (6), 18°C 24h then 37°C 2h (4), 5h (2), 10h (2), 24h (2). Number of cytoplasmic (C) and nuclear (N) samples are the same for each condition except 37°C


Western blot of Flag - tagged REV - ERBα levels ( short and long exposure ) in whole cell extracts from AC16 cells transferred from 37 ° C to 18 ° C for the time periods indicated and also returned after each of these time periods to 37 ° C for 2h . TP53 was used as a loading control as its transcript levels show minimal changes in response to 18 ° C exposure Ponceau stain is shown as an additional loading control .

B-EXP_ASSAY I-EXP_ASSAY O B-GENEPROD O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O O O B-CELL_LINE O O O O O O O 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-GENEPROD O O O O O O O O O O O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O

Western blot of Flag-tagged REV-ERBα levels (short and long exposure) in whole cell extracts from AC16 cells transferred from 37°C to 18°C for the time periods indicated and also returned after each of these time periods to 37°C for 2h. TP53 was used as a loading control as its transcript levels show minimal changes in response to 18°C exposure Ponceau stain is shown as an additional loading control.


Representative maximum intensity z - stack - projected images of DAPI - stained ( blue ) AC16 exposed to 18 ° C and returned to 37 ° C for the time periods indicated , probed by RNA - FISH for REV -

O O B-EXP_ASSAY O O O O O B-EXP_ASSAY O B-SMALL_MOLECULE O B-EXP_ASSAY O O O B-CELL_LINE O O O O O O O O O O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O B-GENEPROD I-GENEPROD

Representative maximum intensity z-stack-projected images of DAPI-stained (blue) AC16 exposed to 18°C and returned to 37°C for the time periods indicated, probed by RNA-FISH for REV-ERBα


Representative maximum intensity z - stack - projected images of DAPI - stained ( blue ) AC16 exposed to 18 ° C and returned to 37 ° C for the time periods indicated , probed by RNA - FISH for CRY2 ( B )

O O B-EXP_ASSAY O O O O O B-EXP_ASSAY O B-SMALL_MOLECULE O B-EXP_ASSAY O O O B-CELL_LINE O O O O O O O O O O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O B-GENEPROD O O O

Representative maximum intensity z-stack-projected images of DAPI-stained (blue) AC16 exposed to 18°C and returned to 37°C for the time periods indicated, probed by RNA-FISH for CRY2 (B)


Representative maximum intensity z - stack - projected images of DAPI - stained ( blue ) AC16 exposed to 18 ° C and returned to 37 ° C for the time periods indicated , probed by RNA - FISH for TP53 ( C )

Representative maximum intensity z-stack-projected images of DAPI-stained (blue) AC16 exposed to 18°C and returned to 37°C for the time periods indicated, probed by RNA-FISH for TP53 (C)


Representative maximum intensity z - stack - projected images of DAPI - stained ( blue ) U2OS cells ( D ) , exposed to 18 ° C and returned to 37 ° C for the time periods indicated , probed by RNA - FISH for REV -

O O B-EXP_ASSAY O O O O O B-EXP_ASSAY O B-SMALL_MOLECULE O B-EXP_ASSAY O O O B-CELL_LINE 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-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O B-GENEPROD I-GENEPROD

Representative maximum intensity z-stack-projected images of DAPI-stained (blue) U2OS cells (D), exposed to 18°C and returned to 37°C for the time periods indicated, probed by RNA-FISH for REV-ERBα


Mean REV - ERBα per AC16 cell nuclear and cytoplasmic area across all images for each condition from quantification of the RNA - FISH data . AC16 cells ( REV - ERBα ) : 37 ° C ( 65 ) , 18 ° C 5h ( 30 ) , 18 ° C 5h then 37 ° C 2h ( 21 ) , 18 ° C 24h ( 63 ) , 18 ° C 24h then 37 ° C 2h ( 64 )

O B-GENEPROD I-GENEPROD I-GENEPROD O B-CELL_LINE O B-SUBCELLULAR O B-SUBCELLULAR I-SUBCELLULAR O O B-EXP_ASSAY O O O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O B-CELL_LINE O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Mean REV-ERBα per AC16 cell nuclear and cytoplasmic area across all images for each condition from quantification of the RNA-FISH data. AC16 cells (REV-ERBα): 37°C (65), 18°C 5h (30), 18°C 5h then 37°C 2h (21), 18°C 24h (63), 18°C 24h then 37°C 2h (64)


Mean CRY2 ( F ) per AC16 cell nuclear and cytoplasmic area across all images for each condition from quantification of the RNA - FISH data . ; AC16 cells ( CRY2 ) : 37 ° C ( 43 ) , 18 ° C 24h ( 42 ) , 18 ° C 24h then 37 ° C 2h ( 42 )

O B-GENEPROD O O O O B-CELL_LINE O B-SUBCELLULAR O B-SUBCELLULAR I-SUBCELLULAR O O B-EXP_ASSAY O O O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O B-CELL_LINE O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Mean CRY2 (F) per AC16 cell nuclear and cytoplasmic area across all images for each condition from quantification of the RNA-FISH data. ; AC16 cells (CRY2): 37°C (43), 18°C 24h (42), 18°C 24h then 37°C 2h (42)


Mean TP53 ( G ) transcripts per AC16 cell nuclear and cytoplasmic area across all images for each condition from quantification of the RNA - FISH data . AC16 cells ( TP53 ) : 37 ° C ( 47 ) , 18 ° C 24h ( 45 ) , 18 ° C 24h then 37 ° C 2h ( 48 )

O B-GENEPROD O O O O O B-CELL_LINE O B-SUBCELLULAR O B-SUBCELLULAR I-SUBCELLULAR O O B-EXP_ASSAY O O O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O B-CELL_LINE O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Mean TP53 (G) transcripts per AC16 cell nuclear and cytoplasmic area across all images for each condition from quantification of the RNA-FISH data. AC16 cells (TP53): 37°C (47), 18°C 24h (45), 18°C 24h then 37°C 2h (48)


Mean REV - ERBα per U2OS ( H ) cell nuclear and cytoplasmic area across all images for each condition from quantification of the RNA - FISH data . U2OS cells ( REV - ERBα ) : 37 ° C ( 61 ) , 18 ° C 5h ( 61 ) , 18 ° C 5h then 37 ° C 2h ( 69 ) , 18 ° C 24h ( 53 ) , 18 ° C 24h then 37 ° C 2h ( 65 )

O B-GENEPROD I-GENEPROD I-GENEPROD O B-CELL_LINE O O O O B-SUBCELLULAR O B-SUBCELLULAR I-SUBCELLULAR O O B-EXP_ASSAY O O O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O B-CELL_LINE O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Mean REV-ERBα per U2OS (H) cell nuclear and cytoplasmic area across all images for each condition from quantification of the RNA-FISH data. U2OS cells (REV-ERBα): 37°C (61), 18°C 5h (61), 18°C 5h then 37°C 2h (69), 18°C 24h (53), 18°C 24h then 37°C 2h (65)


A , B ) Mean baseline - detrended bioluminescence profiles from plate wells containing either PER2 : : LUC U2OS cells with ( WT , left panel ) or without REV - ERBα ( KO , right panel ) recorded at 37 ° C before and after transfer to 18 ° C for 24h ( A ) or 5h ( B ) ( grey region ) from biological repeat 1 . 6 differently colored profiles represent cells synchronized at 6 distinct phases of the circadian period prior to the start of 18 ° C exposure ( time zero ) .

O O O O O O O O B-EXP_ASSAY O O O O O O B-GENEPROD O O B-GENEPROD B-CELL_LINE O O O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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,B) Mean baseline-detrended bioluminescence profiles from plate wells containing either PER2::LUC U2OS cells with (WT, left panel) or without REV-ERBα (KO, right panel) recorded at 37°C before and after transfer to 18°C for 24h (A) or 5h (B) (grey region) from biological repeat 1. 6 differently colored profiles represent cells synchronized at 6 distinct phases of the circadian period prior to the start of 18°C exposure (time zero).


D ) Boxplots and individual points for times for each plate well profile for both WT and KO cells kept at 37 ° C or transferred to 18 ° C for 24h ( top panel ) or 5h ( bottom panel ) from biological repeats 1 and 2 . Points are colored according to their distinct phase prior to 18 ° C exposure . Colored lines ( bottom panel ) show the change in the mean for the points from each phase . Boxplots display median ( central line ) , interquartile range ( IQR , box edges ) and range ( whiskers ) . Number of wells : top panel WT cells : 37 ° C ( 120 ) , 18 ° C 24h ( 119 ) ; top panel KO cells : 37 ° C ( 120 ) , 18 ° C 24h ( 120 ) ; bottom panel WT cells : 37 ° C ( 58 ) , 18 ° C 5h ( 120 ) ; top panel KO cells : 37 ° C ( 60 ) , 18 ° C 5h ( 120 ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Boxplots and individual points for times for each plate well profile for both WT and KO cells kept at 37°C or transferred to 18°C for 24h (top panel) or 5h (bottom panel) from biological repeats 1 and 2. Points are colored according to their distinct phase prior to 18°C exposure. Colored lines (bottom panel) show the change in the mean for the points from each phase. Boxplots display median (central line), interquartile range (IQR, box edges) and range (whiskers). Number of wells: top panel WT cells: 37°C (120), 18°C 24h (119); top panel KO cells: 37°C (120), 18°C 24h (120); bottom panel WT cells: 37°C (58), 18°C 5h (120); top panel KO cells: 37°C (60), 18°C 5h (120).


E ) Bar graphs showing the mean amplitude change ( left axis ) following 18 ° C exposure for 5h for each group of plate well profiles ( grouped according to their distinct phase ( a - f ) prior to 18 ° C exposure , 10 wells per group ) for both WT and KO cells from biological repeats 1 and 2 . Mean changes are plotted to approximately align with the position of the control profile at the point of rewarming within the circadian period that is represented by the PER2 : LUC ( dashed line ) and predicted REV - ERBα expression level ( right axis ) ( grey shaded region ( light grey indicates REV - ERBα deletion ) ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-GENEPROD O B-GENEPROD O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD B-EXP_ASSAY O O O O O O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O

E) Bar graphs showing the mean amplitude change (left axis) following 18°C exposure for 5h for each group of plate well profiles (grouped according to their distinct phase (a-f) prior to 18°C exposure, 10 wells per group) for both WT and KO cells from biological repeats 1 and 2. Mean changes are plotted to approximately align with the position of the control profile at the point of rewarming within the circadian period that is represented by the PER2:LUC (dashed line) and predicted REV-ERBα expression level (right axis) (grey shaded region (light grey indicates REV-ERBα deletion)).


B Spermatocyte chromosomes that underwent condensation ( right ) within a MII oocyte ( MII chromosome mass , left ) . In this study , spermatocytes picked up for injection had tetrad chromosomes ready for condensation . C Chromosomal analysis of MII oocytes that had been injected with primary spermatocytes . In the spermatocyte - injected groups , normality was improved by reducing the ooplasm mass ( * P < 0 . 005 by Fisher ' s exact probability test ) . Arrows in the right figure indicate prematurely separated chromatids . The numbers of oocytes observed are indicated on the top of the bars .

O B-CELL_TYPE B-SUBCELLULAR O O B-EXP_ASSAY O O O O O O B-CELL_TYPE O B-SUBCELLULAR I-SUBCELLULAR O O O O O O O O O B-CELL_TYPE O O O O O O B-SUBCELLULAR O O B-EXP_ASSAY O O B-EXP_ASSAY I-EXP_ASSAY O O B-CELL_TYPE O O O O O B-CELL_TYPE I-CELL_TYPE O O O B-CELL_TYPE O O O O O O O O O O B-SUBCELLULAR O 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-SUBCELLULAR O O O O B-CELL_TYPE O O O O O O O O O O

B Spermatocyte chromosomes that underwent condensation (right) within a MII oocyte (MII chromosome mass, left). In this study, spermatocytes picked up for injection had tetrad chromosomes ready for condensation. C Chromosomal analysis of MII oocytes that had been injected with primary spermatocytes. In the spermatocyte-injected groups, normality was improved by reducing the ooplasm mass (*P < 0.005 by Fisher's exact probability test). Arrows in the right figure indicate prematurely separated chromatids. The numbers of oocytes observed are indicated on the top of the bars.


B Chromosome tracking in 3D . The reconstructed images are viewed from the side of the metaphase plate . Signals are interpolated in the Z axis for visualization . White and red arrowheads , as well as red surfaces , indicate univalent - like chromosomes that underwent unbalanced predivision ( premature segregation of sister chromatids ) . Scale bar = 4 μm . C Halving the recipient ooplasmic mass rescued chromosome alignment . The numbers of misaligned chromosomes and their parental origin were determined in 3D ( n = 39 and 17 oocytes ) . Error bars show the standard deviation . Student ' s t - test was used to compare means . * P < 0 . 05 . N . S . , not significant .

O B-SUBCELLULAR O O O O O O O O O O O O O O B-SUBCELLULAR I-SUBCELLULAR O O 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-SUBCELLULAR O O O O O O O O O B-SUBCELLULAR O O O O O O O O O O O O B-SUBCELLULAR O O B-SUBCELLULAR O O O O O O B-SUBCELLULAR O O O O O O O O O O O O O O B-CELL_TYPE O O O O O O O O O O O O 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 Chromosome tracking in 3D. The reconstructed images are viewed from the side of the metaphase plate. Signals are interpolated in the Z axis for visualization. White and red arrowheads, as well as red surfaces, indicate univalent-like chromosomes that underwent unbalanced predivision (premature segregation of sister chromatids). Scale bar = 4 μm. C Halving the recipient ooplasmic mass rescued chromosome alignment. The numbers of misaligned chromosomes and their parental origin were determined in 3D (n = 39 and 17 oocytes). Error bars show the standard deviation. Student's t-test was used to compare means. *P < 0.05. N.S., not significant.


C Univalent - like chromosomes . Images were 3D - reconstructed and viewed from the top of the metaphase plate . Red surfaces with white arrowheads indicate univalent - like chromosomes . Scale bar = 3 μm .

O O O O B-SUBCELLULAR O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O O O B-SUBCELLULAR I-SUBCELLULAR O O O O O O O O O O B-SUBCELLULAR O O O O O O O

C Univalent-like chromosomes. Images were 3D-reconstructed and viewed from the top of the metaphase plate. Red surfaces with white arrowheads indicate univalent-like chromosomes. Scale bar = 3 μm.


D Halving the ooplasm volume suppressed the premature separation of paternal chromosomes . Oocytes were categorized based on whether the chromosomes exhibited premature separation into univalent - like structures prior to segregation errors . Data from 39 and 17 oocytes , respectively .

O O O B-SUBCELLULAR O O O O O O O B-SUBCELLULAR O B-CELL_TYPE O O O O O O B-SUBCELLULAR O O O O O O O O O O O O O O O O O O B-CELL_TYPE O O O

D Halving the ooplasm volume suppressed the premature separation of paternal chromosomes. Oocytes were categorized based on whether the chromosomes exhibited premature separation into univalent-like structures prior to segregation errors. Data from 39 and 17 oocytes, respectively.


D Effect of forced SAC activation . Oocytes expressing MAD1 - mNeonGreen - CENP - C ( green ) , together with major satellite - mClover ( green ) and H2B - mCherry ( red ) , were imaged . Time after the start of imaging ( h : mm ) is shown . Oocytes were categorized based on anaphase figures ( n = 24 , 23 oocytes ) .

O O O O O O O B-CELL_TYPE O B-GENEPROD O B-GENEPROD O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O B-SUBCELLULAR I-SUBCELLULAR O B-GENEPROD O O O O B-GENEPROD O B-GENEPROD O O O O O O O O O O O O B-EXP_ASSAY O O O O O O O O B-CELL_TYPE O O O O O O O O O O O O B-CELL_TYPE O O

D Effect of forced SAC activation. Oocytes expressing MAD1-mNeonGreen-CENP-C (green), together with major satellite-mClover (green) and H2B-mCherry (red), were imaged. Time after the start of imaging (h:mm) is shown. Oocytes were categorized based on anaphase figures (n = 24, 23 oocytes).


( A ) Western blot analysis for pRb in DM - 2 cultures .

O O O B-EXP_ASSAY I-EXP_ASSAY O O B-GENEPROD O O O O O O

(A) Western blot analysis for pRb in DM-2 cultures.


( B ) Immunostaining for MHC in DM - 3 and DM - 6 differentiating myotubes . Nuclei were stained with DAPI ( blue ) . ( C ) Mean number of myotubes on the indicated days after differentiation . Each time point is mean ± SD of six fields from six independent experiments .

O O O B-EXP_ASSAY O B-SUBCELLULAR O O O O O O O O O B-CELL_TYPE O B-SUBCELLULAR O O O B-EXP_ASSAY O O O O O O O O O O B-CELL_TYPE O O O O O O O O O O O O O O O O O O O O O O

(B) Immunostaining for MHC in DM-3 and DM-6 differentiating myotubes. Nuclei were stained with DAPI (blue). (C) Mean number of myotubes on the indicated days after differentiation. Each time point is mean ± SD of six fields from six independent experiments.


( D ) TUNEL staining . Arrowheads indicate TUNEL - positive nuclei ( green ) .

O O O B-EXP_ASSAY O O O O B-EXP_ASSAY O O B-SUBCELLULAR O O O O

(D) TUNEL staining. Arrowheads indicate TUNEL-positive nuclei (green).


( E ) LC3 - RFP expression in control ( ctrl ) and Rb − / − myotubes . Insets show nuclear DAPI stain ( blue ) .

O O O B-GENEPROD O B-GENEPROD O O O O O O O B-GENEPROD O O O B-CELL_TYPE O O O B-SUBCELLULAR B-EXP_ASSAY O O O O O

(E) LC3-RFP expression in control (ctrl) and Rb−/− myotubes. Insets show nuclear DAPI stain (blue).


( F ) Representative Western blot ( n = 3 ) for LC3 . Chloroquine was added 12 h before cell harvest .

O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O B-GENEPROD O B-SMALL_MOLECULE O O O O O O O O

(F) Representative Western blot (n = 3) for LC3. Chloroquine was added 12 h before cell harvest.


( G ) Representative Western blot for LC3 in E16 . 5 control and mgRb : Rb − / − muscles . ( H ) Quantification of LC3 - II / tubulin ratio in E16 . 5 mgRb : Rb − / − skeletal muscles relative to control as depicted in G ( mean ± SD ; n = 3 ) . * , P = 0 . 016 by Student ' s t test .

O O O O B-EXP_ASSAY I-EXP_ASSAY O B-GENEPROD O O O O O O O O B-GENEPROD O O O B-TISSUE O O O O O O B-GENEPROD O O O B-GENEPROD O O O O O O O B-GENEPROD O O O B-TISSUE I-TISSUE O O O O O O O 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) Representative Western blot for LC3 in E16.5 control and mgRb:Rb−/− muscles. (H) Quantification of LC3-II/tubulin ratio in E16.5 mgRb:Rb−/− skeletal muscles relative to control as depicted in G (mean ± SD; n = 3). *, P = 0.016 by Student's t test.


( I ) Representative Western blot for LC3 - I and LC3 - II expression in whole cell lysates from the indicated myotubes transduced with Ad . GFP or Ad . RbΔK11 . ( J ) Quantification of LC3 - II / tubulin ratio by Western blots as shown in I . Data represent mean ± SD ( n = 7 ) . * , P = 0 . 047 ; * * , P = 0 . 005 by analysis of variance .

O O O O B-EXP_ASSAY I-EXP_ASSAY O B-GENEPROD O O O B-GENEPROD O O O O O O O O O O B-CELL_TYPE O O B-ORGANISM O B-GENEPROD O B-ORGANISM O B-GENEPROD O O O O O O B-GENEPROD O O O B-GENEPROD O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O 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) Representative Western blot for LC3-I and LC3-II expression in whole cell lysates from the indicated myotubes transduced with Ad.GFP or Ad.RbΔK11. (J) Quantification of LC3-II/tubulin ratio by Western blots as shown in I. Data represent mean ± SD (n = 7). *, P = 0.047; **, P = 0.005 by analysis of variance.


( A , left ) MitoTracker staining . ( right ) MitoTracker staining after bezafibrate treatment . ( B ) Quantification of myotubes with perinuclear MitoTracker - positive aggregates in untreated ( vehicle ) or bezafibrate - treated myotubes at DM - 2 . Data represent mean ± SD ( n = 3 ) . * , P < 0 . 001 by Student ' s t test .

O O O O O B-EXP_ASSAY O O O O O B-EXP_ASSAY O O B-SMALL_MOLECULE O O O O O O O B-CELL_TYPE O O B-EXP_ASSAY O O O O O O O O O B-SMALL_MOLECULE O O B-CELL_TYPE O O O O O O 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, left) MitoTracker staining. (right) MitoTracker staining after bezafibrate treatment. (B) Quantification of myotubes with perinuclear MitoTracker-positive aggregates in untreated (vehicle) or bezafibrate-treated myotubes at DM-2. Data represent mean ± SD (n = 3). *, P < 0.001 by Student's t test.


( C ) MitoTracker ( red ) and LC3immunostaining ( green ) of myotubes at DM - 2 . Chloroquine and 3 - MA ( where indicated ) were added 12 h before staining . Note the extensive overlap of MitoTracker and LC3 in Rb − / − myotubes ( yellow ) , which is inhibited by 3 - MA . ( D ) Mean percentage of MitoTracker ( red ) to LC3 ( green ) overlap in 25 myotubes from two independent cultures in control versus Rb − / − myotubes ( total ) or Rb − / − myotubes with perinuclear mitochondrial aggregation ( high ) . * , P = 7 × 10 − 5 by Student ' s t test .

O O O B-EXP_ASSAY O O O O B-GENEPROD O O O O B-CELL_TYPE O O O O O B-SMALL_MOLECULE O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O O O O O O O O O O B-EXP_ASSAY O B-GENEPROD O B-GENEPROD O O O B-CELL_TYPE O O O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O B-EXP_ASSAY O O O O B-GENEPROD O O O O O O B-CELL_TYPE O O O O O O O B-GENEPROD O O O B-CELL_TYPE O O O O B-GENEPROD O O O B-CELL_TYPE 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) MitoTracker (red) and LC3immunostaining (green) of myotubes at DM-2. Chloroquine and 3-MA (where indicated) were added 12 h before staining. Note the extensive overlap of MitoTracker and LC3 in Rb−/−myotubes (yellow), which is inhibited by 3-MA. (D) Mean percentage of MitoTracker (red) to LC3 (green) overlap in 25 myotubes from two independent cultures in control versus Rb−/−myotubes (total) or Rb−/−myotubes with perinuclear mitochondrial aggregation (high). *, P = 7 × 10−5 by Student's t test.


( E ) Ultrastructural analysis of myotubes treated or not with 3 - MA at DM - 2 . Arrowheads point to mitochondria . Asterisks indicate electron - dense autolysosomes . N , nucleus . ( F ) Ratio of mitochondrialDNA / nuclearDNA in the indicated cultures ( n = 3 ) . ( G ) Ratio of mitochondrialDNA / nuclearDNA in E16 . 5 skeletal muscle ( n = 3 ; * , P = 0 . 009 ) . ( H ) Quantification of ATP in E16 . 5 skeletal muscle ( n = 3 ; * , P = 0 . 011 ) . ( F - H ) Error bars represent SD . * , P < 0 . 001 .

O O O O O O B-CELL_TYPE O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O O B-SUBCELLULAR O O O O O O B-SUBCELLULAR O O O B-SUBCELLULAR O O O O O O B-SUBCELLULAR O B-SUBCELLULAR O O O O O O O O O O O O O O O B-SUBCELLULAR O B-SUBCELLULAR O O O O B-TISSUE I-TISSUE 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-TISSUE I-TISSUE O O O O O O O O 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) Ultrastructural analysis of myotubes treated or not with 3-MA at DM-2. Arrowheads point to mitochondria. Asterisks indicate electron-dense autolysosomes. N, nucleus. (F) Ratio of mitochondrialDNA/nuclearDNA in the indicated cultures (n = 3). (G) Ratio of mitochondrialDNA/nuclearDNA in E16.5 skeletal muscle (n = 3; *, P = 0.009). (H) Quantification of ATP in E16.5 skeletal muscle (n = 3; *, P = 0.011). (F-H) Error bars represent SD. *, P < 0.001.


( A ) Mean number of myotubes per field in indicated myoblast cultures induced to differentiate after transduction with Ad . GFP or Ad . Bcl - 2 . Each point represents the mean ± SD of six fields from six independent experiments . Bcl - 2 - rescued , twitching Rb − / − myotubes are shown in Video 2 . ( inset ) Western blot for Bcl - 2 in Ad . Bcl2 - transduced versus uninfected primary myoblasts .

O O O O O O B-CELL_TYPE O O O O B-CELL_TYPE O O O O O O O B-ORGANISM O B-GENEPROD O B-ORGANISM O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O B-GENEPROD O O O B-CELL_TYPE O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O B-GENEPROD I-GENEPROD I-GENEPROD O B-ORGANISM O B-GENEPROD O O O O O O O

(A) Mean number of myotubes per field in indicated myoblast cultures induced to differentiate after transduction with Ad.GFP or Ad.Bcl-2. Each point represents the mean ± SD of six fields from six independent experiments. Bcl-2-rescued, twitching Rb−/− myotubes are shown in Video 2. (inset) Western blot for Bcl-2 in Ad.Bcl2-transduced versus uninfected primary myoblasts.


( B ) Immunostaining for MCK ( green ) in Ad . Bcl - 2 - transduced control ( ctrl ) and Rb − / − myotubes at DM - 6 .

O O O B-EXP_ASSAY O B-GENEPROD O O O O B-ORGANISM O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O B-GENEPROD O O O B-CELL_TYPE O O O O O

(B) Immunostaining for MCK (green) in Ad.Bcl-2-transduced control (ctrl) and Rb−/− myotubes at DM-6.


( C ) Western blot for MCK , myogenin , MHC , and troponin T in indicated cultures at DM - 6 after transduction with Ad . Bcl - 2 . Tubulin was used as a loading control . Black lines indicate that intervening lanes have been spliced out .

O O O B-EXP_ASSAY I-EXP_ASSAY O B-GENEPROD O B-GENEPROD O B-SUBCELLULAR O O B-GENEPROD I-GENEPROD O O O O O O O O O O B-ORGANISM O B-GENEPROD I-GENEPROD I-GENEPROD O B-GENEPROD O O O O O O O O O O O O O O O O O O

(C) Western blot for MCK, myogenin, MHC, and troponin T in indicated cultures at DM-6 after transduction with Ad.Bcl-2. Tubulin was used as a loading control. Black lines indicate that intervening lanes have been spliced out.


( D ) Mean number of Rb − / − myotubes after 3 - MA treatment , bezafibrate , or DMSO / vehicle as indicated . Counts are mean ± SD of six fields ( n = 4 ) .

O O O O O O B-GENEPROD O O O B-CELL_TYPE O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O B-SMALL_MOLECULE O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O

(D) Mean number of Rb−/− myotubes after 3-MA treatment, bezafibrate, or DMSO/vehicle as indicated. Counts are mean ± SD of six fields (n = 4).


( E , left and middle ) Brightfield images of the indicated myotubes treated or not with 3 - MA at DM - 8 . ( right ) Immunostaining for MHC ( red ) at DM - 14 in the presence of 3 - MA . Arrowheads label 3 - MA - rescued Rb − / − myotubes at DM - 8 . 3 - MA - rescued , twitching Rb − / − myotubes are shown in Video 4 .

O O O O O O O O O O O O B-CELL_TYPE O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O O B-EXP_ASSAY O B-SUBCELLULAR O O O O O O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O B-GENEPROD O O O B-CELL_TYPE O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O O B-GENEPROD O O O B-CELL_TYPE O O O O O O

(E, left and middle) Brightfield images of the indicated myotubes treated or not with 3-MA at DM-8. (right) Immunostaining for MHC (red) at DM-14 in the presence of 3-MA. Arrowheads label 3-MA-rescued Rb−/− myotubes at DM-8. 3-MA-rescued, twitching Rb−/− myotubes are shown in Video 4.


( F ) Immunostaining for BrdU ( green ) and MHC ( red ) of 3 - MA - treated cultures at DM - 2 or DM - 10 . Rb − / − myotubes incorporated BrdU at DM - 2 ( arrowheads ) but not DM - 10 .

O O O B-EXP_ASSAY O B-EXP_ASSAY O O O O B-SUBCELLULAR O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O O O O O O B-GENEPROD O O O B-CELL_TYPE O B-EXP_ASSAY O O O O O O O O O O O O O

(F) Immunostaining for BrdU (green) and MHC (red) of 3-MA-treated cultures at DM-2 or DM-10. Rb−/− myotubes incorporated BrdU at DM-2 (arrowheads) but not DM-10.


( G ) Brightfield images ( left and middle ) and MHC staining ( right ) of the indicated cultures treated or not with bezafibrate . Arrowheads point to bezafibrate - rescued Rb − / − myotubes .

O O O O O O O O O O O B-SUBCELLULAR O O O O O O O O O O O O B-SMALL_MOLECULE O O O O B-SMALL_MOLECULE O O B-GENEPROD O O O B-CELL_TYPE O

(G) Brightfield images (left and middle) and MHC staining (right) of the indicated cultures treated or not with bezafibrate. Arrowheads point to bezafibrate-rescued Rb−/− myotubes.


( A ) Immunostaining for MHC ( green ) in the indicated myoblasts induced to differentiate under normoxia or hypoxia treated or not with lonidamine . Note the shortened myotubes in the presence of lonidamine .

O O O B-EXP_ASSAY O B-SUBCELLULAR O O O O O O B-CELL_TYPE O O O O O O O O O O O B-SMALL_MOLECULE O O O O B-CELL_TYPE O O O O B-SMALL_MOLECULE O

(A) Immunostaining for MHC (green) in the indicated myoblasts induced to differentiate under normoxia or hypoxia treated or not with lonidamine. Note the shortened myotubes in the presence of lonidamine.


( B ) Representative immunoblot ( left ) and mean expression of myogenin and MCK normalized for MHC expression ( right ; n = 3 ) .

O O O O B-EXP_ASSAY O O O O O O O B-GENEPROD O B-GENEPROD O O B-SUBCELLULAR O O O O O O O O O

(B) Representative immunoblot (left) and mean expression of myogenin and MCK normalized for MHC expression (right; n = 3).


( C ) Rbf / f myoblasts transduced with Ad . GFP or Ad . Cre and immunoblotted for pRb . Tubulin was used as a loading control .

O O O B-GENEPROD O O B-CELL_TYPE O O B-ORGANISM O B-GENEPROD O B-ORGANISM O B-GENEPROD O B-EXP_ASSAY O B-GENEPROD O B-GENEPROD O O O O O O O

(C) Rbf/f myoblasts transduced with Ad.GFP or Ad.Cre and immunoblotted for pRb. Tubulin was used as a loading control.


( D ) Rbf / f myoblasts transduced with Ad . GFP or Ad . Cre and immunostained for pRb ( red ) . ( insets ) DAPI staining for nuclei ( blue ) .

O O O B-GENEPROD O O B-CELL_TYPE O O B-ORGANISM O B-GENEPROD O B-ORGANISM O B-GENEPROD O B-EXP_ASSAY O B-GENEPROD O O O O O O O B-EXP_ASSAY O O B-SUBCELLULAR O O O O

(D) Rbf/f myoblasts transduced with Ad.GFP or Ad.Cre and immunostained for pRb (red). (insets) DAPI staining for nuclei (blue).


( E ) Immunostaining for MHC in Rbf / f myoblasts transduced with Ad . Cre and induced to differentiate in normoxia ( top and middle ) or hypoxia ( bottom ) .

O O O B-EXP_ASSAY O B-SUBCELLULAR O B-GENEPROD O O B-CELL_TYPE O O B-ORGANISM O B-GENEPROD O O O O O O O O O O O O O O O O O

(E) Immunostaining for MHC in Rbf/f myoblasts transduced with Ad.Cre and induced to differentiate in normoxia (top and middle) or hypoxia (bottom).


( F , top ) Brightfield images of Rbf / f myoblasts transduced with Ad . EV or Ad . Cre and induced to differentiate under hypoxia . ( bottom ) Larger view of the boxed field . Arrowheads point to rescued Rb − / − myotubes . This field is shown in Video 6 .

O O O O O O O O B-GENEPROD O O B-CELL_TYPE O O B-ORGANISM O B-GENEPROD O B-ORGANISM O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O B-GENEPROD O O O B-CELL_TYPE O O O O O O O O O

(F, top) Brightfield images of Rbf/f myoblasts transduced with Ad.EV or Ad.Cre and induced to differentiate under hypoxia. (bottom) Larger view of the boxed field. Arrowheads point to rescued Rb−/− myotubes. This field is shown in Video 6.


( G ) Quantification of myotube formation in Rbf / f myoblasts transduced with Ad . EV or Ad . Cre and induced to differentiate under hypoxia . Counts are mean of six representative fields ( n = 3 ) . * , P = 0 . 004 .

O O O O O B-CELL_TYPE O O B-GENEPROD O O B-CELL_TYPE O O B-ORGANISM O B-GENEPROD O B-ORGANISM O B-GENEPROD O O O O 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) Quantification of myotube formation in Rbf/f myoblasts transduced with Ad.EV or Ad.Cre and induced to differentiate under hypoxia. Counts are mean of six representative fields (n = 3). *, P = 0.004.


( H ) Quantification of myotube formation in the indicated myoblasts induced to differentiate under the indicated conditions . Myotube counts are the mean of six fields ( n = 4 ) .

O O O O O B-CELL_TYPE O O O O B-CELL_TYPE O O O O O O O O B-CELL_TYPE O O O O O O O O O O O O O

(H) Quantification of myotube formation in the indicated myoblasts induced to differentiate under the indicated conditions. Myotube counts are the mean of six fields (n = 4).


( I ) MHC staining ( red ) of the indicated myoblasts transduced with Ad . NF - κB / HIF - 1α and induced to differentiate in normoxia .

O O O B-SUBCELLULAR O O O O O O O B-CELL_TYPE O O B-ORGANISM O B-GENEPROD I-GENEPROD I-GENEPROD O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O

(I) MHC staining (red) of the indicated myoblasts transduced with Ad.NF-κB/HIF-1α and induced to differentiate in normoxia.