Figure 6A ) Comparative gene expression analysis of key enzymes involved in GSH and cysteine synthesis and supply in IDHwt ( n = 220 ) , IDHm with 1p / 19q co - deletion ( n = 151 ) or IDHm without 1p / 19q co - deletion ( n = 226 ) patients based on TCGA RNA - seq data from a cohort of 597 patients . CBS : Cystathionine beta synthase , key enzyme of the transsulfuration pathway . GCLC : catalytic subunit of glutamate - cysteine ligase . SLC7A11 : gene encoding xCT , the cystine - glutamate antiporter . B ) Kaplan - Meier curves of CBS gene expression versus overall survival in patients with IDHwt , IDHm with or without 1p / 19q co - deletion . Statistical significance for the survival analysis is shown as : pv : p - value , ns = non significant .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 6A) Comparative gene expression analysis of key enzymes involved in GSH and cysteine synthesis and supply in IDHwt (n=220), IDHm with 1p/19q co-deletion (n=151) or IDHm without 1p/19q co-deletion (n=226) patients based on TCGA RNA-seq data from a cohort of 597 patients. CBS: Cystathionine beta synthase, key enzyme of the transsulfuration pathway. GCLC: catalytic subunit of glutamate-cysteine ligase. SLC7A11: gene encoding xCT, the cystine-glutamate antiporter.B) Kaplan-Meier curves of CBS gene expression versus overall survival in patients with IDHwt, IDHm with or without 1p/19q co-deletion. Statistical significance for the survival analysis is shown as: pv: p-value, ns= non significant.


Figure 1A . Pull - down experiments using as a bait the biotinylated peptides mimicking the N - terminal cytosolic tail of glycoenzymes performed from the whole HeLa cells lysates ( top blot ) or purified His - tagged GOLPH3 protein ( bottom blot ) . The N - terminal cytosolic tail sequence of each glycoenzyme is indicated on the top of the blot ( LxxR motif in red , biotin at the C - terminus ) . Immunoblotting shows affinity - captured GOLPH3 ( endogenous and recombinant ) . B . Control or GOLPH3 - KD HeLa cells expressing luminal SI - GFP or the chimeras of sucrose - isomaltase containing the N - terminal cytosolic tails of LCS , Gb3S , GM3S and GD3S were fixed and labelled for surface localized GFP ( red ) . Scale bar , 20 μm . C . Pull - down experiments of biotinylated LCS WT and LCS mutant cytosolic tails from HeLa cells lysates ( top blot ) or purified His - tagged GOLPH3 protein ( bottom blot ) . The N - terminal cytosolic tail of LCS WT and LCS mutants ( mut . 1 , 2 and 3 ) is shown on the top of the blot ( mutations are in blue , LxxR motif in red , biotin is at the C - terminus ) . D . HeLa cells expressing luminal SI - GFP chimeras of sucrose - isomaltase ( SI - GFP , green ) containing the WT and mutant N - terminal cytosolic tails of LCS , were fixed and labelled for surface localized GFP ( red ) . Cells expressing LCS - SI - RxR and LCS - SI - LxxR chimeras shown PM staining . Scale bar , 20 μm , ( left ) . PM ( surface ) fluorescence intensity ( in Table S10 are listed the software used in this study ) of each chimera is normalised on the LCS - SI - GFP surface intensity ( data are means ± SEM derived from two biological replicates ; n is indicated in the graph , * * * p < 0 . 001 [ Student ' s t - test ] ) ( right ) . E . Quantitative flow cytometry - based analysis of the experiment reported in D ; n is indicated in the graph ( left ) . Percentage of cells with plasma membrane staining ( surface ) is reported for each chimera ( right ) . F . GOLPH3 - KD or OE HeLa cells were processed for ShTxB ( upper panels ) or ChTxB ( lower panels ) staining followed by flow cytometry analysis . Flow cytometry distributions and the relative scatter plots reporting the percentage of positive cells are shown for each condition .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 1A. Pull-down experiments using as a bait the biotinylated peptides mimicking the N-terminal cytosolic tail of glycoenzymes performed from the whole HeLa cells lysates (top blot) or purified His-tagged GOLPH3 protein (bottom blot). The N-terminal cytosolic tail sequence of each glycoenzyme is indicated on the top of the blot (LxxR motif in red, biotin at the C-terminus). Immunoblotting shows affinity-captured GOLPH3 (endogenous and recombinant).B. Control or GOLPH3-KD HeLa cells expressing luminal SI-GFP or the chimeras of sucrose-isomaltase containing the N-terminal cytosolic tails of LCS, Gb3S, GM3S and GD3S were fixed and labelled for surface localized GFP (red). Scale bar, 20 μm.C. Pull-down experiments of biotinylated LCS WT and LCS mutant cytosolic tails from HeLa cells lysates (top blot) or purified His-tagged GOLPH3 protein (bottom blot). The N-terminal cytosolic tail of LCS WT and LCS mutants (mut. 1, 2 and 3) is shown on the top of the blot (mutations are in blue, LxxR motif in red, biotin is at the C-terminus).D. HeLa cells expressing luminal SI-GFP chimeras of sucrose-isomaltase (SI-GFP, green) containing the WT and mutant N-terminal cytosolic tails of LCS, were fixed and labelled for surface localized GFP (red). Cells expressing LCS-SI-RxR and LCS-SI-LxxR chimeras shown PM staining. Scale bar, 20 μm, (left). PM (surface) fluorescence intensity (in Table S10 are listed the software used in this study) of each chimera is normalised on the LCS-SI-GFP surface intensity (data are means ± SEM derived from two biological replicates; n is indicated in the graph, ***p <0.001 [Student's t-test]) (right).E. Quantitative flow cytometry-based analysis of the experiment reported in D; n is indicated in the graph (left). Percentage of cells with plasma membrane staining (surface) is reported for each chimera (right).F. GOLPH3-KD or OE HeLa cells were processed for ShTxB (upper panels) or ChTxB (lower panels) staining followed by flow cytometry analysis. Flow cytometry distributions and the relative scatter plots reporting the percentage of positive cells are shown for each condition.


Figure 2A . HeLa cells mock - treated or GOLPH3 - KD were transiently transfected with LCS _ GFP _ RUSH ( for simplicity , referred in the text as LCS - GR ) and subjected to the synchronisation protocol described in Materials and Methods . Cells were then subjected to IF at each indicated time point : LCS - GR ( green ) , cis Golgi marker GM130 ( red ) , and TGN marker TGN46 ( blue ) . Representative individual Golgi mini - stacks are shown . Scale bar , 1 µm . LCS - GR intra - Golgi trafficking in mock or GOLPH3 - KD conditions it has been quantified ( right graph ) by computational coalescence of line scans , and was performed by normalized line scan analysis ( normalisation of the distances was performed considering the GM130 peak as 0 , and the TGN46 peak as 1 ) . The n indicated in the graph refers to the number of Golgi stacks analysed in each condition . B . Mock and GOLPH3 - KD HeLa cells co - expressing LCS - GR and VSV - G were subjected to the co - synchronisation protocol described in Materials and Methods . Cells were then subjected to IF : LCS - GR or VSV - G ( green ) , GM130 ( blue ) , and TGN46 ( red ) . Representative individual Golgi mini - stacks are shown . Scale bar , 1 µm . C . Quantification of the experiment in ( B ) was performed by normalized line scan analysis ( normalisation of the distances was performed considering the GM130 peak as 0 , and the TGN46 peak as 1 ) ; n indicated in the graph refers to the number of Golgi stacks analysed in each condition , individual data points and means ± SEM are shown , * * * p < 0 . 001 ( Student ' s t test ) ; ns , not significant . D D . HeLa cells were transfected with LCS - GR , subjected to the synchronisation protocol ( see Materials and Methods ) , fixed and processed for cryoimmunolabeling with an anti - GFP antibody ( 15 nm gold particles ) and anti - GM130 antibody ( 10 nm gold particles , black arrowheads ) peri - Golgi vesicles containing LCS - GR are marked by red arrowheads . Scale bar , 150 nm . E , F . Quantification of the distribution of LCS - GR as measured by Frequency Distribution Analysis ( E ) and linear density ( LD , in F ) ( data are means ± SEM , number of Golgi stacks is n = 15 at 0 min , n = 14 at 10 min , n = 17 at 20 min . ( E ) The red arrows indicate the fraction of LCS - GR gold particles ( % ) in vesicles at each indicated time - point . G . HeLa cells expressing LCS - GR mock or GOLPH3 - KD were treated and stained as in D . Black arrowheads indicate GM130 ; peri - Golgi vesicles containing LCS - GR are marked by red arrowheads . LCS - GR in the TGN is marked by blue arrowheads . Scale bar , 150 nm . H . Quantification of LCS - GR distribution ( linear density , LD ) across the stack and in peri - Golgi vesicles ( data are means ± SEM , number of Golgi stacks is n = 17 at 20 min for the CTRL , n = 13 at 20 min for the GOLPH3 KD ; * p < 0 . 05 , * * p < 0 . 01 ; * * * p < 0 . 001 [ Student ' s t - test ] ) .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 2A. HeLa cells mock-treated or GOLPH3-KD were transiently transfected with LCS_GFP_RUSH (for simplicity, referred in the text as LCS-GR) and subjected to the synchronisation protocol described in Materials and Methods. Cells were then subjected to IF at each indicated time point: LCS-GR (green), cis Golgi marker GM130 (red), and TGN marker TGN46 (blue). Representative individual Golgi mini-stacks are shown. Scale bar, 1 µm. LCS-GR intra-Golgi trafficking in mock or GOLPH3-KD conditions it has been quantified (right graph) by computational coalescence of line scans, and was performed by normalized line scan analysis (normalisation of the distances was performed considering the GM130 peak as 0, and the TGN46 peak as 1). The n indicated in the graph refers to the number of Golgi stacks analysed in each condition.B. Mock and GOLPH3-KD HeLa cells co-expressing LCS-GR and VSV-G were subjected to the co-synchronisation protocol described in Materials and Methods. Cells were then subjected to IF: LCS-GR or VSV-G (green), GM130 (blue), and TGN46 (red). Representative individual Golgi mini-stacks are shown. Scale bar, 1 µm. C. Quantification of the experiment in (B) was performed by normalized line scan analysis (normalisation of the distances was performed considering the GM130 peak as 0, and the TGN46 peak as 1); n indicated in the graph refers to the number of Golgi stacks analysed in each condition, individual data points and means ± SEM are shown, ***p < 0.001 (Student's t test); ns, not significant. D D. HeLa cells were transfected with LCS-GR, subjected to the synchronisation protocol (see Materials and Methods), fixed and processed for cryoimmunolabeling with an anti-GFP antibody (15 nm gold particles) and anti-GM130 antibody (10 nm gold particles, black arrowheads) peri-Golgi vesicles containing LCS-GR are marked by red arrowheads. Scale bar, 150 nm.E, F. Quantification of the distribution of LCS-GR as measured by Frequency Distribution Analysis (E) and linear density (LD, in F) (data are means± SEM, number of Golgi stacks is n=15 at 0 min, n=14 at 10 min, n=17 at 20 min. (E) The red arrows indicate the fraction of LCS-GR gold particles (%) in vesicles at each indicated time-point.G. HeLa cells expressing LCS-GR mock or GOLPH3-KD were treated and stained as in D. Black arrowheads indicate GM130; peri-Golgi vesicles containing LCS-GR are marked by red arrowheads. LCS-GR in the TGN is marked by blue arrowheads. Scale bar, 150 nm.H. Quantification of LCS-GR distribution (linear density, LD) across the stack and in peri-Golgi vesicles (data are means ± SEM, number of Golgi stacks is n=17 at 20 min for the CTRL, n=13 at 20 min for the GOLPH3 KD; *p <0.05, **p < 0.01; ***p <0.001 [Student's t-test]).


Figure 3A . PHFs GOLPH3 OE ( left ) , or KD ( right ) , were fixed and processed for IF : Endogenous LCS ( green ) , GOLPH3 ( red ) , TGN46 or DAPI ( blue ) . Dashed lines indicate cells overexpressing or interfered for GOLPH3 . Scale bar , 20 µm . Quantification of the amount of endogenous LCS ( right graph ) at the Golgi ; data are means ± SEM derived from two biological replicates , n = 42 in CTRL , n = 49 in GOLPH3 KD and n = 27 in GOLPH3 OE ; * * * p < 0 . 001 [ Student ' s t - test ] ) . B . GOLPH3 - OE HeLa cells were fixed and processed for IF : Endogenous LCS ( green ) , GOLPH3 ( red ) , and TGN46 ( blue ) . Dashed lines indicate cells overexpressing GOLPH3 . Scale bar , 20 µm . Quantification of the amount of endogenous LCS ( right graph ) at the Golgi ( data are means ± SEM derived from two biological replicates , n = 21 in CTRL , n = 11 in GOLPH3 OE ; * * * p < 0 . 001 [ Student ' s t - test ] ) . C . GOLPH3 OE or KD HeLa cells expressing LCS - HA were fixed and processed for IF : HA ( green ) . Asterisks indicate GOLPH3 OE cells . Scale bar , 20 µm . D . Assessment of LCS - GR synchronized transport in GOLPH3 OE or KD HeLa cells . Micrographs show cells at the indicated trafficking time points . Scale bar , 20 µm . E . Representative WB of cells treated as in ( D ) ( n = 3 ) . F . Plots of the densitometric quantification of the amount of LCS - GR ( Golgi mature band ) over time , starting from 2 hr chase ( data are means of at least 3 experiments ± SD ) . G . GOLPH3 OE or KD HeLa cells expressing LCS - HA were treated with bafilomycin A1 ( BafA1 , 10 nM ) for 16 hr , fixed , and processed for IF labelling : LCS ( green ) , LAMP1 ( red ) , and DAPI ( blue ) . Arrowheads indicate LCS and LAMP1 co - localisation . Scale bar , 20 µm . H . WB of cells treated as in ( G ) . Densitometric quantification of the blot is plotted in the top panel ( data are means of at least 3 experiments ± SD ) . I . GOLPH3 OE or KD HeLa cells expressing the indicated HA - tagged glycoenzymes were fixed and processed for IF . Asterisks indicate GOLPH3 OE cells . Scale bar , 20 µm . J . Number of glycoenzymes - HA positive spots per cell in GOLPH3 - KD cells ( left panel ) and percentage of cells with glycoenzymes - HA in the ER in GOLPH3 - OE cells ( right panel ) are plotted ( n is indicated in the graph ; data are means ± SEM ; * * * p < 0 . 001 [ Student ' s t - test ] ) .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O 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-PANEL_START O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O 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-PANEL_START O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 3A. PHFs GOLPH3 OE (left), or KD (right), were fixed and processed for IF: Endogenous LCS (green), GOLPH3 (red), TGN46 or DAPI (blue). Dashed lines indicate cells overexpressing or interfered for GOLPH3. Scale bar, 20 µm. Quantification of the amount of endogenous LCS (right graph) at the Golgi; data are means ± SEM derived from two biological replicates, n=42 in CTRL, n=49 in GOLPH3 KD and n=27 in GOLPH3 OE; ***p <0.001 [Student's t-test]).B. GOLPH3-OE HeLa cells were fixed and processed for IF: Endogenous LCS (green), GOLPH3 (red), and TGN46 (blue). Dashed lines indicate cells overexpressing GOLPH3. Scale bar, 20 µm. Quantification of the amount of endogenous LCS (right graph) at the Golgi (data are means ± SEM derived from two biological replicates, n=21 in CTRL, n=11 in GOLPH3 OE; ***p <0.001 [Student's t-test]).C. GOLPH3 OE or KD HeLa cells expressing LCS-HA were fixed and processed for IF: HA (green). Asterisks indicate GOLPH3 OE cells. Scale bar, 20 µm.D. Assessment of LCS-GR synchronized transport in GOLPH3 OE or KD HeLa cells. Micrographs show cells at the indicated trafficking time points. Scale bar, 20 µm.E. Representative WB of cells treated as in (D) (n = 3).F. Plots of the densitometric quantification of the amount of LCS-GR (Golgi mature band) over time, starting from 2 hr chase (data are means of at least 3 experiments ± SD).G. GOLPH3 OE or KD HeLa cells expressing LCS-HA were treated with bafilomycin A1 (BafA1, 10 nM) for 16 hr, fixed, and processed for IF labelling: LCS (green), LAMP1 (red), and DAPI (blue). Arrowheads indicate LCS and LAMP1 co-localisation. Scale bar, 20 µm.H. WB of cells treated as in (G). Densitometric quantification of the blot is plotted in the top panel (data are means of at least 3 experiments ± SD).I. GOLPH3 OE or KD HeLa cells expressing the indicated HA-tagged glycoenzymes were fixed and processed for IF. Asterisks indicate GOLPH3 OE cells. Scale bar, 20 µm.J. Number of glycoenzymes-HA positive spots per cell in GOLPH3-KD cells (left panel) and percentage of cells with glycoenzymes-HA in the ER in GOLPH3-OE cells (right panel) are plotted (n is indicated in the graph; data are means ± SEM; ***p <0.001 [Student's t-test]).


Figure 4A . Effects of GOLPH3 KD or OE on glycosphingolipid synthetic flux as assessed by [ 3H ] - sphingosine pulse ( 2 hr ) and chase ( indicated times ) , lipid extraction and HPTLC separation and radioactive counting ( data are means ± SD of at least 3 independent experiments ; * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 [ Student ' s t - test ] ) . B . Effects of GOLPH3 KD or OE on sphingolipid levels as assessed by LC / MS of HeLa cells ( top ) and PHFs ( middle ) . Effects of LCS KD or OE on sphingolipid levels as assessed by LC / MS of HeLa cells ( bottom ) ( data are means ± SD of at least 3 independent experiments ; * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 [ Student ' s t - test ] ) . Targeted lipidomics data from this study are available at Metabolomics Workbench database with the following IDs : ST001673 for HeLa cells , and ST001672 for PHFs . C . Effects of GOLPH3 KD or OE on Gb3 as assessed by MALDI - MS after lipid extraction . Profiles are representative of 3 independent experiments . Untargeted lipidomics data from this study are available at Metabolomics Workbench database with the following ID : ST001674 . D . Histograms reporting the percentage of ShtxB or ChTxB positive cells in each condition as in Fig . 1F ( data are means ± SD of at least 3 independent experiments ; * p < 0 . 05 , * * * p < 0 . 001 [ Student ' s t - test ] ) . E . Effects of GOLPH3 KD or OE on ceramide levels as assessed by LC / MS of HeLa cells ( top ) and PHFs ( middle ) . Effects of LCS KD or OE on ceramide levels as assessed by LC / MS of HeLa cells ( bottom ) . The dotted black box highlights the effects on C16 : 0 Cer ( data are means ± SD of at least 3 independent experiments ; * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 [ Student ' s t - test ] ) . Targeted lipidomics data from this study are available at Metabolomics Workbench database with the following IDs : ST001673 for HeLa cells , and ST001672 for PHFs .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 4A. Effects of GOLPH3 KD or OE on glycosphingolipid synthetic flux as assessed by [3H]-sphingosine pulse (2 hr) and chase (indicated times), lipid extraction and HPTLC separation and radioactive counting (data are means ± SD of at least 3 independent experiments; *p <0.05, **p <0.01, ***p <0.001 [Student's t-test]).B. Effects of GOLPH3 KD or OE on sphingolipid levels as assessed by LC/MS of HeLa cells (top) and PHFs (middle). Effects of LCS KD or OE on sphingolipid levels as assessed by LC/MS of HeLa cells (bottom) (data are means ± SD of at least 3 independent experiments; *p <0.05, **p <0.01, ***p <0.001 [Student's t-test]). Targeted lipidomics data from this study are available at Metabolomics Workbench database with the following IDs: ST001673 for HeLa cells, and ST001672 for PHFs.C. Effects of GOLPH3 KD or OE on Gb3 as assessed by MALDI-MS after lipid extraction. Profiles are representative of 3 independent experiments. Untargeted lipidomics data from this study are available at Metabolomics Workbench database with the following ID: ST001674.D. Histograms reporting the percentage of ShtxB or ChTxB positive cells in each condition as in Fig. 1F (data are means ± SD of at least 3 independent experiments; *p <0.05, ***p <0.001 [Student's t-test]).E. Effects of GOLPH3 KD or OE on ceramide levels as assessed by LC/MS of HeLa cells (top) and PHFs (middle). Effects of LCS KD or OE on ceramide levels as assessed by LC/MS of HeLa cells (bottom). The dotted black box highlights the effects on C16:0 Cer (data are means ± SD of at least 3 independent experiments; *p <0.05, **p <0.01, ***p <0.001 [Student's t-test]). Targeted lipidomics data from this study are available at Metabolomics Workbench database with the following IDs: ST001673 for HeLa cells, and ST001672 for PHFs.


Figure 5A . Cell lysates from HeLa cells KD or OE for either GOLPH3 or LCS were processed for SDS - PAGE and Western blotting with antibodies aimed at evaluating the activation status of the mTOR pathway . Data are representative of at least 3 independent experiments . B . Clonogenic assay of HeLa cells in adherence . 250 HeLa cells KD for either GOLPH3 or LCS were allowed to form colonies for 10 days . Afterward , colonies were fixed and stained as detailed in Materials and Methods . The graph on right reports the number of colonies per well in each indicated condition ( data are means ± SEM of 3 independent experiments ; * p < 0 . 05 , * * p < 0 . 01 [ Student ' s t - test ] ) . C . Soft agar colony formation assay of HeLa cells transfected with different concentration of either GOLPH3 ( left upper graph ) or LCS ( left bottom graph ) encoding plasmids . Soft agar colony formation assay of HeLa cells KD for either GOLPH3 or LCS ( right upper graph ) . Soft agar colony formation assay of HeLa cells KD for LCS and GOLPH3 OE alone or in combination with LCS KD ( right bottom graph ) . Colonies were stained and quantified as described in Materials and Methods ; ( individual data points are shown in the graphs ; data are means ± SD of at least 3 independent experiments ; * p < 0 . 05 , * * * p < 0 . 001 , n . s . = not significant [ Student ' s t - test ] ) . D . Protein lysates from parental ( left ) , GCS - KO ( middle ) , and LCS - KO ( right ) HeLa cells , CTRL or GOLPH3 - OE were processed for SDS - PAGE and WB with the indicated antibodies . Data are representative of at least 3 independent experiments . E . Soft agar colony formation assay of parental and LCS - KO HeLa cells expressing LCS - HA or GOLPH3 . Colony number was quantified ; ( individual data points are shown in the graphs ; data are means ± SD of at least 3 independent experiments ; * * * p < 0 . 001 [ Student ' s t - test ] ) . F . Quantification of soft agar colony formation assay of LNCaP and DU145 prostatic cancer cell lines treated with increasing concentrations of siRNA targeting GOLPH3 or LCS ( data are means ± SEM of at least 3 independent experiments ; * * * p < 0 . 001 [ Student ' s t - test ] ) .

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Figure 5A. Cell lysates from HeLa cells KD or OE for either GOLPH3 or LCS were processed for SDS-PAGE and Western blotting with antibodies aimed at evaluating the activation status of the mTOR pathway. Data are representative of at least 3 independent experiments.B. Clonogenic assay of HeLa cells in adherence. 250 HeLa cells KD for either GOLPH3 or LCS were allowed to form colonies for 10 days. Afterward, colonies were fixed and stained as detailed in Materials and Methods. The graph on right reports the number of colonies per well in each indicated condition (data are means ± SEM of 3 independent experiments; *p <0.05, **p <0.01 [Student's t-test]).C. Soft agar colony formation assay of HeLa cells transfected with different concentration of either GOLPH3 (left upper graph) or LCS (left bottom graph) encoding plasmids. Soft agar colony formation assay of HeLa cells KD for either GOLPH3 or LCS (right upper graph). Soft agar colony formation assay of HeLa cells KD for LCS and GOLPH3 OE alone or in combination with LCS KD (right bottom graph). Colonies were stained and quantified as described in Materials and Methods; (individual data points are shown in the graphs; data are means ± SD of at least 3 independent experiments; *p <0.05, ***p <0.001, n.s. = not significant [Student's t-test]).D. Protein lysates from parental (left), GCS-KO (middle), and LCS-KO (right) HeLa cells, CTRL or GOLPH3-OE were processed for SDS-PAGE and WB with the indicated antibodies. Data are representative of at least 3 independent experiments.E. Soft agar colony formation assay of parental and LCS-KO HeLa cells expressing LCS-HA or GOLPH3. Colony number was quantified; (individual data points are shown in the graphs; data are means ± SD of at least 3 independent experiments; ***p <0.001 [Student's t-test]).F. Quantification of soft agar colony formation assay of LNCaP and DU145 prostatic cancer cell lines treated with increasing concentrations of siRNA targeting GOLPH3 or LCS (data are means ± SEM of at least 3 independent experiments; ***p <0.001 [Student's t-test]).


Figure 6D . Representative images of NSCLC sections stained for GOLPH3 and LCS . Scale bar , 100 µm .

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Figure 6D. Representative images of NSCLC sections stained for GOLPH3 and LCS. Scale bar, 100 µm.


Figure 1 ( A ) Expression of all rate - limiting glycolytic isoenzymes in R1 cells after knockdown of Stat3 using three independent shRNAs compared to an empty vector control shRNA ( sh - ctrl ) , as measured by Western blot . β - actin was employed as a loading control throughout . ( B - I ) Expression of the pluripotency genes c - Myc , Oct - 4 , Nanog , Klf4 and Sox - 2 in R1 cells after knockdown of Hk2 ( B ) , Pfkl ( C ) , Hk1 ( D ) , Hk3 ( E ) , Pfkm ( F ) , Pkm2 ( G ) , Pklr ( H ) and Pfkp ( I ) glycolytic enzymes using two or three independent shRNAs , as measured by Western blot .

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Figure 1(A) Expression of all rate-limiting glycolytic isoenzymes in R1 cells after knockdown of Stat3 using three independent shRNAs compared to an empty vector control shRNA (sh-ctrl), as measured by Western blot. β-actin was employed as a loading control throughout. (B-I) Expression of the pluripotency genes c-Myc, Oct-4, Nanog, Klf4 and Sox-2 in R1 cells after knockdown of Hk2 (B), Pfkl (C), Hk1 (D), Hk3 (E), Pfkm (F), Pkm2 (G), Pklr (H) and Pfkp (I) glycolytic enzymes using two or three independent shRNAs, as measured by Western blot. D


Figure 2 ( A - C ) Expression of Pfkp mRNA levels ( top panels ) and Stat3 and Pfkp protein expression ( bottom panels ) after knockdown of Stat3 using three independent shRNAs in either R1 cells ( A ) or E14 cells ( B ) or after overexpression of Stat3 in R1 cells ( C ) , as measured by qPCR and Western blot , respectively . β - actin was employed as a loading control throughout . ChIP assays against Stat3 p - Stat3 ( Y705 ) or isotype - matched immunoglobulin G ( IgG ) ( F ) , as measured by PCR , and images were representative of three independent experiments . Nanog promoter is shown as a positive control . ( G ) Transcriptional activity of the indicated pGL3 - based Pfkp promoter or mutant constructs expressed in HEK293T cells with or without overexpression of Stat3 , as measured by luciferase reporter assay ( top panel ) . Western blotting was used to verify Stat3 expression ( bottom panel ) .

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Figure 2(A-C) Expression of Pfkp mRNA levels (top panels) and Stat3 and Pfkp protein expression (bottom panels) after knockdown of Stat3 using three independent shRNAs in either R1 cells (A) or E14 cells (B) or after overexpression of Stat3 in R1 cells (C), as measured by qPCR and Western blot, respectively. β-actin was employed as a loading control throughout.ChIP assays against Stat3 p-Stat3 (Y705) or isotype-matched immunoglobulin G (IgG) (F), as measured by PCR, and images were representative of three independent experiments. Nanog promoter is shown as a positive control.(G) Transcriptional activity of the indicated pGL3-based Pfkp promoter or mutant constructs expressed in HEK293T cells with or without overexpression of Stat3, as measured by luciferase reporter assay (top panel). Western blotting was used to verify Stat3 expression (bottom panel).


Figure 3 ( B ) Representative images of alkaline phosphatase - stained colonies formed by R1 cells after overexpression of Pfkp . Images were representative of three independent experiments . ( K , L ) Representative images of immunofluorescence staining in control and Pfkp knockdown R1 cells for Sox17 following definitive endoderm differentiation ( K ) or Foxa2 following definitive endoderm differentiation ( L ) . Green staining indicates positive cells with nuclei counterstained with DAPI . Images were representative of three independent experiments . ( M , N ) Quantitative analysis of Sox17 ( M ) and Foxa2 ( N ) in R1 cells from ( K ) and ( L ) , respectively as determined by flow cytometry . Representative profiles ( left panels ) and quantitation ( right panels ) as a percentage of total cells .

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Figure 3(B) Representative images of alkaline phosphatase-stained colonies formed by R1 cells after overexpression of Pfkp. Images were representative of three independent experiments.(K, L) Representative images of immunofluorescence staining in control and Pfkp knockdown R1 cells for Sox17 following definitive endoderm differentiation (K) or Foxa2 following definitive endoderm differentiation (L). Green staining indicates positive cells with nuclei counterstained with DAPI. Images were representative of three independent experiments. (M, N) Quantitative analysis of Sox17 (M) and Foxa2 (N) in R1 cells from (K) and (L), respectively as determined by flow cytometry. Representative profiles (left panels) and quantitation (right panels) as a percentage of total cells.


Figure 4 ( A , B ) Protein interactome of Pfkp in R1 cells . SDS - PAGE gel comparing Pfkp and control ( IgG ) immunoprecipitates ( left ) with candidate Pfkp - interacting proteins identified by mass spectrometry ( right ) ( A ) . Validation of candidate Pfkp - interacting proteins using Western blotting ( B ) . ( E - F ) Co - IP assays mapping interactions between Pfkp and fragments of Lin41 ( E ) , interactions between Lin41 and fragments of Pfkp ( F ) in HEK293T cells . ( J , K ) Expression of Lin41 in control versus Pfkp knockdown R1 cells without or with MG132 treatment ( J ) or with Lactacyctin treatment ( K ) , as determined by Western blot .

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Figure 4(A, B) Protein interactome of Pfkp in R1 cells. SDS-PAGE gel comparing Pfkp and control (IgG) immunoprecipitates (left) with candidate Pfkp-interacting proteins identified by mass spectrometry (right) (A). Validation of candidate Pfkp-interacting proteins using Western blotting (B).(E-F) Co-IP assays mapping interactions between Pfkp and fragments of Lin41 (E), interactions between Lin41 and fragments of Pfkp (F) in HEK293T cells.(J, K) Expression of Lin41 in control versus Pfkp knockdown R1 cells without or with MG132 treatment (J) or with Lactacyctin treatment (K), as determined by Western blot.


Figure 5 ( F ) HEK293T cells were co - transfected with the indicated vectors to overexpress Pfkp in combination with HA - ubiquitin ( Ub ) and the individual wildtype Flag - Lin41 or serine substitution mutants . Lin41 ubiquitination levels were determined using Western blotting against HA ( top panels ) with input samples analyzed in parallel to verify transfection efficiency ( bottom panels ) . ( G ) Control or Pfkp knockdown R1 cells were co - transfected with the individual wildtype Flag - Lin41 or serine substitution mutants and Lin41 ubiquitination levels determined as per ( F ) using Western blotting against Flag . ( H ) In vitro ubiquitination assays employing the products from in vitro kinase assays employing recombinant GST - Lin41 and Flag - Pfkp , with Lin41 ubiquitination measured by Western blotting against Lin41 . (

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Figure 5(F) HEK293T cells were co-transfected with the indicated vectors to overexpress Pfkp in combination with HA-ubiquitin (Ub) and the individual wildtype Flag-Lin41 or serine substitution mutants. Lin41 ubiquitination levels were determined using Western blotting against HA (top panels) with input samples analyzed in parallel to verify transfection efficiency (bottom panels).(G) Control or Pfkp knockdown R1 cells were co-transfected with the individual wildtype Flag-Lin41 or serine substitution mutants and Lin41 ubiquitination levels determined as per (F) using Western blotting against Flag. (H) In vitro ubiquitination assays employing the products from in vitro kinase assays employing recombinant GST-Lin41 and Flag-Pfkp, with Lin41 ubiquitination measured by Western blotting against Lin41. (


Figure 6 ( C - E ) Expression of selected expression of pluripotency and lineage specification markers in the cells from ( B ) determined by qPCR ( C ) with parallel Western blotting analysis of a panel of ectodermal ( D ) and endodermal ( E ) markers . ( G - J ) RNA pull - down assays measuring interactions between Fgf5 ( G ) , Otx2 ( H ) , Sox1 ( I ) , and Pax2 ( J ) mRNAs and endogenous Lin41 in R1 cells . Recovery of each mRNA was compared in sense and anti - sense probes using qPCR ( top ) with Western blotting used to detect Lin41 ( bottom ) . ( K ) Relative stability of mRNAs for the ectodermal genes Fgf5 , Otx2 , Sox1 , and Pax2 R1 cells in control and Pfkp knockdown EBs at Day 6 , as determined in actinomycin D chase assays . ( L ) Relative stability of ectodermal gene mRNAs measured as per ( K ) in Day 6 Pfkp knockdown EBs transfected with or without Lin41 .

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Figure 6(C-E) Expression of selected expression of pluripotency and lineage specification markers in the cells from (B) determined by qPCR (C) with parallel Western blotting analysis of a panel of ectodermal (D) and endodermal (E) markers.(G-J) RNA pull-down assays measuring interactions between Fgf5 (G), Otx2 (H), Sox1 (I), and Pax2 (J) mRNAs and endogenous Lin41 in R1 cells. Recovery of each mRNA was compared in sense and anti-sense probes using qPCR (top) with Western blotting used to detect Lin41(bottom).(K) Relative stability of mRNAs for the ectodermal genes Fgf5, Otx2, Sox1, and Pax2 R1 cells in control and Pfkp knockdown EBs at Day 6, as determined in actinomycin D chase assays. (L) Relative stability of ectodermal gene mRNAs measured as per (K) in Day 6 Pfkp knockdown EBs transfected with or without Lin41.


Figure 1 Decreasing ∆ ψm from ∆ III to ρ0 cells . WT and mutant yeast cells were cultured to log phase and stained with TMRM for FACS analysis . Decreasing proliferation rates from ∆ III to ρ0 cells . Cells were cultured from 0 . 05 OD600 in SCD . OD600 was measured every 2 hours . Transcriptome analysis of OXPHOS mutants . WT and mutant yeast cells were cultured to log phase . RNAseq was performed with three biological replicates per genotype ( Dataset EV1 ) . All the transcripts and four categories of transcripts ( gene lists in Table EV1B ) are shown . Significantly changed genes ( P value < 0 . 05 ; fold change > 1 . 5 or < 0 . 67 ) are highlighted with dashed lines ; numbers of significantly changed genes are indicated . qRT - PCR analysis of selected transcripts in ρ0 cells .

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Figure 1 Decreasing ∆ψm from ∆III to ρ0 cells. WT and mutant yeast cells were cultured to log phase and stained with TMRM for FACS analysis.  Decreasing proliferation rates from ∆III to ρ0 cells. Cells were cultured from 0.05 OD600 in SCD. OD600 was measured every 2 hours.  Transcriptome analysis of OXPHOS mutants. WT and mutant yeast cells were cultured to log phase. RNAseq was performed with three biological replicates per genotype (Dataset EV1). All the transcripts and four categories of transcripts (gene lists in Table EV1B) are shown. Significantly changed genes (P value < 0.05; fold change > 1.5 or < 0.67) are highlighted with dashed lines; numbers of significantly changed genes are indicated. qRT-PCR analysis of selected transcripts in ρ0 cells.


Figure 2 SILAC analysis of mitochondrial proteome of ∆ IV and ρ0 cells . Equal amount of WT cells labeled with heavy amino acids and unlabeled mutant cells were mixed to extract mitochondrial fraction for mass - spectrometry analysis . Three biological replicates were prepared for each pair of strains ( Dataset EV2 ) . 568 high fidelity mitochondrial proteins ( gene / protein list in Table EV1A ) in ∆ IV cells and 572 such proteins in ρ0 cells were quantitatively measured . Significantly changed proteins ( P value < 0 . 05 ; fold change > 1 . 5 or < 0 . 67 ) are highlighted with dashed lines ; numbers of significantly changed proteins are indicated . Western blot analysis of representative mitochondrial proteins endogenously tagged with HA . The selection markers were removed by the Cre - loxP system . Whole cell lysates were extracted and equal amount of proteins was loaded for western blot . Polysome fractionation analysis of OXPHOS mutants . Polysomes were fractionated by sucrose gradients ( 10 % - 50 % ) , and the gradients were separated and measured at A254 .

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Figure 2 SILAC analysis of mitochondrial proteome of ∆IV and ρ0 cells. Equal amount of WT cells labeled with heavy amino acids and unlabeled mutant cells were mixed to extract mitochondrial fraction for mass-spectrometry analysis. Three biological replicates were prepared for each pair of strains (Dataset EV2). 568 high fidelity mitochondrial proteins (gene/protein list in Table EV1A) in ∆IV cells and 572 such proteins in ρ0 cells were quantitatively measured. Significantly changed proteins (P value < 0.05; fold change > 1.5 or < 0.67) are highlighted with dashed lines; numbers of significantly changed proteins are indicated.  Western blot analysis of representative mitochondrial proteins endogenously tagged with HA. The selection markers were removed by the Cre-loxP system. Whole cell lysates were extracted and equal amount of proteins was loaded for western blot. Polysome fractionation analysis of OXPHOS mutants. Polysomes were fractionated by sucrose gradients (10%-50%), and the gradients were separated and measured at A254.


Figure 3 qRT - PCR analysis of INH1 mRNA levels . Data are mean ± SD from three biological replicates . Western blot analysis of Inh1 protein levels . Whole cell lysates were extracted and equal amount of proteins was loaded for western blot . Deletion of INH1 benefits the proliferation of ρ0 cells at 37 ℃ . Serial dilutions ( tenfold dilution ) of the indicated strains were analyzed on SCD plates at 30 ℃ and 37 ℃ for 2 days . Western blot analysis of copper - inducible overexpression of Inh1 . A cassette expressing Inh1 under the control of CUP1 promoter was inserted into the HO locus . Inh1 overexpression was induced by 100 μM CuSO4 for 12 hours . Overexpression of Inh1 decreases ∆ ψm in ρ0 cells . Cells were cultured in SCD or SCD plus 100 μM CuSO4 for 12 hours to mid - log phase . Cells were then stained with 125 nM TMRM for FACS analysis . Overexpression of Inh1 represses the proliferation of ρ0 cells . Serial dilutions ( tenfold dilution ) of the indicated strains were analyzed on SCD or SCD plus 100 μM CuSO4 plates at 30 ℃ for 2 days . Overexpression of Inh1 reduces the protein levels of mitochondrial ISC biosynthesis proteins ( Nfs1 and Yah1 ) and nuclear ISC - containing protein Pol3 . Cells were cultured in SCD or SCD plus 100 μM CuSO4 for 12 hours to mid - log phase . Whole cell lysates were extracted and equal amount of proteins was loaded for western blot . Nfs1 , Yah1 were endogenously tagged with FLAG , and Pol3 was endogenously tagged with HA .

O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 3 qRT-PCR analysis of INH1 mRNA levels. Data are mean ± SD from three biological replicates. Western blot analysis of Inh1 protein levels. Whole cell lysates were extracted and equal amount of proteins was loaded for western blot.Deletion of INH1 benefits the proliferation of ρ0 cells at 37 ℃. Serial dilutions (tenfold dilution) of the indicated strains were analyzed on SCD plates at 30℃ and 37 ℃ for 2 days. Western blot analysis of copper-inducible overexpression of Inh1. A cassette expressing Inh1 under the control of CUP1 promoter was inserted into the HO locus. Inh1 overexpression was induced by 100 μM CuSO4 for 12 hours.  Overexpression of Inh1 decreases ∆ψm in ρ0 cells. Cells were cultured in SCD or SCD plus 100 μM CuSO4 for 12 hours to mid-log phase. Cells were then stained with 125 nM TMRM for FACS analysis.  Overexpression of Inh1 represses the proliferation of ρ0 cells. Serial dilutions (tenfold dilution) of the indicated strains were analyzed on SCD or SCD plus 100 μM CuSO4 plates at 30℃ for 2 days.  Overexpression of Inh1 reduces the protein levels of mitochondrial ISC biosynthesis proteins (Nfs1 and Yah1) and nuclear ISC-containing protein Pol3. Cells were cultured in SCD or SCD plus 100 μM CuSO4 for 12 hours to mid-log phase. Whole cell lysates were extracted and equal amount of proteins was loaded for western blot. Nfs1, Yah1 were endogenously tagged with FLAG, and Pol3 was endogenously tagged with HA.


Figure 4 The changes of Puf3 - associated transcripts encoding mitochondrial proteins in OXPHOS mutants . The RNAseq results in Figure 1C ( Dataset EV1 ) are re - analyzed . Transcripts encoding high fidelity mitochondrial proteins ( gene list in Table EV1A ) and the Puf3 - associated transcripts encoding mitochondrial proteins ( gene list in Table EV1D ) are shown . The significantly changed transcripts ( P value < 0 . 05 ; fold change > 1 . 5 or < 0 . 67 ) are highlighted with dashed lines ; numbers of significantly changed transcripts are indicated . B and C , Deletion of PUF3 upregulates mitochondrial transcripts in WT cells ( B ) , whereas downregulates mitochondrial transcripts in ρ0 cells ( C ) . WT and mutant yeast cells were cultured to log phase . RNAseq was performed with three biological replicates per genotype ( Dataset EV4 ) . All the transcripts and the Puf3 - associated transcripts encoding mitochondrial proteins are shown . The significantly changed transcripts ( P value < 0 . 05 ; fold change > 1 . 5 or < 0 . 67 ) are highlighted with dashed lines ; numbers of significantly changed transcripts are indicated . D . Deletion of PUF3 in ρ0 cells downregulates mitochondrial proteins . SILAC analysis of mitochondrial fractions from ρ0 and ρ0 puf3 ∆ cells are shown . Equal amount of ρ0 cells labeled with heavy amino acids and unlabeled ρ0 puf3 ∆ cells were mixed to extract mitochondrial fraction for mass - spectrometry analysis . Three biological replicates were prepared for each pair of strains ( Dataset EV5 ) . Mitochondrial proteins encoded by Puf3 - associated mRNAs are highlighted in red . Mia40 substrates are highlighted in green ( gene list in Table EV1E ) . Mia40 substrates encoded by Puf3 associated transcripts are highlighted in green with red circle . The significantly changed proteins ( P value < 0 . 05 ; fold change > 1 . 5 or < 0 . 67 ) are highlighted with dashed lines ; numbers of significantly changed proteins are indicated . E . Correlation plots of fold changes ( ρ0 puf3 ∆ versus ρ0 ) between mitochondrial proteins and their transcripts . Puf3 - associated transcripts encoding mitochondrial proteins are highlighted in red . Mia40 substrates are highlighted in green . Mia40 substrates encoded by Puf3 associated transcripts are highlighted in green with red circle . F . Deletion of PUF3 decreases ∆ ψm in ρ0 cells . Cells were cultured to mid - log phase , and stained with 125 nM TMRM for FACS analysis G . Deletion of PUF3 decreases the proliferation of ρ0 cells . Serial dilutions ( tenfold dilution ) of the indicated strains were analyzed on SCD plates at 30 ℃ for 2 days . H . Schematic illustration of the consensus Puf3 - binding motif Lapointe et al . , 2015 ( ) and the motif at Mia40 3 ' - UTR . Mutating the Puf3 - binding motif of MIA40 mRNA abolishes Mia40 upregulation in ρ0 cells . Mia40 was endogenously tagged with FLAG and its 3 ' - UTR was edited as indicated . The selection markers were removed by the Cre - loxP system . Whole cell lysates were extracted and equal amount of proteins was loaded for western blot . RC : reverse complement . DE : deletion . I . Mutating the Puf3 - binding motif of MIA40 mRNA decreases ∆ ψm of ρ0 cells . J . Mutating the Puf3 - binding motif of MIA40 mRNA impairs the proliferation of ρ0 cells . Cells were cultured from 0 . 05 OD in SCD , and OD600 was measured every 2 hours . Cells were cultured to mid - log phase , and stained with 125 nM TMRM for FACS analysis K . Overexpression of MIA40 rescues ∆ ψm of ρ0 puf3 ∆ cells . Single copy plasmid expressing Mia40 driven by the MIA40 promoter was transformed into the indicated strains . L . Overexpression of MIA40 rescues the proliferation of ρ0 puf3 ∆ cells . Strains were prepared as in ( K ) . Cells were cultured from 0 . 05 OD in SCD , and OD600 was measured every 2 hours . Cells were cultured to mid - log phase , and stained with 125 nM TMRM for FACS

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

Figure 4 The changes of Puf3-associated transcripts encoding mitochondrial proteins in OXPHOS mutants. The RNAseq results in Figure 1C (Dataset EV1) are re-analyzed. Transcripts encoding high fidelity mitochondrial proteins (gene list in Table EV1A) and the Puf3-associated transcripts encoding mitochondrial proteins (gene list in Table EV1D) are shown. The significantly changed transcripts (P value < 0.05; fold change > 1.5 or < 0.67) are highlighted with dashed lines; numbers of significantly changed transcripts are indicated.  B and C, Deletion of PUF3 upregulates mitochondrial transcripts in WT cells (B), whereas downregulates mitochondrial transcripts in ρ0 cells (C). WT and mutant yeast cells were cultured to log phase. RNAseq was performed with three biological replicates per genotype (Dataset EV4). All the transcripts and the Puf3-associated transcripts encoding mitochondrial proteins are shown. The significantly changed transcripts (P value < 0.05; fold change > 1.5 or < 0.67) are highlighted with dashed lines; numbers of significantly changed transcripts are indicated.  D. Deletion of PUF3 in ρ0 cells downregulates mitochondrial proteins. SILAC analysis of mitochondrial fractions from ρ0 and ρ0 puf3∆ cells are shown. Equal amount of ρ0 cells labeled with heavy amino acids and unlabeled ρ0 puf3∆ cells were mixed to extract mitochondrial fraction for mass-spectrometry analysis. Three biological replicates were prepared for each pair of strains (Dataset EV5). Mitochondrial proteins encoded by Puf3-associated mRNAs are highlighted in red. Mia40 substrates are highlighted in green (gene list in Table EV1E). Mia40 substrates encoded by Puf3 associated transcripts are highlighted in green with red circle. The significantly changed proteins (P value < 0.05; fold change > 1.5 or < 0.67) are highlighted with dashed lines; numbers of significantly changed proteins are indicated.  E. Correlation plots of fold changes (ρ0 puf3∆ versus ρ0) between mitochondrial proteins and their transcripts. Puf3-associated transcripts encoding mitochondrial proteins are highlighted in red. Mia40 substrates are highlighted in green. Mia40 substrates encoded by Puf3 associated transcripts are highlighted in green with red circle.  F. Deletion of PUF3 decreases ∆ψm in ρ0 cells. Cells were cultured to mid-log phase, and stained with 125 nM TMRM for FACS analysis  G. Deletion of PUF3 decreases the proliferation of ρ0 cells. Serial dilutions (tenfold dilution) of the indicated strains were analyzed on SCD plates at 30℃ for 2 days.  H. Schematic illustration of the consensus Puf3-binding motif Lapointe et al., 2015() and the motif at Mia40 3'-UTR. Mutating the Puf3-binding motif of MIA40 mRNA abolishes Mia40 upregulation in ρ0 cells. Mia40 was endogenously tagged with FLAG and its 3'-UTR was edited as indicated. The selection markers were removed by the Cre-loxP system. Whole cell lysates were extracted and equal amount of proteins was loaded for western blot. RC: reverse complement. DE: deletion.  I. Mutating the Puf3-binding motif of MIA40 mRNA decreases ∆ψm of ρ0 cells. J. Mutating the Puf3-binding motif of MIA40 mRNA impairs the proliferation of ρ0 cells. Cells were cultured from 0.05 OD in SCD, and OD600 was measured every 2 hours. Cells were cultured to mid-log phase, and stained with 125 nM TMRM for FACS analysis K. Overexpression of MIA40 rescues ∆ψm of ρ0 puf3∆ cells. Single copy plasmid expressing Mia40 driven by the MIA40 promoter was transformed into the indicated strains. L. Overexpression of MIA40 rescues the proliferation of ρ0 puf3∆ cells. Strains were prepared as in (K). Cells were cultured from 0.05 OD in SCD, and OD600 was measured every 2 hours. Cells were cultured to mid-log phase, and stained with 125 nM TMRM for FACS analysis


Figure 5 Puf3 is phosphorylated in WT cells and is hyper - phosphorylated in ρ0 cells . Puf3 - FLAG was immunoprecipitated from the indicated strains and treated with / without λ - phosphatase , and analyzed by regular and Phos - tag SDS - PAGE . Puf3 phosphorylation in response to OXPHOS inhibitors and in OXPHOS mutants . WT cells were treated with antimycin ( 10 μM ) , KCN ( 10 μM ) or oligomycin ( 10 μM ) for 3 hours , or shifted from SCD to SCEG medium for 3 hours . Whole cell lysates were extracted and equal amount of proteins was loaded for western blot . Treatments and strains with Puf3 hyper - phosphorylation was highlighted in red . Glucose depletion was used as a positive control . Asterisk indicates a non - specific band . Puf3 - 15A mutation inhibits Puf3 hyper - phosphorylation in ρ0 cells . A cassette expressing WT or mutant Puf3 - FLAG ( 11A or 15A ) under the control of PUF3 promoter was inserted into the HO site in puf3 ∆ cells . The 15A mutant that blocks Puf3 hyper - phosphorylation is highlighted in red . Puf3 - FLAG was immunoprecipitated from the indicated strains and analyzed by regular and Phos - tag SDS - PAGE . Puf3 - 15A mutation inhibits Puf3 hyper - phosphorylation upon oligomycin treatment and in ∆ V and ρ0 cells . WT cells were treated with antimycin ( 10 μM ) , KCN ( 10 μM ) or oligomycin ( 10 μM ) for 3 hours , or shifted from SCD to SCEG medium for 3 hours . Whole cell lysates were extracted and equal amount of proteins was loaded for western blot . Asterisk indicates a non - specific band . Puf3 - 15A mutation downregulates Puf3 - associated mitochondrial transcripts in ρ0 cells . ρ0 and ρ0 puf3 - 15A cells were cultured to log phase . RNAseq was performed with three biological replicates per genotype ( Dataset EV7 ) . All the transcripts and the Puf3 - associated transcripts encoding mitochondrial proteins ( gene lists in Table EV1D ) are shown . The significantly changed transcripts ( P value < 0 . 05 ; fold change > 1 . 5 or < 0 . 67 ) are highlighted with dashed lines ; numbers of significantly changed transcripts are indicated . Puf3 - 15A mutation inhibits the upregulation of Mia40 in ∆ V and ρ0 cells . Mia40 was endogenously tagged with FLAG . The selection markers were removed by the Cre - loxP system . Whole cell lysates were extracted and equal amount of proteins was loaded for western blot . Puf3 - 15A mutation decreases ∆ ψm in ρ0 cells . Cells were cultured to mid - log phase and stained with 125 nM TMRM for FACS analysis . The red ] highlight two cell lines for comparison . Puf3 - 15A mutation has no effect on the proliferation of WT cells . Puf3 - 15A mutation inhibits the proliferation of ρ0 cells . Mia40 overexpression rescues the proliferation defect of ρ0 puf3 - 15A cells . Single copy plasmid expressing Mia40 driven by MIA40 promoter was transformed into the indicated strains .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 5 Puf3 is phosphorylated in WT cells and is hyper-phosphorylated in ρ0 cells. Puf3-FLAG was immunoprecipitated from the indicated strains and treated with/without λ-phosphatase, and analyzed by regular and Phos-tag SDS-PAGE.  Puf3 phosphorylation in response to OXPHOS inhibitors and in OXPHOS mutants. WT cells were treated with antimycin (10 μM), KCN (10 μM) or oligomycin (10 μM) for 3 hours, or shifted from SCD to SCEG medium for 3 hours. Whole cell lysates were extracted and equal amount of proteins was loaded for western blot. Treatments and strains with Puf3 hyper-phosphorylation was highlighted in red. Glucose depletion was used as a positive control. Asterisk indicates a non-specific band.  Puf3-15A mutation inhibits Puf3 hyper-phosphorylation in ρ0 cells. A cassette expressing WT or mutant Puf3-FLAG (11A or 15A) under the control of PUF3 promoter was inserted into the HO site in puf3∆ cells. The 15A mutant that blocks Puf3 hyper-phosphorylation is highlighted in red. Puf3-FLAG was immunoprecipitated from the indicated strains and analyzed by regular and Phos-tag SDS-PAGE.  Puf3-15A mutation inhibits Puf3 hyper-phosphorylation upon oligomycin treatment and in ∆V and ρ0 cells. WT cells were treated with antimycin (10 μM), KCN (10 μM) or oligomycin (10 μM) for 3 hours, or shifted from SCD to SCEG medium for 3 hours. Whole cell lysates were extracted and equal amount of proteins was loaded for western blot. Asterisk indicates a non-specific band.  Puf3-15A mutation downregulates Puf3-associated mitochondrial transcripts in ρ0 cells. ρ0 and ρ0 puf3-15A cells were cultured to log phase. RNAseq was performed with three biological replicates per genotype (Dataset EV7). All the transcripts and the Puf3-associated transcripts encoding mitochondrial proteins (gene lists in Table EV1D) are shown. The significantly changed transcripts (P value < 0.05; fold change > 1.5 or < 0.67) are highlighted with dashed lines; numbers of significantly changed transcripts are indicated. Puf3-15A mutation inhibits the upregulation of Mia40 in ∆V and ρ0 cells. Mia40 was endogenously tagged with FLAG. The selection markers were removed by the Cre-loxP system. Whole cell lysates were extracted and equal amount of proteins was loaded for western blot. Puf3-15A mutation decreases ∆ψm in ρ0 cells. Cells were cultured to mid-log phase and stained with 125 nM TMRM for FACS analysis. The red ] highlight two cell lines for comparison.  Puf3-15A mutation has no effect on the proliferation of WT cells.   Puf3-15A mutation inhibits the proliferation of ρ0 cells.   Mia40 overexpression rescues the proliferation defect of ρ0 puf3-15A cells. Single copy plasmid expressing Mia40 driven by MIA40 promoter was transformed into the indicated strains.


Figure 6 Snf1 maintains glycolysis and glucose transporter gene expression in both WT and ρ0 cells , and represses ribosome and Ribi gene expression in ρ0 cells . ρ0 snf1 ∆ cells were prepared by acute EtBr ( 25 µg / ml ) treatment of snf1 ∆ cells in SCD for 2 days . RNAseq was performed with three biological replicates per genotype ( Dataset EV8 ) . All the transcripts and two categories of transcripts ( gene lists in Table EV1B ) are shown . Significantly changed genes ( P value < 0 . 05 ; fold change > 1 . 5 or < 0 . 67 ) are highlighted with dashed lines ; numbers of significantly changed genes are indicated . Snf1 represses ribosome biogenesis in WT and ρ0 cells . Polysomes of the indicated strains were fractionated by sucrose gradients ( 10 % - 50 % ) , and the gradients were separated and measured at A254 . Deletion of SNF1 decreases ∆ ψm in both ∆ V and ρ0 cells . Cells were cultured to mid - log phase and stained with 125 nM TMRM for FACS analysis Deletion of SNF1 decreases the proliferation of both ∆ V and ρ0 cells . Serial dilutions of the indicated strains were analyzed on SCD plates at 30 ℃ for 2 days . Deletion of MIG1 in ρ0 snf1 ∆ cells rescues ∆ ψm . Cells were cultured to mid - log phase and stained with 125 nM TMRM for FACS analysis Deletion of MIG1 in ρ0 snf1 ∆ cells partially rescues cell proliferation . A plasmid overexpressing MIG1 under the control of MIG1 promoter was transformed into the indicated strains . Serial dilutions ( tenfold dilution ) were analyzed on SCD plates at 30 ℃ for 2 days . V : empty vector . Combining MIG1 deletion and NOG2 overexpression in ρ0 snf1 ∆ cells fully rescues cell proliferation . A plasmid overexpressing NOG2 under the control of TEF1 promoter was transformed into the indicated strains . Serial dilutions ( tenfold dilution ) were analyzed on SCD plates at 30 ℃ for 2 days . V : empty vector .

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

Figure 6 Snf1 maintains glycolysis and glucose transporter gene expression in both WT and ρ0 cells, and represses ribosome and Ribi gene expression in ρ0 cells. ρ0 snf1∆ cells were prepared by acute EtBr (25 µg/ml) treatment of snf1∆ cells in SCD for 2 days. RNAseq was performed with three biological replicates per genotype (Dataset EV8). All the transcripts and two categories of transcripts (gene lists in Table EV1B) are shown. Significantly changed genes (P value < 0.05; fold change > 1.5 or < 0.67) are highlighted with dashed lines; numbers of significantly changed genes are indicated.  Snf1 represses ribosome biogenesis in WT and ρ0 cells. Polysomes of the indicated strains were fractionated by sucrose gradients (10%-50%), and the gradients were separated and measured at A254.  Deletion of SNF1 decreases ∆ψm in both ∆V and ρ0 cells. Cells were cultured to mid-log phase and stained with 125 nM TMRM for FACS analysis  Deletion of SNF1 decreases the proliferation of both ∆V and ρ0 cells. Serial dilutions of the indicated strains were analyzed on SCD plates at 30℃ for 2 days.  Deletion of MIG1 in ρ0 snf1∆ cells rescues ∆ψm. Cells were cultured to mid-log phase and stained with 125 nM TMRM for FACS analysis  Deletion of MIG1 in ρ0 snf1∆ cells partially rescues cell proliferation. A plasmid overexpressing MIG1 under the control of MIG1 promoter was transformed into the indicated strains. Serial dilutions (tenfold dilution) were analyzed on SCD plates at 30℃ for 2 days. V: empty vector.  Combining MIG1 deletion and NOG2 overexpression in ρ0 snf1∆ cells fully rescues cell proliferation. A plasmid overexpressing NOG2 under the control of TEF1 promoter was transformed into the indicated strains. Serial dilutions (tenfold dilution) were analyzed on SCD plates at 30℃ for 2 days. V: empty vector.


Figure 7 Heat map plots of the mRNA level changes of chaperones , ubiquitin - proteasome pathway , and autophagy genes ( gene list in Table EV1F ) in the indicated strains ( data from Dataset EV1 and Dataset EV10 ) . Selected genes for growth analysis in B are highlighted in red . Deletion of HSC82 selectively impairs the proliferation of ρ0 puf3 ∆ cells . Cells were cultured from 0 . 05 OD in SCD , and OD600 was measured every 2 hours . Data are mean ± SD from three biological replicates . Deletion of HSC82 selectively decreases ∆ ψm in ρ0 puf3 ∆ cells . Cells were cultured to mid - log phase and stained with 125 nM TMRM for FACS analysis . The red ] highlight two cell lines for comparison .

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

Figure 7 Heat map plots of the mRNA level changes of chaperones, ubiquitin-proteasome pathway, and autophagy genes (gene list in Table EV1F) in the indicated strains (data from Dataset EV1 and Dataset EV10). Selected genes for growth analysis in B are highlighted in red.  Deletion of HSC82 selectively impairs the proliferation of ρ0 puf3∆ cells. Cells were cultured from 0.05 OD in SCD, and OD600 was measured every 2 hours. Data are mean ± SD from three biological replicates.  Deletion of HSC82 selectively decreases ∆ψm in ρ0 puf3∆ cells. Cells were cultured to mid-log phase and stained with 125 nM TMRM for FACS analysis. The red ] highlight two cell lines for comparison.


Figure 1A Quantification and representative immunoblots of lamin B1 in the striatum ( 8w , 12w and 20w ; N = 6 for both genotypes ; 30w ; N = 4 and 5 for WT and R6 / 1 mice , respectively ) , hippocampus ( 8w ; N = 8 and 6 for WT and R6 / 1 mice , respectively ; 12w ; N = 7 and 6 for WT and R6 / 1 mice , respectively ; 20w N = 5 and 6 for WT and R6 / 1 mice , respectively ; 30w ; N = 8 and 5 for WT and R6 / 1 mice , respectively ) and cortex ( 8w ; N = 5 and 4 for WT and R6 / 1 mice , respectively ; 12w and 20w ; N = 6 for both genotypes ; 30w ; N = 6 and 5 for WT and R6 / 1 mice , respectively ) . Data information : Data is expressed as a percentage of controls . Each point corresponds to the value from an individual sample . Bars represent the mean ± S . E . M . * P < 0 . 05 , * * P < 0 . 01 and * * * P < 0 . 001 as compared with WT mice ( two - tailed unpaired Student ' s t test ) . Tubulin was used as loading control . Exact P values are reported in Appendix Table S3 . B Quantification and representative immunoblots of lamin B2 in the striatum ( 8w , 12w and 20w ; N = 6 for both genotypes , 30w ; N = 5 for both genotypes ) , hippocampus ( 8w ; N = 8 and 6 for WT and R6 / 1 mice , respectively ; 12w ; N = 6 and 7 for WT and R6 / 1 mice , respectively ; 20w ; N = 5 for both genotypes ; 30w ; N = 8 and 5 for WT and R6 / 1 mice , respectively ) and cortex ( 8w and 12w ; N = 6 for both genotypes ; 20w ; N = 5 for both genotypes ; 30w ; N = 8 and 5 for WT and R6 / 1 mice , respectively ) . Data information : Data is expressed as a percentage of controls . Each point corresponds to the value from an individual sample . Bars represent the mean ± S . E . M . * P < 0 . 05 , * * P < 0 . 01 and * * * P < 0 . 001 as compared with WT mice ( two - tailed unpaired Student ' s t test ) . Tubulin was used as loading control . Exact P values are reported in Appendix Table S3 .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 1A Quantification and representative immunoblots of lamin B1 in the striatum (8w, 12w and 20w; N = 6 for both genotypes; 30w; N = 4 and 5 for WT and R6/1 mice, respectively), hippocampus (8w; N = 8 and 6 for WT and R6/1 mice, respectively; 12w; N = 7 and 6 for WT and R6/1 mice, respectively; 20w N = 5 and 6 for WT and R6/1 mice, respectively; 30w; N = 8 and 5 for WT and R6/1 mice, respectively) and cortex (8w; N = 5 and 4 for WT and R6/1 mice, respectively; 12w and 20w; N = 6 for both genotypes; 30w; N = 6 and 5 for WT and R6/1 mice, respectively). Data information: Data is expressed as a percentage of controls. Each point corresponds to the value from an individual sample. Bars represent the mean ± S.E.M. *P < 0.05, **P < 0.01 and ***P < 0.001 as compared with WT mice (two-tailed unpaired Student's t test). Tubulin was used as loading control. Exact P values are reported in Appendix Table S3. B Quantification and representative immunoblots of lamin B2 in the striatum (8w, 12w and 20w; N = 6 for both genotypes, 30w; N = 5 for both genotypes), hippocampus (8w; N = 8 and 6 for WT and R6/1 mice, respectively; 12w; N = 6 and 7 for WT and R6/1 mice, respectively; 20w; N = 5 for both genotypes; 30w; N = 8 and 5 for WT and R6/1 mice, respectively) and cortex (8w and 12w; N = 6 for both genotypes; 20w; N = 5 for both genotypes; 30w; N = 8 and 5 for WT and R6/1 mice, respectively). Data information: Data is expressed as a percentage of controls. Each point corresponds to the value from an individual sample. Bars represent the mean ± S.E.M. *P < 0.05, **P < 0.01 and ***P < 0.001 as compared with WT mice (two-tailed unpaired Student's t test). Tubulin was used as loading control. Exact P values are reported in Appendix Table S3.


Figure 2A Mouse brain tissue was processed for immunohistochemistry by combining anti - lamin B1 ( red ) and anti - NeuN ( green ) antibodies . Representative images ( maximal Z - projections ) showing the distribution of lamin B1 in the striatum of 30 - week - old wild - type ( WT ) and R6 / 1 mice . Yellow arrowheads show co - localization between lamin B1 and NeuN , and white arrowheads show NeuN negative lamin B1 positive nuclei . Scale bar 50 μm and 25 μm for low and high magnification , respectively . B Lamin B1 distribution in human putamen was analysed by immunohistochemistry . Antibody against lamin B1 ( red ) was combined with DAPI - Fluoromount G ( blue ) to label nuclei . Representative images show the distribution of lamin B1 in the putamen of non - affected individuals ( CTL ) and HD patients at different stages of the disease ( VS II - IV : Vonsattel grades ) . Yellow and white arrowheads indicate MSNs and glial cells , respectively . Scale bar 50 μm and 20 μm for low and high magnification , respectively . C The distribution of lamin B1 in the putamen of HD patients was analysed by immunohistochemistry . Anti - lamin B1 antibody ( red ) was combined with anti - GFAP antibody ( green ) and nuclei were labelled with DAPI - Fluoromount G ( blue ) . Representative images show the distribution of lamin B1 at Vonsattel grade III . Yellow arrowheads indicate MSNs and white arrowheads indicate GFAP - positive cells . Scale bar 10 μm .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 2A Mouse brain tissue was processed for immunohistochemistry by combining anti-lamin B1 (red) and anti-NeuN (green) antibodies. Representative images (maximal Z-projections) showing the distribution of lamin B1 in the striatum of 30-week-old wild-type (WT) and R6/1 mice. Yellow arrowheads show co-localization between lamin B1 and NeuN, and white arrowheads show NeuN negative lamin B1 positive nuclei. Scale bar 50 μm and 25 μm for low and high magnification, respectively.B Lamin B1 distribution in human putamen was analysed by immunohistochemistry. Antibody against lamin B1 (red) was combined with DAPI-Fluoromount G (blue) to label nuclei. Representative images show the distribution of lamin B1 in the putamen of non-affected individuals (CTL) and HD patients at different stages of the disease (VS II-IV: Vonsattel grades). Yellow and white arrowheads indicate MSNs and glial cells, respectively. Scale bar 50 μm and 20 μm for low and high magnification, respectively. C The distribution of lamin B1 in the putamen of HD patients was analysed by immunohistochemistry. Anti-lamin B1 antibody (red) was combined with anti-GFAP antibody (green) and nuclei were labelled with DAPI-Fluoromount G (blue). Representative images show the distribution of lamin B1 at Vonsattel grade III. Yellow arrowheads indicate MSNs and white arrowheads indicate GFAP-positive cells. Scale bar 10 μm.


Figure 3A Representative images showing the distribution of lamin B1 . On the left , images showing maximal Z - projection . Scale bar 150 μm . Small images correspond to CA1 and DG magnified images showing independent DAPI , GFAP and Lamin B1 channels , and merge , of representative confocal Z - stack images . Scale bar 50 μm . B Left , representative images showing the distribution of lamin B1 in the nuclei of CA - 1 hippocampal neurons from WT and R6 / 1 mice along different Z axis planes ( 1 - 6 ) . On the right , a 3D reconstruction of the Z - stack confocal images generated using ImageJ . Scale bar 20 μm .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 3A Representative images showing the distribution of lamin B1. On the left, images showing maximal Z-projection. Scale bar 150 μm. Small images correspond to CA1 and DG magnified images showing independent DAPI, GFAP and Lamin B1 channels, and merge, of representative confocal Z-stack images. Scale bar 50 μm. B Left, representative images showing the distribution of lamin B1 in the nuclei of CA-1 hippocampal neurons from WT and R6/1 mice along different Z axis planes (1-6). On the right, a 3D reconstruction of the Z-stack confocal images generated using ImageJ. Scale bar 20 μm.


Figure 4A Graphs show the quantification of different parameters in mice striatal MSNs nuclei ( Ctip2 + / NeuN + ) . N = 4 for both genotypes . An average of 5 , 000 nuclei was analysed for each sample . Representative images are shown . Scale bar 7 μm . B Graphs show the quantification of different parameters in mice striatal interneurons ( Ctip2 - / NeuN + ) . N = 4 for both genotypes . An average of 5 , 000 nuclei was analysed for each sample . Representative images are shown . Scale bar 7 μm . C Graphs show the quantification of lamin B1 intensity and circularity in 30 - week - old R6 / 1 mouse striatal neurons nuclei with ( mHtt + ) or without ( mHtt - ) nuclear inclusions . N = 6 . EM48 antibody was used to label mHtt inclusions . Representative images are shown . Scale bar 7 μm . D Graphs showing lamin B1 intensity and circularity in MSNs nuclei from the putamen of HD patients at different stages of the disease ( VS : Vonsattel grade ) and corresponding controls ( CTL : non - affected individuals ) . N = 6 for CTL , N = 3 for VS I - II and N = 4 for VS III - IV . An average of 50 nuclei was examined for each sample . Representative images ( maximal Z - projections ) are shown . Scale bar 7μm . E Graphs show the quantification of different parameters in hippocampal CA1 ( Ctip2 + / Prox1 - ) and DG ( Ctip2 + / Prox1 + ) neurons nuclei . N = 4 for each genotype . Representative images are shown . Scale bar 7 μm .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 4A Graphs show the quantification of different parameters in mice striatal MSNs nuclei (Ctip2+/NeuN+). N = 4 for both genotypes. An average of 5,000 nuclei was analysed for each sample. Representative images are shown. Scale bar 7 μm.B Graphs show the quantification of different parameters in mice striatal interneurons (Ctip2-/NeuN+). N = 4 for both genotypes. An average of 5,000 nuclei was analysed for each sample. Representative images are shown. Scale bar 7 μm.C Graphs show the quantification of lamin B1 intensity and circularity in 30-week-old R6/1 mouse striatal neurons nuclei with (mHtt +) or without (mHtt -) nuclear inclusions. N = 6. EM48 antibody was used to label mHtt inclusions. Representative images are shown. Scale bar 7 μm. D Graphs showing lamin B1 intensity and circularity in MSNs nuclei from the putamen of HD patients at different stages of the disease (VS: Vonsattel grade) and corresponding controls (CTL: non-affected individuals). N = 6 for CTL, N = 3 for VS I-II and N = 4 for VS III-IV. An average of 50 nuclei was examined for each sample. Representative images (maximal Z-projections) are shown. Scale bar 7μm. E Graphs show the quantification of different parameters in hippocampal CA1 ( Ctip2+/Prox1-) and DG (Ctip2+/Prox1+) neurons nuclei. N = 4 for each genotype. Representative images are shown. Scale bar 7 μm.


Figure 5 A Graphs show the quantification of lamin B1 intensity and circularity . N = 3 . An average of 12 nuclei was examined in each culture . Data are expressed as a percentage of controls . Bars represent the mean ± S . E . M . * P < 0 . 05 and * * P < 0 . 01 as compared to mApple - C1 control neurons ( two - tailed unpaired Student ' s t test ) . Exact P values are reported in Appendix Table S3 . B Representative images showing primary striatal neurons transfected with mApple - C1 or with mApple - LB1 , both in red . Neuronal nuclei were stained with DAPI - Fluoromount G ( blue ) . Lamin B1 is shown in white . Scale bar 10 μm .

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

Figure 5 A Graphs show the quantification of lamin B1 intensity and circularity. N = 3. An average of 12 nuclei was examined in each culture. Data are expressed as a percentage of controls. Bars represent the mean ± S.E.M. *P < 0.05 and **P < 0.01 as compared to mApple-C1 control neurons (two-tailed unpaired Student's t test). Exact P values are reported in Appendix Table S3. B Representative images showing primary striatal neurons transfected with mApple-C1 or with mApple-LB1, both in red. Neuronal nuclei were stained with DAPI-Fluoromount G (blue). Lamin B1 is shown in white. Scale bar 10 μm.


Figure 6 B - D Nuclear permeability in 30 - week - old wild - type ( WT ) and R6 / 1 mice striatal MSNs , CA1 and DG neurons nuclei , respectively . ( B and C ) N = 4 for each condition ; ( D ) N = 5 for each condition . An average of 25 nuclei were analysed for each sample

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

Figure 6 B-D Nuclear permeability in 30-week-old wild-type (WT) and R6/1 mice striatal MSNs, CA1 and DG neurons nuclei, respectively.(B and C) N = 4 for each condition; (D) N = 5 for each condition. An average of 25 nuclei were analysed for each sample


Figure 7A UCSC genome browser capture of lamin B1 ChIP - seq signal ( log ( LB1 / Input ) ) and LADs discovered in wild - type ( WT LAD ) and R6 / 1 ( R6 / 1 LADs ) mice in combination with NPC lamin B1 DamID ( top ) . Black arrows highlight differences in identified LADs between WT and R6 / 1 mice ChIP - seq data . Boxplots of LADs size ( log10 ( LADs size + 1 ) ) and of LADs score ( log10 ( LADs score + 1 ) ) obtained by EDD ( bottom ) from WT ( N = 3 ) and R6 / 1 ( N = 3 ) lamin B1 ChIP - seq data . Hipp , hippocampus . The bottom and top of the boxes are the first and third quartiles , and the line within represents the median . The whiskers denote the interval within 1 . 5 times the interquartile range ( IQR ) from the median . C UCSC genome browser capture of lamin B1 ChIP - seq signal ( log ( LB1 / Input ) ) LADs discovered in wild - type ( WT LADs ) and R6 / 1 ( R6 / 1 LADs ) mice ; and common ( green ) , wild - type ( WT ) specific ( WT spec . LADs , red ) and R6 / 1 specific ( R6 / 1 spec . LADs , blue ) LADs identified by EDD ( top , WT ( N = 3 ) , R6 / 1 ( N = 3 ) ) . Black arrows highlight WT specific LADs not identified in R6 / 1 mice . Boxplot of average size ( log10 ( LADs size + 1 ) ) for common , WT specific and R6 / 1 specific LADs ( right ) . The bottom and top of the boxes are the first and third quartiles , and the line within represents the median . The whiskers denote the interval within 1 . 5 times the interquartile range ( IQR ) from the median . D Lamin B1 enrichment in common , wild - type ( WT , N = 3 ) specific ( spec ) and R6 / 1 ( N = 3 ) specific LADs ( log10 ( LB1 / Input reads + 1 ) in hippocampus ( Hipp ; left ) . Bar graphs of significant ( Benjamini ' s adjusted P - value < 0 . 05 ) Biological Processes terms from DAVID for genes within common and WT specific LADs ( right ) . Gene - term enrichment was estimated by DAVID using a modified Fisher ' s exact test and Benjamini multiple correction test . Bars represents the - log10 ( Benjamini ' s adjusted P - value ) . Exact P values are reported in Appendix Table S3 . E Representaive immunoblot showing lamin B1 levels in the nucleoplasm ( Nucleop ) and chromatin ( Chrom ) in the hippocampus of 30 - week - old wild - type ( WT , N = 7 ) and R6 / 1 ( N = 7 ) mice . TBP , TATA binding protein .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 7A UCSC genome browser capture of lamin B1 ChIP-seq signal (log(LB1/Input)) and LADs discovered in wild-type (WT LAD) and R6/1 (R6/1 LADs) mice in combination with NPC lamin B1 DamID (top). Black arrows highlight differences in identified LADs between WT and R6/1 mice ChIP-seq data. Boxplots of LADs size (log10(LADs size + 1)) and of LADs score (log10(LADs score + 1)) obtained by EDD (bottom) from WT (N=3) and R6/1 (N=3) lamin B1 ChIP-seq data. Hipp, hippocampus. The bottom and top of the boxes are the first and third quartiles, and the line within represents the median. The whiskers denote the interval within 1.5 times the interquartile range (IQR) from the median. C UCSC genome browser capture of lamin B1 ChIP-seq signal (log(LB1/Input)) LADs discovered in wild-type (WT LADs) and R6/1 (R6/1 LADs) mice; and common (green), wild-type (WT) specific (WT spec. LADs, red) and R6/1 specific (R6/1 spec. LADs, blue) LADs identified by EDD (top, WT (N=3), R6/1 (N=3)). Black arrows highlight WT specific LADs not identified in R6/1 mice. Boxplot of average size (log10(LADs size + 1)) for common, WT specific and R6/1 specific LADs (right). The bottom and top of the boxes are the first and third quartiles, and the line within represents the median. The whiskers denote the interval within 1.5 times the interquartile range (IQR) from the median.  D Lamin B1 enrichment in common, wild-type (WT, N=3) specific (spec) and R6/1 (N=3) specific LADs (log10(LB1/Input reads +1) in hippocampus (Hipp; left). Bar graphs of significant (Benjamini's adjusted P-value < 0.05) Biological Processes terms from DAVID for genes within common and WT specific LADs (right). Gene-term enrichment was estimated by DAVID using a modified Fisher's exact test and Benjamini multiple correction test. Bars represents the -log10 (Benjamini's adjusted P-value). Exact P values are reported in Appendix Table S3. E Representaive immunoblot showing lamin B1 levels in the nucleoplasm (Nucleop) and chromatin (Chrom) in the hippocampus of 30-week-old wild-type (WT, N = 7) and R6/1 (N = 7) mice. TBP, TATA binding protein.


Figure 8 B UCSC genome browser capture of wild - type ( WT , N = 3 ) and R6 / 1 ( N = 3 ) mice hippocampus ATAC - seq data , hippocampal H3K9ac and unchanged , closed in R6 / 1 and open in R6 / 1 accessible detected regions ( left ) in Tuba1α , Gabra2α and Dlx2 gene locus . Arrows in blue ( closed in R6 / 1 ) and red ( open in R6 / 1 ) indicate differential accessible regions . Bar graphs of significant ( Benjamini ' s adjusted P - value < 0 . 05 ) Biological Processes terms from DAVID for genes associated with decreased ( right top ) or increased ( right bottom ) chromatin accessibility regions . Gene - term enrichment westimated by DAVID using a modified Fisher ' s exact test and Benjamini multiple correction test . Bars represents the - log10 ( Benjamini ' s adjusted P - value ) . Exact P values are reported in Appendix Table S3 . C Boxplots showing average gene expression ( log10 ( FPKMs + 1 ) for genes up or down - regulated ( adjusted P - value < 0 . 001 , | FC | > 2 ) in R6 / 1 ( N = 9 ) versus WT ( N = 9 ) mice ( left ) . The bottom and top of the boxes are the first and third quartiles , and the line within represents the median . The whiskers denote the interval within 1 . 5 times the interquartile range ( IQR ) from the median . Heatmap showing expression profile ( log10 ( FPKMs + 1 ) ) of genes up - or down - regulated ( adjusted P - value < 0 . 001 , | FC | > 2 , N = 9 ) in R6 / 1 mice ( mid ) . Genes are ranked by the degree of expression . Numbers in colour scale show the correspondence between gene expression values and colours . Boxplots showing average TSS chromatin accessibility ( log10 ( ATAC reads + 1 ) , N = 3 ) for genes up or down - regulated ( adjusted P - value < 0 . 001 , | FC | > 2 , N = 9 ) in R6 / 1 versus WT mice hippocampus ( right ) . Exact P values are reported in Appendix Table S3 . The bottom and top of the boxes are the first and third quartiles , and the line within represents the median . The whiskers denote the interval within 1 . 5 times the interquartile range ( IQR ) from the median . D Average gene expression ( closed or open regions FPKMs / unchanged regions FPKMs ) for genes associated with differential accessible regions in R6 / 1 ( N = 9 ) versus WT ( N = 9 ) mice ( adjusted P - value < 0 . 05 , N = 3 ) . Each point corresponds to the value from an individual sample . Data is shown as the mean ± S . E . M . * P < 0 . 05 as compared with WT mice ( two - tailed unpaired Student ' s t test ) . Exact P values are reported in Appendix Table S3 .

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

Figure 8 B UCSC genome browser capture of wild-type (WT, N=3) and R6/1 (N=3) mice hippocampus ATAC-seq data, hippocampal H3K9ac and unchanged, closed in R6/1 and open in R6/1 accessible detected regions (left) in Tuba1α, Gabra2α and Dlx2 gene locus. Arrows in blue (closed in R6/1) and red (open in R6/1) indicate differential accessible regions. Bar graphs of significant (Benjamini's adjusted P-value < 0.05) Biological Processes terms from DAVID for genes associated with decreased (right top) or increased (right bottom) chromatin accessibility regions. Gene-term enrichment westimated by DAVID using a modified Fisher's exact test and Benjamini multiple correction test. Bars represents the -log10 (Benjamini's adjusted P-value). Exact P values are reported in Appendix Table S3.  C Boxplots showing average gene expression (log10(FPKMs +1) for genes up or down-regulated (adjusted P-value < 0.001, |FC|>2) in R6/1 (N=9) versus WT (N=9) mice (left). The bottom and top of the boxes are the first and third quartiles, and the line within represents the median. The whiskers denote the interval within 1.5 times the interquartile range (IQR) from the median. Heatmap showing expression profile (log10 (FPKMs +1)) of genes up- or down-regulated (adjusted P-value<0.001, |FC|>2, N=9) in R6/1 mice (mid). Genes are ranked by the degree of expression. Numbers in colour scale show the correspondence between gene expression values and colours. Boxplots showing average TSS chromatin accessibility (log10(ATAC reads +1), N=3) for genes up or down-regulated (adjusted P-value < 0.001, |FC|>2, N=9) in R6/1 versus WT mice hippocampus (right). Exact P values are reported in Appendix Table S3. The bottom and top of the boxes are the first and third quartiles, and the line within represents the median. The whiskers denote the interval within 1.5 times the interquartile range (IQR) from the median. D Average gene expression (closed or open regions FPKMs/ unchanged regions FPKMs) for genes associated with differential accessible regions in R6/1 (N=9) versus WT (N=9) mice (adjusted P-value < 0.05, N=3). Each point corresponds to the value from an individual sample. Data is shown as the mean ± S.E.M. *P < 0.05 as compared with WT mice (two-tailed unpaired Student's t test). Exact P values are reported in Appendix Table S3.