( E - F ) Serum antibodies collected pre - immunization and 7 - or 14 - days post NP - CGG immunization , detected by NP7 - BSA ELISA using isotype - specific secondary antibodies . Data show mean ± SEM from N = 3 experiments with statistical significance from 2 - way ANOVA with multiple comparisons .

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(E-F) Serum antibodies collected pre-immunization and 7- or 14-days post NP-CGG immunization, detected by NP7-BSA ELISA using isotype-specific secondary antibodies. Data show mean ± SEM from N = 3 experiments with statistical significance from 2-way ANOVA with multiple comparisons.


( H ) Numbers of Tfh cells 14 days post - immunization , identified as a CD4 + CD44 + PD - 1 + CXCR5 + population . Mean and SEM of N = 4 mice .

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(H) Numbers of Tfh cells 14 days post-immunization, identified as a CD4+CD44+PD-1+CXCR5+ population. Mean and SEM of N = 4 mice.


( A ) Soluble anti - IgM internalization in CRISPR - targeted follicular B cells . N = 11 - 12 mice . Data show mean ± SEM . P , statistical significance from two - way ANOVA . ( B ) Soluble anti - IgD internalization . N = 3 mice . Data show mean ± SEM . P , statistical significance from two - way ANOVA . ( C ) Soluble anti - IgG internalization in GC B cells from SRBC - immunized mice . N = 4 mice . Data show mean ± SEM . P , statistical significance from two - way ANOVA .

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(A) Soluble anti-IgM internalization in CRISPR-targeted follicular B cells. N = 11- 12 mice. Data show mean ± SEM. P, statistical significance from two-way ANOVA. (B) Soluble anti-IgD internalization. N = 3 mice. Data show mean ± SEM. P, statistical significance from two-way ANOVA. (C) Soluble anti-IgG internalization in GC B cells from SRBC-immunized mice. N = 4 mice. Data show mean ± SEM. P, statistical significance from two-way ANOVA.


( F ) IgM degradation following total cell surface biotinylation , measured as loss of biotin - labelled membrane IgM ( mIgM ) in cell lysates over time . ( G ) Western densitometry quantifying IgM degradation assay . Proportion of biotin detected is normalized to the initial WT condition ( left ) or to the initial amount for each genotype ( right ) . Data show mean ± SEM from three independent tests . P , statistical significance from two - way ANOVA .

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(F) IgM degradation following total cell surface biotinylation, measured as loss of biotin-labelled membrane IgM (mIgM) in cell lysates over time. (G) Western densitometry quantifying IgM degradation assay. Proportion of biotin detected is normalized to the initial WT condition (left) or to the initial amount for each genotype (right). Data show mean ± SEM from three independent tests. P, statistical significance from two-way ANOVA.


( K ) Proliferation ( CFSE dilution ) of OTII CD4 cells in co - cultures with anti - Igκ - OVA pulsed B cells ; and quantification from 6 - 8 mice . Data show mean ± SEM .

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(K) Proliferation (CFSE dilution) of OTII CD4 cells in co-cultures with anti-Igκ-OVA pulsed B cells; and quantification from 6-8 mice. Data show mean ± SEM.


( C - D ) Isolated naïve B cells from CRISPR - targeted chimeras after 3 - day culture in stated cytokines . ( C ) mCherry percentage in targeted primary cell cultures . N = 15 mice . * p < 0 . 05 , * * p < 0 . 01 , * * * * p < 0 . 0001 using two - way ANOVA . ( D ) AnnexinV surface stain in targeted cells . N = 3 - 4 mice . Data information : All data show means ± SEM .

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(C-D) Isolated naïve B cells from CRISPR-targeted chimeras after 3-day culture in stated cytokines. (C) mCherry percentage in targeted primary cell cultures. N = 15 mice. *p<0.05, **p<0.01, ****p<0.0001 using two-way ANOVA. (D) AnnexinV surface stain in targeted cells. N = 3-4 mice. Data information: All data show means ± SEM.


( G ) Oxygen consumption rate ( OCR ) in CRISPR - targeted Ramos cells . N = 2 independent infections . P , two - way ANOVA of basal and maximum OCR . ( H ) OCR in B cells isolated from Sh3gl1 - / - and WT littermates . N = 9 mice . P , two - way ANOVA of basal and maximum OCR .

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(G) Oxygen consumption rate (OCR) in CRISPR-targeted Ramos cells. N = 2 independent infections. P, two-way ANOVA of basal and maximum OCR. (H) OCR in B cells isolated from Sh3gl1-/- and WT littermates. N = 9 mice. P, two-way ANOVA of basal and maximum OCR.


Cell cultures with full RPMI media supplemented with 50 μg / ml FeSO4 or 5 μM hemin chloride . ( J ) SH3GL1 - targeted Ramos cells . N = 4 independent infections . P , statistical significance from two - way ANOVA from both treatments versus RPMI . ( K ) Control sgRNA - targeted Ramos cells .

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Cell cultures with full RPMI media supplemented with 50 μg/ml FeSO4 or 5 μM hemin chloride. (J) SH3GL1-targeted Ramos cells. N = 4 independent infections. P, statistical significance from two-way ANOVA from both treatments versus RPMI. (K) Control sgRNA-targeted Ramos cells.


( O ) TfR internalization assay in Ramos cells using biotinylated anti - CD71 . 4 independent CRISPR infections in 2 experiments . P , significance calculated using two - way ANOVA and indicated in corresponding color .

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(O) TfR internalization assay in Ramos cells using biotinylated anti-CD71. 4 independent CRISPR infections in 2 experiments. P, significance calculated using two-way ANOVA and indicated in corresponding color.


( P , Q ) Mouse transferrin internalization in B cells from SRBC - immunized WT and Sh3gl1 - / - littermates . N = 7 mice per genotype in 2 experiments . P , statistical significance analyzed using two - way ANOVA . ( P ) Naïve B cells . ( Q ) GC B cells .

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(P,Q) Mouse transferrin internalization in B cells from SRBC-immunized WT and Sh3gl1-/- littermates. N = 7 mice per genotype in 2 experiments. P, statistical significance analyzed using two-way ANOVA. (P) Naïve B cells. (Q) GC B cells.


A Robust immunofluorescence detection of ubiquitination on LAMP1 - positive compartments with pan - ubiquitin FK2 or K48 chain - specific antibodies . HeLa cells were treated with 250 µM LLOMe or EtOH ( untreated ) for 3 h , fixed and stained with indicated antibodies . Confocal microscopy was followed by automated image analysis . Graphs represent data from three independent experiments with ≥ 50 cells per condition ( mean ± SD ) . Scale bar : 20 µm .

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A Robust immunofluorescence detection of ubiquitination on LAMP1-positive compartments with pan-ubiquitin FK2 or K48 chain-specific antibodies. HeLa cells were treated with 250 µM LLOMe or EtOH (untreated) for 3 h, fixed and stained with indicated antibodies. Confocal microscopy was followed by automated image analysis. Graphs represent data from three independent experiments with ≥ 50 cells per condition (mean ± SD). Scale bar: 20 µm.


C Primary screen using pools of four single siRNAs targeting 37 human E2 enzymes . HeLa cells were seeded in 384 - well plates , transfected with a siRNA library in quadruplicates for 72 h . Cells were LLOMe - treated and processed for imaging Plates were imaged automatically with a spinning disk confocal microscope . Automated image analysis was done using the Acapella software and the percentage of ubiquitinated lysosomes was determined . Robust z - scores were calculated for LLOMe - treated samples for each depletion against the values of the whole plate . Samples exceeding the threshold of - 2 of robust z - score ( highlighted in red ) were considered for the secondary screen .

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C Primary screen using pools of four single siRNAs targeting 37 human E2 enzymes. HeLa cells were seeded in 384-well plates, transfected with a siRNA library in quadruplicates for 72 h. Cells were LLOMe-treated and processed for imaging Plates were imaged automatically with a spinning disk confocal microscope. Automated image analysis was done using the Acapella software and the percentage of ubiquitinated lysosomes was determined. Robust z-scores were calculated for LLOMe-treated samples for each depletion against the values of the whole plate. Samples exceeding the threshold of -2 of robust z-score (highlighted in red) were considered for the secondary screen.


D Selected candidates were re - screened with the same work pipeline but with the four single siRNAs of pools of each candidate from the primary screen . Candidates exceeding the threshold in both stainings are highlighted in red .

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D Selected candidates were re-screened with the same work pipeline but with the four single siRNAs of pools of each candidate from the primary screen. Candidates exceeding the threshold in both stainings are highlighted in red.


E Damage - induced translocation of UBE2QL1 to lysosomes . The three best candidates ( from D ) were transiently expressed with N - terminal HA - tag in HeLa cells . Cells were treated with LLOMe or EtOH ( untreated ) for 3 h , fixed and stained for HA and LAMP1 as indicated and imaged by confocal laser scanning microscopy . Scale bar : 10 µm . F Automated quantification of ( E ) . Shown are Pearson correlation coefficients ( P . C . C ) of HA and LAMP1 signals . Graph represents data from three independent experiments with ≥ 30 cells per condition ( mean ± SD ) . * * * * P < 0 . 0001 ( One - way - ANOVA with Bonferroni ' s multiple comparison test ) .

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E Damage-induced translocation of UBE2QL1 to lysosomes. The three best candidates (from D) were transiently expressed with N-terminal HA-tag in HeLa cells. Cells were treated with LLOMe or EtOH (untreated) for 3 h, fixed and stained for HA and LAMP1 as indicated and imaged by confocal laser scanning microscopy. Scale bar: 10 µm. F Automated quantification of (E). Shown are Pearson correlation coefficients (P.C.C) of HA and LAMP1 signals. Graph represents data from three independent experiments with ≥ 30 cells per condition (mean ± SD). ****P<0.0001 (One-way-ANOVA with Bonferroni's multiple comparison test).


G Western blot detection of endogenous UBE2QL1 and depletion efficiency . Cells were transfected with control siRNA ( Ctrl ) , two UBE2QL1 siRNAs from the screen ( # 2 or # 4 ) or an additional siRNA ( # 5 ) , and lysates probed with an antibody to UBE2QL1 as indicated . GAPDH was probed as loading control .

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G Western blot detection of endogenous UBE2QL1 and depletion efficiency. Cells were transfected with control siRNA (Ctrl), two UBE2QL1 siRNAs from the screen (#2 or #4) or an additional siRNA (#5), and lysates probed with an antibody to UBE2QL1 as indicated. GAPDH was probed as loading control.


A HeLa cells were transfected with different UBE2QL1 siRNAs for 60 h , and LLOMe or control - treated ( untreated ) for 3 h . Cells were fixed and immuno - stained for LAMP1 along with antibodies specific for K63 or K48 - linked ubiquitin chains as indicated and imaged with a laser scanning confocal microscope . Note the strong reduction of damage - induced K48 ubiquitination and decreased intensity of the K63 signal . Scale bar : 20 µm . B , C Automated image quantification of ( A ) . Shown are percentages of K48 or K63 - positive LAMP1 vesicles per cell . Graphs represent data from three independent experiments with ≥ 70 cells per condition ( mean ± SD ) . * * P < 0 . 01 ; * * * P < 0 . 001 ; * * * * P < 0 . 0001 ( One - way - ANOVA with Bonferroni ' s multiple comparison test ) .

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A HeLa cells were transfected with different UBE2QL1 siRNAs for 60 h, and LLOMe or control-treated (untreated) for 3 h. Cells were fixed and immuno-stained for LAMP1 along with antibodies specific for K63 or K48-linked ubiquitin chains as indicated and imaged with a laser scanning confocal microscope. Note the strong reduction of damage-induced K48 ubiquitination and decreased intensity of the K63 signal. Scale bar: 20 µm. B, C Automated image quantification of (A). Shown are percentages of K48 or K63-positive LAMP1 vesicles per cell. Graphs represent data from three independent experiments with ≥ 70 cells per condition (mean ± SD). **P<0.01; ***P<0.001; ****P<0.0001 (One-way-ANOVA with Bonferroni's multiple comparison test).


A HeLa cells expressing UBE2QL1 - HA were fixed at indicated time points after LLOMe treatment and processed for confocal microscopy . Cells were stained with antibodies specific for LAMP1 ( not shown ) , and combinations of HA with K48 chains , K63 chains or p62 as indicated . Note the delayed co - emergence of K48 chains and UBE2QL1 vesicles , compared to the earlier appearance of K63 chains and p62 . Arrows indicate colocalizing or non - colocalizing vesicles . Scale bars : 10 µm , 2 µm for inlays . B Automated quantification of ( A ) . Shown are the Pearson correlation coefficients ( P . C . C . ) representing colocalization of UBE2QL1 - HA , K48 chains , K63 chains or p62 with LAMP1 - positive vesicles . Graphs represent data from three independent experiments with ≥ 30 cells per condition ( mean ± SD ) . C Automated quantification of ( A ) . P . C . C . ' s representing colocalization of K48 chains , K63 chains or p62 with UBE2QL1 - HA in cells treated with LLOMe or vehicle alone ( untreated ) for 180 min . Graphs represent data from three independent experiments with ≥ 30 cells per condition ( mean ± SD ) .

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A HeLa cells expressing UBE2QL1-HA were fixed at indicated time points after LLOMe treatment and processed for confocal microscopy. Cells were stained with antibodies specific for LAMP1 (not shown), and combinations of HA with K48 chains, K63 chains or p62 as indicated. Note the delayed co-emergence of K48 chains and UBE2QL1 vesicles, compared to the earlier appearance of K63 chains and p62. Arrows indicate colocalizing or non-colocalizing vesicles. Scale bars: 10 µm, 2 µm for inlays. B Automated quantification of (A). Shown are the Pearson correlation coefficients (P.C.C.) representing colocalization of UBE2QL1-HA, K48 chains, K63 chains or p62 with LAMP1-positive vesicles. Graphs represent data from three independent experiments with ≥ 30 cells per condition (mean ± SD). C Automated quantification of (A). P.C.C.'s representing colocalization of K48 chains, K63 chains or p62 with UBE2QL1-HA in cells treated with LLOMe or vehicle alone (untreated) for 180 min. Graphs represent data from three independent experiments with ≥ 30 cells per condition (mean ± SD).


Immuno - electron microscopy of LLOMe - treated HeLa cells . Cells in ( A ) overexpress mCherry - Gal3 and a dominant - negative C160S mutant of GFP - YOD1 , and were immunostained for p62 ( A ) p62 ( 10 nm gold particles ) localizes as a cap ( arrowheads ) to the outer limiting membrane of lysosomes ( L ) . Scale bars : 200 nm .

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Immuno-electron microscopy of LLOMe-treated HeLa cells. Cells in (A) overexpress mCherry-Gal3 and a dominant-negative C160S mutant of GFP-YOD1, and were immunostained for p62 (A) p62 (10 nm gold particles) localizes as a cap (arrowheads) to the outer limiting membrane of lysosomes (L). Scale bars: 200 nm.


Immuno - electron microscopy of LLOMe - treated HeLa cells . Cells in ( B ) overexpress mCherry - Gal3 and a dominant - negative C160S mutant of GFP - YOD1 , and were immunostained for p62 and K48 . ( B ) K48 ubiquitin chains ( 15 nm gold particles , arrows ) are found in the lumen of lysosomes , that also contain p62 ( 10 nm gold particles , arrowheads ) at their outer membrane . Scale bars : 200 nm .

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Immuno-electron microscopy of LLOMe-treated HeLa cells. Cells in (B) overexpress mCherry-Gal3 and a dominant-negative C160S mutant of GFP-YOD1, and were immunostained for p62 and K48. (B) K48 ubiquitin chains (15 nm gold particles, arrows) are found in the lumen of lysosomes, that also contain p62 (10 nm gold particles, arrowheads) at their outer membrane. Scale bars: 200 nm.


Immuno - electron microscopy of LLOMe - treated HeLa cells . ( C ) Overexpressed UBE2QL1 - GFP ( 10 nm gold particles ) is found in late endosomes ( LE , Inset ) and lysosomes . M = mitochondrion . Scale bars : 200 nm .

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Immuno-electron microscopy of LLOMe-treated HeLa cells. (C) Overexpressed UBE2QL1-GFP (10 nm gold particles) is found in late endosomes (LE, Inset) and lysosomes. M = mitochondrion. Scale bars: 200 nm.


D , E Comparative UBE2QL1 proximity mapping before and after lysosome damage using SILAC mass spectrometry . HeLa cells expressing a UBE2QL1 - APEX2 fusion were EtOH ( light - labelled , L ) - or LLOMe ( heavy - labelled , H ) - treated and pulsed with biotin phenol ( 30 min ) and H2O2 ( 1 min ) . Proteins detected in replicative streptavidin purifications are depicted in a Venn diagram ( D ) and displayed high correlation coefficients ( E ) .

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D, E Comparative UBE2QL1 proximity mapping before and after lysosome damage using SILAC mass spectrometry. HeLa cells expressing a UBE2QL1-APEX2 fusion were EtOH (light-labelled, L)- or LLOMe (heavy-labelled, H)-treated and pulsed with biotin phenol (30 min) and H2O2 (1 min). Proteins detected in replicative streptavidin purifications are depicted in a Venn diagram (D) and displayed high correlation coefficients (E).


, F Comparative UBE2QL1 proximity mapping before and after lysosome damage using SILAC mass spectrometry . HeLa cells expressing a UBE2QL1 - APEX2 fusion were EtOH ( light - labelled , L ) - or LLOMe ( heavy - labelled , H ) - treated and pulsed with biotin phenol ( 30 min ) and H2O2 ( 1 min ) . ( F ) Results from the four experiments are summarized in a volcano plot . Horizontal dotted line represents the significance threshold ( p > 0 . 05 ) . The vertical lines indicate the fold change cutoff ( log2 ( H / L ) ≥ 1 . 5 ) . Red dots indicate proteins above the significance and fold - change thresholds . Protein names are color - coded in blue ( endosome / lysosome - associated ) , green ( endolysosomal damage / autophagy - associated ) and orange ( VCP / p97 and its cofactors ) .

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, F Comparative UBE2QL1 proximity mapping before and after lysosome damage using SILAC mass spectrometry. HeLa cells expressing a UBE2QL1-APEX2 fusion were EtOH (light-labelled, L)- or LLOMe (heavy-labelled, H)-treated and pulsed with biotin phenol (30 min) and H2O2 (1 min). (F) Results from the four experiments are summarized in a volcano plot. Horizontal dotted line represents the significance threshold (p > 0.05). The vertical lines indicate the fold change cutoff (log2 (H/L) ≥ 1.5). Red dots indicate proteins above the significance and fold-change thresholds. Protein names are color-coded in blue (endosome/lysosome-associated), green (endolysosomal damage/autophagy-associated) and orange (VCP/p97 and its cofactors).


A HeLa cells stably expressing p97 - GFP were control or UBE2QL1 - depleted for 48 h and expression of p97 - GFP was induced for the last 24 h . Cells were LLOMe or control treated for 1 h , fixed after a chase of 2 h , and stained with antibodies to K48 ubiquitin chains and LAMP1 as indicated . Note the reduction of the K48 and p97 - GFP signals on damaged lysosomes in UBE2QL1 - depleted cells . Arrows indicate colocalizing or non - colocalizing vesicles . Scale bars : 20 µm , 10 µm for inlays .

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A HeLa cells stably expressing p97-GFP were control or UBE2QL1-depleted for 48 h and expression of p97-GFP was induced for the last 24 h. Cells were LLOMe or control treated for 1 h, fixed after a chase of 2 h, and stained with antibodies to K48 ubiquitin chains and LAMP1 as indicated. Note the reduction of the K48 and p97-GFP signals on damaged lysosomes in UBE2QL1-depleted cells. Arrows indicate colocalizing or non-colocalizing vesicles. Scale bars: 20 µm, 10 µm for inlays.


B Automated quantification Shown are fold increases of Pearson correlation coefficients ( P . C . C . ) of p97 - GFP and LAMP1 signals , normalized to untreated siCtrl . Data are from four independent experiments with ≥ 30 cells per condition . Data information : data are presented as mean ± SD . * P < 0 . 05 ; * * * P < 0 . 001 ( One - way - ANOVA with Bonferroni ' s multiple comparison test ) .

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B Automated quantification Shown are fold increases of Pearson correlation coefficients (P.C.C.) of p97-GFP and LAMP1 signals, normalized to untreated siCtrl. Data are from four independent experiments with ≥ 30 cells per condition. Data information: data are presented as mean ± SD. *P<0.05; ***P<0.001 (One-way-ANOVA with Bonferroni's multiple comparison test).


C HeLa cells were control or UBE2QL1 - depleted for 60 h , fixed after 1 h of LLOMe or control treatment and stained for LAMP1 and p62 and analyzed by confocal microscopy . Arrows indicate colocalizing or non - colocalizing vesicles . Scale bars : 20 µm , 5 µm for inlays .

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C HeLa cells were control or UBE2QL1-depleted for 60 h, fixed after 1 h of LLOMe or control treatment and stained for LAMP1 and p62 and analyzed by confocal microscopy. Arrows indicate colocalizing or non-colocalizing vesicles. Scale bars: 20 µm, 5 µm for inlays.


D Quantification Shown are fold increases of Pearson correlation coefficients ( P . C . C . ) of p62 on LAMP1 signals , normalized to untreated siCtrl . Graph represents data from three independent experiments with ≥ 30 cells per condition . Data information : data are presented as mean ± SD . * P < 0 . 05 ; * * * P < 0 . 001 ( One - way - ANOVA with Bonferroni ' s multiple comparison test ) .

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D Quantification Shown are fold increases of Pearson correlation coefficients (P.C.C.) of p62 on LAMP1 signals, normalized to untreated siCtrl. Graph represents data from three independent experiments with ≥ 30 cells per condition. Data information: data are presented as mean ± SD. *P<0.05; ***P<0.001 (One-way-ANOVA with Bonferroni's multiple comparison test).


E HeLa cells were treated but fixed after additional 2 h of chase . Samples were stained for Galectin - 3 ( Gal3 ) to detect damaged lysosomes , and LC3B as marker for autophagosomes . Note the reduction of LC3B colocalization with Gal3 indicating impaired autophagosome formation . Arrows indicate colocalizing or non - colocalizing vesicles . Scale bars : 20 µm , 5 µm for inlays .

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E HeLa cells were treated but fixed after additional 2 h of chase. Samples were stained for Galectin-3 (Gal3) to detect damaged lysosomes, and LC3B as marker for autophagosomes. Note the reduction of LC3B colocalization with Gal3 indicating impaired autophagosome formation. Arrows indicate colocalizing or non-colocalizing vesicles. Scale bars: 20 µm, 5 µm for inlays.


F Quantification Shown are fold increases of Pearson correlation coefficients ( P . C . C . ) of LC3B and Gal3 signals , normalized to untreated siCtrl , representing LC3B colocalization with Gal3 . Graph represents data from three independent experiments with ≥ 50 cells per condition . Data information : data are presented as mean ± SD . * P < 0 . 05 ; * * * P < 0 . 001 ( One - way - ANOVA with Bonferroni ' s multiple comparison test ) .

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F Quantification Shown are fold increases of Pearson correlation coefficients (P.C.C.) of LC3B and Gal3 signals, normalized to untreated siCtrl, representing LC3B colocalization with Gal3. Graph represents data from three independent experiments with ≥ 50 cells per condition. Data information: data are presented as mean ± SD. *P<0.05; ***P<0.001 (One-way-ANOVA with Bonferroni's multiple comparison test).


G Western blot analysis of lysates of cells transfected with UBE2QL1 or control siRNA for 72 h , treated with 200 nM Bafilomycin A1 for 5 h or 250 µM LLOMe for 3 h as indicated and probed with an antibody specific for LC3A / B . GAPDH was probed as loading control .

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G Western blot analysis of lysates of cells transfected with UBE2QL1 or control siRNA for 72 h, treated with 200 nM Bafilomycin A1 for 5 h or 250 µM LLOMe for 3 h as indicated and probed with an antibody specific for LC3A/B. GAPDH was probed as loading control.


A Gal3 puncta assay for damaged lysosomes . HeLa cells were depleted of UBE2QL1 with two siRNAs for 60 h and treated with LLOMe or EtOH alone ( untreated ) for 1 h . Cells were fixed at indicated times after washout , stained with DAPI and Gal3 and LAMP1 antibodies , and processed for confocal microscopy . Note increased number of Gal3 puncta in untreated UBE2QL1 - depleted cells and their persistence 10 h after LLOMe - induced damage . Scale bars : 10 µm , 2 µm for inlays . Arrows indicate Gal3 and LAMP1 colocalizing vesicles .

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A Gal3 puncta assay for damaged lysosomes. HeLa cells were depleted of UBE2QL1 with two siRNAs for 60 h and treated with LLOMe or EtOH alone (untreated) for 1 h. Cells were fixed at indicated times after washout, stained with DAPI and Gal3 and LAMP1 antibodies, and processed for confocal microscopy. Note increased number of Gal3 puncta in untreated UBE2QL1-depleted cells and their persistence 10 h after LLOMe-induced damage. Scale bars: 10 µm, 2 µm for inlays. Arrows indicate Gal3 and LAMP1 colocalizing vesicles.


B Automated quantification The percentage of cells with more than three Gal3 puncta is shown . Graph represents data from three independent experiments with ≥ 50 cells per condition ( mean ± SD ) . * P < 0 . 05 ; * * P < 0 . 01 ( One - way - ANOVA with Dunnett ' s multiple comparison test ) .

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B Automated quantification The percentage of cells with more than three Gal3 puncta is shown. Graph represents data from three independent experiments with ≥ 50 cells per condition (mean ± SD). *P<0.05; **P<0.01 (One-way-ANOVA with Dunnett's multiple comparison test).


C Survival assay . Control or UBE2QL1 - depleted HeLa cells ( 48 h ) were treated with increasing concentrations of LLOMe as indicated . Cell viability was measured with the MTS assay . Graph represents data from one experiment including three replicates ( mean ± SD ) . * P < 0 . 05 ; * * * * P < 0 . 0001 ( One - way - ANOVA with Bonferroni ' s multiple comparison test ) .

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C Survival assay. Control or UBE2QL1-depleted HeLa cells (48 h) were treated with increasing concentrations of LLOMe as indicated. Cell viability was measured with the MTS assay. Graph represents data from one experiment including three replicates (mean ± SD). *P<0.05; ****P<0.0001 (One-way-ANOVA with Bonferroni's multiple comparison test).


A Control or UBE2QL1 - depleted ( for 60 h ) HeLa cells were stained with DAPI and an antibody against TFEB and automatically analyzed by confocal microscopy . Note nuclear translocation of TFEB upon loss of UBE2QL1 . Scale bar : 20µm .

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A Control or UBE2QL1-depleted (for 60 h) HeLa cells were stained with DAPI and an antibody against TFEB and automatically analyzed by confocal microscopy. Note nuclear translocation of TFEB upon loss of UBE2QL1. Scale bar: 20µm.


B Automated quantification The intensity ratio of the cytoplasmic and nuclear TFEB signals was determined in both conditions . Graph represents data from four independent experiments with ≥ 100 cells per condition ( mean ± SD ) . * * * P < 0 . 001 ; * * * * P < 0 . 0001 ( One - way - ANOVA with Bonferroni ' s multiple comparison test ) .

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B Automated quantification The intensity ratio of the cytoplasmic and nuclear TFEB signals was determined in both conditions. Graph represents data from four independent experiments with ≥ 100 cells per condition (mean ± SD). ***P<0.001; ****P<0.0001 (One-way-ANOVA with Bonferroni's multiple comparison test).


C Cells were stained for LAMP1 and mTOR . Note the increase in lysosomes and lysosomal dissociation of mTOR in UBE2QL1 depleted cells . Scale bar : 20 µm .

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C Cells were stained for LAMP1 and mTOR. Note the increase in lysosomes and lysosomal dissociation of mTOR in UBE2QL1 depleted cells. Scale bar: 20 µm.


D Automated quantification The colocalization of mTOR and LAMP1 is shown as Pearson correlation coefficients ( P . C . C . ) of the signals . Graph represents data from three independent experiments with ≥ 70 cells per condition ( mean ± SD ) . * P < 0 . 05 ( One - way - ANOVA with Bonferroni ' s multiple comparison test ) .

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D Automated quantification The colocalization of mTOR and LAMP1 is shown as Pearson correlation coefficients (P.C.C.) of the signals. Graph represents data from three independent experiments with ≥ 70 cells per condition (mean ± SD). *P<0.05 (One-way-ANOVA with Bonferroni's multiple comparison test).


E Automated quantification Number of LAMP1 - positive vesicles per cell is shown . Graph represents data from three independent experiments with ≥ 70 cells per condition ( mean ± SD ) .

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E Automated quantification Number of LAMP1-positive vesicles per cell is shown. Graph represents data from three independent experiments with ≥ 70 cells per condition (mean ± SD).


F Western blot analysis of lysates of cells treated As a positive control , cells were treated with the mTOR - inhibitor Torin1 ( 1 µM ) for 2 h . GAPDH was probed as loading control .

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F Western blot analysis of lysates of cells treated As a positive control, cells were treated with the mTOR-inhibitor Torin1 (1 µM) for 2 h. GAPDH was probed as loading control.


G C . elegans wild - type ( N2 ) and mutant strains for ubc - 25 ( ok1732 ) , for scav - 3 ( qx193 ) or the ubc - 25 and scav - 3 double mutant ( ok1732 ; qx193 ) carrying a EGFP - Gal3 transgene were imaged in adult worms ( L4 + 24 h ) . 3D reconstructions of the hypodermis . Small and big units represent 1 and 5 µm , respectively .

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G C.elegans wild-type (N2) and mutant strains for ubc-25(ok1732), for scav-3(qx193) or the ubc-25 and scav-3 double mutant (ok1732;qx193) carrying a EGFP-Gal3 transgene were imaged in adult worms (L4 + 24 h). 3D reconstructions of the hypodermis. Small and big units represent 1 and 5 µm, respectively.


H Average volume of permeabilized lysosomes within the region of hypodermis corresponding to 100 µm of body length was quantified . At least 20 animals were scored in each strain . * P < 0 . 05 ; * * P < 0 . 01 ; * * * * P < 0 . 0001 ( One - way - ANOVA with Dunnett ' s multiple comparison test or Student ' s two - tailed unpaired t test ) .

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H Average volume of permeabilized lysosomes within the region of hypodermis corresponding to 100 µm of body length was quantified. At least 20 animals were scored in each strain. *P<0.05; **P<0.01; ****P<0.0001 (One-way-ANOVA with Dunnett's multiple comparison test or Student's two-tailed unpaired t test).


I Populations of worms with different extent of lysosome permeabilization were analyzed . Graph represents percentage of worms with less than 5 , 5 to 20 or more than 20 permeabilized lysosomes in the region of hypodermis corresponding to 100 µm of body length .

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I Populations of worms with different extent of lysosome permeabilization were analyzed. Graph represents percentage of worms with less than 5, 5 to 20 or more than 20 permeabilized lysosomes in the region of hypodermis corresponding to 100 µm of body length.


RT - PCR analysis ( top ) and representative direct sequencing ( bottom ) of Ttn exon 326 transcripts from HL - 1 cardiomyocytes transiently transfected with different 2OMePS AONs . Only 2OMePS - AON1 and 2OMePS - AON1 + 3 ( * ) lead to a correct excision of exon 326 as confirmed by direct sequencing .

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RT-PCR analysis (top) and representative direct sequencing (bottom) of Ttn exon 326 transcripts from HL-1 cardiomyocytes transiently transfected with different 2OMePS AONs. Only 2OMePS-AON1 and 2OMePS-AON1 + 3 (*) lead to a correct excision of exon 326 as confirmed by direct sequencing.


Immunofluorescence images of sarcomeric α - actinin in untransfected and 2OMePS - AON1 + 3 - transfected HL - 1 cardiomyocytes . Scale bars , 10 μm .

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Immunofluorescence images of sarcomeric α-actinin in untransfected and 2OMePS-AON1 + 3-transfected HL-1 cardiomyocytes. Scale bars, 10 μm.


RT - PCR analysis of TTN exon 326 transcripts from DCM cardiomyocytes transiently transfected with 2OMePS - AON1 , 2OMePS - AON3 , and 2OMePS - AON1 + 3 .

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RT-PCR analysis of TTN exon 326 transcripts from DCM cardiomyocytes transiently transfected with 2OMePS-AON1, 2OMePS-AON3, and 2OMePS-AON1 + 3.


RT - PCR analysis ( left ) and representative direct sequencing ( right ) of TTN exon 326 transcripts from DCM cardiomyocytes infected with the U7snRNA - TTNAONs - IRES - GFP lentiviral vector carrying the AON1 and 3 sequences ( TTN - AON ) or with a control vector ( U7snRNA - ScrAONs - IRES - GFP ) .

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RT-PCR analysis (left) and representative direct sequencing (right) of TTN exon 326 transcripts from DCM cardiomyocytes infected with the U7snRNA-TTNAONs-IRES-GFP lentiviral vector carrying the AON1 and 3 sequences (TTN-AON) or with a control vector (U7snRNA-ScrAONs-IRES-GFP).


Mass spectrometry - based analysis of titin peptides in cells infected with the U7snRNA - ScrAONs - IRES - GFP ( Scr - AON ) and U7snRNA - TTNAONs - IRES - GFP ( TTN - AON ) vectors . Unsupervised hierarchical clustering identified a cluster significantly enriched in peptides mapping to exon 326 that was down - regulated in DCM Scr - AON cardiomyocytes compared to CTR Scr - AON cardiomyocytes ( n = 3 , P = 9 . 03E − 8 , Fisher ' s exact test , FDR = 0 . 04 , top ) . Down - regulation of exon 326 was also detected in DCM TTN - AON cells when compared to DCM Scr - AON cells ( n = 3 , P = 0 . 02 , Fisher ' s exact test , bottom ) .

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Mass spectrometry-based analysis of titin peptides in cells infected with the U7snRNA-ScrAONs-IRES-GFP (Scr-AON) and U7snRNA-TTNAONs-IRES-GFP (TTN-AON) vectors. Unsupervised hierarchical clustering identified a cluster significantly enriched in peptides mapping to exon 326 that was down-regulated in DCM Scr-AON cardiomyocytes compared to CTR Scr-AON cardiomyocytes (n = 3, P = 9.03E−8, Fisher's exact test, FDR = 0.04, top). Down-regulation of exon 326 was also detected in DCM TTN-AON cells when compared to DCM Scr-AON cells (n = 3, P = 0.02, Fisher's exact test, bottom).


Percentage of iPSC - derived cardiomyocytes with cross - striated myofibrils occupying a fraction ( partially organized , dark gray ) or the whole cytoplasm ( fully organized , light gray ) from two control and two patient clones . Statistical difference was tested using the two - sided chi - squared test ( CTR1 basal : n = 283 , CTR2 basal : n = 250 , DCM1 basal : n = 255 , DCM2 basal : n = 236 ; * * * P = 1 . 93E − 16 ) .

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Percentage of iPSC-derived cardiomyocytes with cross-striated myofibrils occupying a fraction (partially organized, dark gray) or the whole cytoplasm (fully organized, light gray) from two control and two patient clones. Statistical difference was tested using the two-sided chi-squared test (CTR1 basal: n = 283, CTR2 basal: n = 250, DCM1 basal: n = 255, DCM2 basal: n = 236; ***P = 1.93E−16).


Immunofluorescence images of titin ( Z - disk portion ) and cardiac troponin T ( cTNT , A - band ) in well - organized sarcomeres from CTR and DCM single cardiomyocytes under basal conditions . Scale bars , 5 μm .

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Immunofluorescence images of titin (Z-disk portion) and cardiac troponin T (cTNT, A-band) in well-organized sarcomeres from CTR and DCM single cardiomyocytes under basal conditions. Scale bars, 5 μm.


Immunofluorescence images ( left ) of α - actinin and cTNT in CTR and DCM single cardiomyocytes , illustrating 3 different levels of sarcomeric organization ( perinuclear , fully and peripherally organized ) . Percentage of cells with different levels of sarcomeric organization ( right ) under basal and stress conditions . Statistical difference was tested using the chi - squared test ( CTR1 basal : n = 283 , CTR2 basal : n = 250 , DCM1 basal : n = 255 , DCM2 basal : n = 236 , CTR1 Iso : n = 245 , CTR2 Iso : n = 230 ; DCM1 Iso : n = 242 and DCM2 Iso : n = 269 ; * * * P = 1 . 93E − 16 CTR basal versus DCM basal ; * * * P = 9 . 61E − 34 CTR Iso versus DCM Iso , * * P = 0 . 001 DCM basal versus DCM Iso ) . No significant differences were observed comparing CTR basal and CTR Iso groups . Scale bars , 25 μm .

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Immunofluorescence images (left) of α-actinin and cTNT in CTR and DCM single cardiomyocytes, illustrating 3 different levels of sarcomeric organization (perinuclear, fully and peripherally organized). Percentage of cells with different levels of sarcomeric organization (right) under basal and stress conditions. Statistical difference was tested using the chi-squared test (CTR1 basal: n = 283, CTR2 basal: n = 250, DCM1 basal: n = 255, DCM2 basal: n = 236, CTR1 Iso: n = 245, CTR2 Iso: n = 230; DCM1 Iso: n = 242 and DCM2 Iso: n = 269; ***P = 1.93E−16 CTR basal versus DCM basal; ***P = 9.61E−34 CTR Iso versus DCM Iso, **P = 0.001 DCM basal versus DCM Iso). No significant differences were observed comparing CTR basal and CTR Iso groups. Scale bars, 25 μm.


Percentage of perinuclear , fully , and peripherally organized single cardiomyocytes from two CTR and two DCM iPSC clones after infection with the U7snRNA - ScrAONs - IRES - GFP and U7snRNA - TTNAONs - IRES - GFP lentiviruses . Statistical difference was tested using the two - sided chi - squared test ( CTR1 Scr - AON : n = 190 , CTR2 Scr - AON : n = 200 , DCM1 Scr - AON : n = 221 , DCM2 Scr - AON : n = 115 , CTR1 TTN - AON : n = 223 , CTR2 TTN - AON : n = 187 , DCM1 TTN - AON : n = 171 , DCM2 TTN - AON : n = 243 ; * * * P = 4 . 22E − 15 CTR Scr - AON versus DCM Scr - AON ; * * * P = 4 . 61E − 02 DCM Scr - AON versus DCM TTN - AON ) . No significant differences were observed comparing CTR Scr - AON and CTR TTN - AON groups .

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Percentage of perinuclear, fully, and peripherally organized single cardiomyocytes from two CTR and two DCM iPSC clones after infection with the U7snRNA-ScrAONs-IRES-GFP and U7snRNA-TTNAONs-IRES-GFP lentiviruses. Statistical difference was tested using the two-sided chi-squared test (CTR1 Scr-AON: n = 190, CTR2 Scr-AON: n = 200, DCM1 Scr-AON: n = 221, DCM2 Scr-AON: n = 115, CTR1 TTN-AON: n = 223, CTR2 TTN-AON: n = 187, DCM1 TTN-AON: n = 171, DCM2 TTN-AON: n = 243; ***P = 4.22E−15 CTR Scr-AON versus DCM Scr-AON; ***P = 4.61E−02 DCM Scr-AON versus DCM TTN-AON). No significant differences were observed comparing CTR Scr-AON and CTR TTN-AON groups.


qRT - PCR analysis of SRF target genes ( MYH6 , MYH7 and ACTC1 ) in CTR and DCM single cardiomyocytes under basal condition ( no infection , NI ) and after infection with control U7snRNA - ScrAONs - IRES - GFP ( Scr - AON ) and the U7snRNA - TTNAONs - IRES - GFP ( TTN - AON ) lentiviruses . Statistical difference was tested using the two - sided Student ' s t - test ( * * P = 0 . 009 , CTR Scr - AON versus DCM Scr - AON ; * P = 0 . 04 , DCM Scr - AON versus DCM TTN - AON for MYH6 ; * * P = 0 . 002 , CTR Scr - AON versus DCM Scr - AON ; * * P = 0 . 002 , DCM Scr - AON versus DCM TTN - AON for MYH7 ; * * P = 0 . 004 , CTR Scr - AON versus DCM Scr - AON ; * P = 0 . 02 , DCM Scr - AON versus DCM TTN - AON for ACTC1 ) . No significant differences were observed comparing the CTR NI , CTR Scr - AON and CTR TTN - AON groups and comparing the DCM NI and DCM Scr - AON groups . Expression values were relative to CTR Scr - AON , normalized to GAPDH , and presented as mean ± SEM , n = 3 .

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qRT-PCR analysis of SRF target genes (MYH6, MYH7 and ACTC1) in CTR and DCM single cardiomyocytes under basal condition (no infection, NI) and after infection with control U7snRNA-ScrAONs-IRES-GFP (Scr-AON) and the U7snRNA-TTNAONs-IRES-GFP (TTN-AON) lentiviruses. Statistical difference was tested using the two-sided Student's t-test (**P = 0.009, CTR Scr-AON versus DCM Scr-AON; *P = 0.04, DCM Scr-AON versus DCM TTN-AON for MYH6; **P = 0.002, CTR Scr-AON versus DCM Scr-AON; **P = 0.002, DCM Scr-AON versus DCM TTN-AON for MYH7; **P = 0.004, CTR Scr-AON versus DCM Scr-AON; *P = 0.02, DCM Scr-AON versus DCM TTN-AON for ACTC1). No significant differences were observed comparing the CTR NI, CTR Scr-AON and CTR TTN-AON groups and comparing the DCM NI and DCM Scr-AON groups. Expression values were relative to CTR Scr-AON, normalized to GAPDH, and presented as mean ± SEM, n = 3.


Immunofluorescence images showing normal ( a , b , e , f , i , l , o , p ) and altered ( c , d , g , h , m , n , q , r ) intracellular distribution of SRF ( a and b , nuclear ; c and d , cytoplasmic ) , MURF2 ( e and f , sarcomeric ; g and h , nuclear ) , Nbr1 ( i and l , sarcomeric ; m and n , diffused ) , and SQSTM1 / p62 ( o and p , sarcomeric ; q and r , diffused ) in representative single cardiomyocytes ( left ) . Sarcomeres are marked by α - actinin . On the right , percentage of CTR and DCM cardiomyocytes showing cytoplasmic expression of SRF , nuclear accumulation of MURF2 , and diffused expression of Nbr1 and of SQSTM1 / p62 after infection with control U7snRNA - ScrambleAONs - IRES - GFP ( Scr - AON ) and the U7snRNA - TTNAONs - IRES - GFP ( TTN - AON ) lentiviruses ( right ) . Data represent mean values ± SEM from two control and two DCM clones . Statistical difference was tested using the two - sided chi - squared test ( CTR Scr - AON : n = 874 , n = 874 , n = 882 and n = 890 , CTR TTN - AON : n = 880 , n = 990 , n = 878 and n = 890 , DCM Scr - AON : n = 890 , n = 887 , n = 884 and n = 886 , DCM TTN - AON : n = 900 , n = 875 , n = 899 and n = 891 for SRF , MURF2 , Nbr1 and SQSTM1 / p62 , respectively ; * P = 0 . 01 , CTR Scr - AON versus DCM Scr - AON ; * P = 0 . 04 , DCM Scr - AON versus DCM TTN - AON for SRF ; * P = 0 . 02 , CTR Scr - AON versus DCM Scr - AON ; * P = 0 . 04 , DCM Scr - AON versus DCM TTN - AON for MURF2 ; * P = 0 . 03 , CTR Scr - AON versus DCM Scr - AON ; * P = 0 . 03 , DCM Scr - AON versus DCM TTN - AON for Nbr1 ; * * P = 0 . 009 , CTR Scr - AON versus DCM Scr - AON ; * P = 0 . 01 , DCM Scr - AON versus DCM TTN - AON for SQSTM1 / p62 ) . No significant differences were observed comparing CTR Scr - AON and CTR TTN - AON groups . Scale bars , 50 μm .

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Immunofluorescence images showing normal (a, b, e, f, i, l, o, p) and altered (c, d, g, h, m, n, q, r) intracellular distribution of SRF (a and b, nuclear; c and d, cytoplasmic), MURF2 (e and f, sarcomeric; g and h, nuclear), Nbr1 (i and l, sarcomeric; m and n, diffused), and SQSTM1/p62 (o and p, sarcomeric; q and r, diffused) in representative single cardiomyocytes (left). Sarcomeres are marked by α-actinin. On the right, percentage of CTR and DCM cardiomyocytes showing cytoplasmic expression of SRF, nuclear accumulation of MURF2, and diffused expression of Nbr1 and of SQSTM1/p62 after infection with control U7snRNA-ScrambleAONs-IRES-GFP (Scr-AON) and the U7snRNA-TTNAONs-IRES-GFP (TTN-AON) lentiviruses (right). Data represent mean values ± SEM from two control and two DCM clones. Statistical difference was tested using the two-sided chi-squared test (CTR Scr-AON: n = 874, n = 874, n = 882 and n = 890, CTR TTN-AON: n = 880, n = 990, n = 878 and n = 890, DCM Scr-AON: n = 890, n = 887, n = 884 and n = 886, DCM TTN-AON: n = 900, n = 875, n = 899 and n = 891 for SRF, MURF2, Nbr1 and SQSTM1/p62, respectively; *P = 0.01, CTR Scr-AON versus DCM Scr-AON; *P = 0.04, DCM Scr-AON versus DCM TTN-AON for SRF; *P = 0.02, CTR Scr-AON versus DCM Scr-AON; *P = 0.04, DCM Scr-AON versus DCM TTN-AON for MURF2; *P = 0.03, CTR Scr-AON versus DCM Scr-AON; *P = 0.03, DCM Scr-AON versus DCM TTN-AON for Nbr1; **P = 0.009, CTR Scr-AON versus DCM Scr-AON; *P = 0.01, DCM Scr-AON versus DCM TTN-AON for SQSTM1/p62). No significant differences were observed comparing CTR Scr-AON and CTR TTN-AON groups. Scale bars, 50 μm.


B , C Immunofluorescence images of sarcomeric α - actinin and myosin ( B ) and assessment of cardiac function ( C ) in homozygous Ttn knock - in embryos cultured for 24 h after mScrAON and mTtnAON transient transfection ( n = 3 per group ) or lentivirus delivery ( n = 7 per group ) . Scale bars , 10 μm .

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B, C Immunofluorescence images of sarcomeric α-actinin and myosin (B) and assessment of cardiac function (C) in homozygous Ttn knock-in embryos cultured for 24 h after mScrAON and mTtnAON transient transfection (n = 3 per group) or lentivirus delivery (n = 7 per group). Scale bars, 10 μm.


D Electron microscopy analysis in Ttn knock - in embryos cultured for 24 h after mScrAON and mTtnAON transient transfection . mTtnAON treatment of homozygous embryos rescued myofibril formation ( left , black arrows ) , resulting in thicker filaments ( right ) .

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D Electron microscopy analysis in Ttn knock-in embryos cultured for 24 h after mScrAON and mTtnAON transient transfection. mTtnAON treatment of homozygous embryos rescued myofibril formation (left, black arrows), resulting in thicker filaments (right).


Echocardiographic analysis of adult knock - in mice . Statistical difference was tested using the two - sided Student ' s t - test ( WT : n = 6 , HET : n = 6 ; HET + vPMO - mScrAON : n = 10 and HET + vPMO - mTtnAON : n = 14 ; * P = 0 . 013 , HET versus HET + vPMO - mTtnAON and * P = 0 . 02 , HET + vPMO - mScrAON versus HET + vPMO - mTtnAON for LVEDD differences ; * * P = 0 . 006 , HET versus HET + vPMO - mTtnAON and * P = 0 . 02 , HET + vPMO - mScrAON versus HET + vPMO - mTtnAON for LV - EF differences ; * P = 0 . 03 , HET versus HET + vPMO - mTtnAON and * P = 0 . 04 , HET + vPMO - mScrAON versus HET + vPMO - mTtnAON for IVSd differences ; * * P = 0 . 001 , HET versus HET + vPMO - mTtnAON and * P = 0 . 03 , HET + vPMO - mScrAON versus HET + vPMO - mTtnAON and for PWd differences ) . No significant differences were observed among groups at day 0 and day 7 . Data represent mean values ± SEM .

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Echocardiographic analysis of adult knock-in mice. Statistical difference was tested using the two-sided Student's t-test (WT: n = 6, HET: n = 6; HET + vPMO-mScrAON: n = 10 and HET + vPMO-mTtnAON: n = 14; *P = 0.013, HET versus HET + vPMO-mTtnAON and *P = 0.02, HET + vPMO-mScrAON versus HET + vPMO-mTtnAON for LVEDD differences; **P = 0.006, HET versus HET + vPMO-mTtnAON and *P = 0.02, HET + vPMO-mScrAON versus HET + vPMO-mTtnAON for LV-EF differences; *P = 0.03, HET versus HET + vPMO-mTtnAON and *P = 0.04, HET + vPMO-mScrAON versus HET + vPMO-mTtnAON for IVSd differences; **P = 0.001, HET versus HET + vPMO-mTtnAON and *P = 0.03, HET + vPMO-mScrAON versus HET + vPMO-mTtnAON and for PWd differences). No significant differences were observed among groups at day 0 and day 7. Data represent mean values ± SEM.


Masson ' s trichrome staining of heart sections from HET animals injected with vPMO - mScrAONs and vPMO - mTtnAON . Scale bars , 1 mm .

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Masson's trichrome staining of heart sections from HET animals injected with vPMO-mScrAONs and vPMO-mTtnAON. Scale bars, 1 mm.


Fluorescence in situ hybridization ( FISH ) of heart muscle tissue from adult knock - in mice with a probe complementary to vPMO - mTtnAON . Scale bars , 250 and 50 μm ( magnification ) .

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Fluorescence in situ hybridization (FISH) of heart muscle tissue from adult knock-in mice with a probe complementary to vPMO-mTtnAON. Scale bars, 250 and 50 μm (magnification).


RT - PCR analysis of Ttn exon 326 transcripts from heart tissue of untreated WT and HET animals and vPMO - mScrAON - and vPMO - mTtnAON - treated mice ( left ) . Representative direct sequencing of Ttn exon 326 transcripts from vPMO - mTtnAON - treated HET heart tissue ( right ) .

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RT-PCR analysis of Ttn exon 326 transcripts from heart tissue of untreated WT and HET animals and vPMO-mScrAON- and vPMO-mTtnAON-treated mice (left). Representative direct sequencing of Ttn exon 326 transcripts from vPMO-mTtnAON-treated HET heart tissue (right).


Mass spectrometry - based analysis of titin peptides in adult knock - in mice injected with vPMO - mScrAONs and vPMO - mTtnAONs . Unsupervised hierarchical clustering identified a cluster enriched in exon 326 peptides that was down - regulated in vPMO - mTtnAON - treated animals compared to vPMO - mScrAON - treated littermates . Another cluster enriched in C - terminal peptides was up - regulated in the vPMO - mTtnAON group compared to the vPMO - mScrAON group ( n = 3 , P = 0 . 02 , Fisher ' s exact test , FDR = 0 . 04 ) .

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Mass spectrometry-based analysis of titin peptides in adult knock-in mice injected with vPMO-mScrAONs and vPMO-mTtnAONs. Unsupervised hierarchical clustering identified a cluster enriched in exon 326 peptides that was down-regulated in vPMO-mTtnAON-treated animals compared to vPMO-mScrAON-treated littermates. Another cluster enriched in C-terminal peptides was up-regulated in the vPMO-mTtnAON group compared to the vPMO-mScrAON group (n = 3, P = 0.02, Fisher's exact test, FDR = 0.04).


A - B ) Western - blot analysis for GAPDH and LC3 of protein lysates obtained from HeLa cells treated with DMSO or 1 µM rapamycin ( Rapa ) for the indicated time periods ( A ) ( n = 7 ) or for 5 h with the indicated concentrations ( B ) ( n = 6 ) . One hour before harvesting , 100 nM bafilomycin A1 was added . Upper panels : representative Western blots ; lower panels : quantification of the LC3 - II / GAPDH ratio .

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A-B) Western-blot analysis for GAPDH and LC3 of protein lysates obtained from HeLa cells treated with DMSO or 1 µM rapamycin (Rapa) for the indicated time periods (A) (n = 7) or for 5 h with the indicated concentrations (B) (n = 6). One hour before harvesting, 100 nM bafilomycin A1 was added. Upper panels: representative Western blots; lower panels: quantification of the LC3-II/GAPDH ratio.


C ) GFP - LC3 - punctae quantification in HeLa cells treated for 5 h with different concentrations of rapamycin . Left : representative pictures . The scale bar represents 10 µm . Concentrations are mentioned in the right lower corner . Right : Quantification of the number of punctae per cell ( n = 3 ) . * p < 0 . 05 , repeated measurements ANOVA .

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C) GFP-LC3-punctae quantification in HeLa cells treated for 5 h with different concentrations of rapamycin. Left: representative pictures. The scale bar represents 10 µm. Concentrations are mentioned in the right lower corner. Right: Quantification of the number of punctae per cell (n = 3). * p<0.05, repeated measurements ANOVA.


A ) Representative measurements ( n = 4 ) of cytosolic Ca2 + signals , displayed as Fura2 ratio ( F340 / F380 ) , showing the effect of 0 . 3 µM and 100 µM ATP , 1 µM thapsigargin ( Tg ) or 10 µM ionomycin ( Iono ) in intact HeLa cells treated with different concentrations of rapamycin ( Rapa ) for 5 h . 45 s prior to the addition of ATP , Tg or Iono , EGTA ( 3 mM ) was given to buffer extracellular Ca2 + as indicated .

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A) Representative measurements (n = 4) of cytosolic Ca2+ signals, displayed as Fura2 ratio (F340/F380), showing the effect of 0.3 µM and 100 µM ATP, 1 µM thapsigargin (Tg) or 10 µM ionomycin (Iono) in intact HeLa cells treated with different concentrations of rapamycin (Rapa) for 5 h. 45 s prior to the addition of ATP, Tg or Iono, EGTA (3 mM) was given to buffer extracellular Ca2+ as indicated.


B ) Quantification of the average amplitude of the response ( F − F0 ) ( n = 4 ) . * p < 0 . 05 ; * * p < 0 . 01 ; * * * p < 0 . 001 , repeated measurements ANOVA .

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B) Quantification of the average amplitude of the response (F−F0) (n = 4). * p<0.05; ** p<0.01; *** p<0.001, repeated measurements ANOVA.


C ) Mean resting cytosolic [ Ca2 + ] , measured in Fura2 - loaded HeLa cells treated with the indicated concentrations of rapamycin for 5 h , as well as in the absence ( Ctrl ) or presence of DMSO ( n = 3 ) . * p < 0 . 05 , repeated measurements ANOVA .

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C) Mean resting cytosolic [Ca2+], measured in Fura2-loaded HeLa cells treated with the indicated concentrations of rapamycin for 5 h, as well as in the absence (Ctrl) or presence of DMSO (n = 3). * p<0.05, repeated measurements ANOVA.


D ) Unidirectional 45Ca2 + - flux experiments in permeabilized cells pretreated with 1 µM rapamycin for 5 h or with DMSO ( Ctrl ) . Mean fractional 45Ca2 + release ( % / 2 min ) is shown as a function of time with the effect of 0 . 7 µM IP3 ( circles ) or no addition ( squares ) . The horizontal bar indicates the presence of IP3 .

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D) Unidirectional 45Ca2+-flux experiments in permeabilized cells pretreated with 1 µM rapamycin for 5 h or with DMSO (Ctrl). Mean fractional 45Ca2+ release (%/2 min) is shown as a function of time with the effect of 0.7 µM IP3 (circles) or no addition (squares). The horizontal bar indicates the presence of IP3.


E ) Quantitative analysis of the IP3 - induced 45Ca2 + release in cells pretreated for 5 h with 1 µM rapamycin or DMSO ( Ctrl ) ( n = 8 ) . * * * p < 0 . 001 , paired Student ' s t - test .

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E) Quantitative analysis of the IP3-induced 45Ca2+ release in cells pretreated for 5 h with 1 µM rapamycin or DMSO (Ctrl) (n = 8). *** p<0.001, paired Student's t-test.


A - B ) Western - blot analysis for luminal Ca2 + - binding proteins in HeLa cells treated with the indicated concentrations of rapamycin ( Rapa ) for 5 h : calreticulin ( CRT ) ( A ) and BiP / Grp78 ( BiP ) ( B ) . Upper panels : representative Western blots ; lower panels : quantification of the protein / GAPDH ratio ( n = 4 ) .

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A-B) Western-blot analysis for luminal Ca2+-binding proteins in HeLa cells treated with the indicated concentrations of rapamycin (Rapa) for 5 h: calreticulin (CRT) (A) and BiP/Grp78 (BiP) (B). Upper panels: representative Western blots; lower panels: quantification of the protein/GAPDH ratio (n = 4).


C ) Western - blot analysis for SERCA2 in HeLa cells treated with the indicated concentrations of rapamycin for 5 h . Upper panels : representative Western blots ; lower panel : quantification of the SERCA2 / GAPDH ratio ( n = 4 ) .

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C) Western-blot analysis for SERCA2 in HeLa cells treated with the indicated concentrations of rapamycin for 5 h. Upper panels: representative Western blots; lower panel: quantification of the SERCA2/GAPDH ratio (n = 4).


D ) Representative plot showing the decrease in ER 45Ca2 + content ( logarithmic scale ) in a Ca2 + - free efflux medium without ATP as a function of time in permeabilized HeLa cells pretreated for 5 h with 1 µM rapamycin or with DMSO . The passively bound Ca2 + was determined by loading the cells with 45Ca2 + in the presence of 10 µM of the Ca2 + ionophore A23187 and then subtracted from the stored 45Ca2 + . The ER Ca2 + - leak rate can be estimated as the rate of decline of the ER 45Ca2 + - store content as a function of time .

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D) Representative plot showing the decrease in ER 45Ca2+ content (logarithmic scale) in a Ca2+-free efflux medium without ATP as a function of time in permeabilized HeLa cells pretreated for 5 h with 1 µM rapamycin or with DMSO. The passively bound Ca2+ was determined by loading the cells with 45Ca2+ in the presence of 10 µM of the Ca2+ ionophore A23187 and then subtracted from the stored 45Ca2+. The ER Ca2+-leak rate can be estimated as the rate of decline of the ER 45Ca2+-store content as a function of time.


E ) Quantification of the mean 45Ca2 + - store content at the beginning of the measurement ( t0 ) ( n = 5 ) .

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E) Quantification of the mean 45Ca2+-store content at the beginning of the measurement (t0) (n = 5).


F ) Quantification of the mean slope of the curve in D after transformation to a linear scale , which is a measure of the 45Ca2 + - leak rate ( n = 5 ) . * p < 0 . 05 ; * * p < 0 . 01 , paired Student ' s t - test .

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F) Quantification of the mean slope of the curve in D after transformation to a linear scale, which is a measure of the 45Ca2+-leak rate (n = 5). * p<0.05; ** p<0.01, paired Student's t-test.


A ) Representative Western - blot analysis for Atg12 ( showing the autophagic Atg12 - Atg5 complex ) , GAPDH and LC3 of protein lysates obtained from MEF cells pretreated with ( + Dox ) or without ( - Dox ) doxycycline and treated with DMSO or 0 . 1 , 1 or 5 µM rapamycin ( Rapa ) for 5 h ( n = 3 ) .

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A) Representative Western-blot analysis for Atg12 (showing the autophagic Atg12-Atg5 complex), GAPDH and LC3 of protein lysates obtained from MEF cells pretreated with (+Dox) or without (-Dox) doxycycline and treated with DMSO or 0.1, 1 or 5 µM rapamycin (Rapa) for 5 h (n = 3).


B - C ) Representative measurements of cytosolic Ca2 + signals , displayed as Fura2 ratio ( F340 / F380 ) , showing the effect of 1 mM ATP ( B ) or 10 µM ionomycin ( Iono ) in intact MEF cells pretreated with or without doxycycline and treated with different concentrations of rapamycin for 5 h . Prior to the addition of ATP or Iono , EGTA ( 3 mM ) was added to chelate the extracellular Ca2 + as indicated .

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B-C) Representative measurements of cytosolic Ca2+ signals, displayed as Fura2 ratio (F340/F380), showing the effect of 1 mM ATP (B) or 10 µM ionomycin (Iono) in intact MEF cells pretreated with or without doxycycline and treated with different concentrations of rapamycin for 5 h. Prior to the addition of ATP or Iono, EGTA (3 mM) was added to chelate the extracellular Ca2+ as indicated.


D ) Quantification of the average amplitude of the response ( F − F0 ) ( n = 3 , 4 , 5 and 6 for ATP - Dox , ATP + Dox , Iono - Dox and Iono + Dox , resp . ) * p < 0 . 05 ; * * p < 0 . 01 ; * * * p < 0 . 001 , repeated measurements ANOVA .

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D) Quantification of the average amplitude of the response (F−F0) (n = 3, 4, 5 and 6 for ATP-Dox, ATP+Dox, Iono-Dox and Iono+Dox, resp.) * p<0.05; ** p<0.01; *** p<0.001, repeated measurements ANOVA.


2 + - dependent . Western - blot analysis for GAPDH and LC3 of protein lysates obtained from HeLa cells treated for 5 h with DMSO , 1 µM rapamycin ( Rapa ) , 10 µM BAPTA - AM or both . One hour before harvesting , 100 nM bafilomycin A1 was added . Left : representative Western blots ; right : quantification of the LC3 - II / GAPDH ratio ( n = 6 ) . * p < 0 . 05 , repeated measurements ANOVA .

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2+-dependent. Western-blot analysis for GAPDH and LC3 of protein lysates obtained from HeLa cells treated for 5 h with DMSO, 1 µM rapamycin (Rapa), 10 µM BAPTA-AM or both. One hour before harvesting, 100 nM bafilomycin A1 was added. Left: representative Western blots; right: quantification of the LC3-II/GAPDH ratio (n = 6). * p<0.05, repeated measurements ANOVA.


( b ) Temperature - sensitive growth assay . Yeast strains ( wild - type or deletion mutants , indicated on the left ) were transformed with either an empty vector or a plasmid carrying an artificial guide RNA gene targeting U2 pseudouridylation at position 42 and 44 ( indicated on the top ) , and grown at various temperatures ( indicated at the bottom ) .

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(b) Temperature-sensitive growth assay. Yeast strains (wild-type or deletion mutants, indicated on the left) were transformed with either an empty vector or a plasmid carrying an artificial guide RNA gene targeting U2 pseudouridylation at position 42 and 44 (indicated on the top), and grown at various temperatures (indicated at the bottom).


( c ) Blockage of pseudouridylation by deletion of specific pseudouridylase genes . RNAs isolated from wild - type and pseudouridylase - deletion strains ( indicated on the top ) were used for U2 pseudouridylation assay ( CMC modification followed by primer - extension ) . Primer - extension pauses / stops correspond to ψ sites ( indicated on the left and right ) . Note : The above - background signal of ψ42 in the snr81Δ lane ( and to some extent , in the snr81Δ pus7Δ lane ) is likely caused by the presence of strong ψ44 band . This is a primer - extension artifact , which often occurs when there is a strong ψ signal in the neighboring position . Alternatively , it is possible that having ψ at position 44 allows for an unidentified enzyme other than snR81 to modify U42 ( albeit inefficiently ) ( Perhaps Pus1 , which is responsible for ψ44 formation , can be a bit " processive " ) . Lanes 1 - 8 and lanes 9 - 16 are from two separate gels .

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(c) Blockage of pseudouridylation by deletion of specific pseudouridylase genes. RNAs isolated from wild-type and pseudouridylase-deletion strains (indicated on the top) were used for U2 pseudouridylation assay (CMC modification followed by primer-extension). Primer-extension pauses/stops correspond to ψ sites (indicated on the left and right). Note: The above-background signal of ψ42 in the snr81Δ lane (and to some extent, in the snr81Δ pus7Δ lane) is likely caused by the presence of strong ψ44 band. This is a primer-extension artifact, which often occurs when there is a strong ψ signal in the neighboring position. Alternatively, it is possible that having ψ at position 44 allows for an unidentified enzyme other than snR81 to modify U42 (albeit inefficiently) (Perhaps Pus1, which is responsible for ψ44 formation, can be a bit "processive"). Lanes 1-8 and lanes 9-16 are from two separate gels.


( d ) Restoration of U2 pseudouridylation by an artificial box H / ACA RNA . RNAs isolated from various yeast strains ( indicated on the top ) , including the snr81Δ pusΔ strain that was transformed with an artificial guide RNA targeting positions 42 and 44 ( lanes 9 and 10 ) , were assayed for pseudouridylation [ see legend to ( c ) ] .

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(d) Restoration of U2 pseudouridylation by an artificial box H/ACA RNA. RNAs isolated from various yeast strains (indicated on the top), including the snr81Δ pusΔ strain that was transformed with an artificial guide RNA targeting positions 42 and 44 (lanes 9 and 10), were assayed for pseudouridylation [see legend to (c)].


( a ) Growth assay using the ACT1 - CUP1 reporter system . Yeast cells carrying the ACT1 - CUP1 reporter were deleted of pseudouridylase genes ( indicated on the left ) , and were then assayed for growth at 30 ° C on media containing various concentrations of [ Cu2 + ] ( indicated at the bottom ) .

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(a) Growth assay using the ACT1-CUP1 reporter system. Yeast cells carrying the ACT1-CUP1 reporter were deleted of pseudouridylase genes (indicated on the left), and were then assayed for growth at 30°C on media containing various concentrations of [Cu2+] (indicated at the bottom).


( b ) Growth rescue by restoration of pseudouridylation . The wild - type strain [ from ( a ) ] or the snr81Δ pusΔ strain [ also from ( a ) ] , which exhibited the most severe growth - deficiency phenotype , was transformed with an empty vector or a plasmid carrying a pseudouridylation guide RNA gene targeting either position 42 , position 44 , or both ( indicated ) . The resulting cells were assayed for growth at 30 ° C on media containing various concentrations of [ Cu2 + ] ( indicated at the bottom ) .

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(b) Growth rescue by restoration of pseudouridylation. The wild-type strain [from (a)] or the snr81Δ pusΔ strain [also from (a)], which exhibited the most severe growth-deficiency phenotype, was transformed with an empty vector or a plasmid carrying a pseudouridylation guide RNA gene targeting either position 42, position 44, or both (indicated). The resulting cells were assayed for growth at 30°C on media containing various concentrations of [Cu2+] (indicated at the bottom).


( d ) Splicing assay using the wild - type and mutant ACT1 - CUP1 reporters . RNAs isolated from the wild - type and the snr81Δ pusΔ strains ( indicated on the top ) , which carry the wild - type ACT1 - CUP1 reporter pre - mRNA or any of the mutant ACT1 - CUP1 reporter pre - mRNAs [ indicated , also see ( c ) ] , were used for splicing assay ( primer - extension analysis ) . The un - spliced pre - mRNA , lariat intermediate , and spliced mRNA are indicated . A primer complementary to U6 was also used ( as an internal control ) in the assay , and the U6 band is indicated as well . In addition , the growth phenotype of each strain ( in various concentrations of [ Cu2 + ] ) is also shown . Lanes 1 - 12 , lanes 13 and 14 , and lanes 15 and 16 are from separate gels .

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(d) Splicing assay using the wild-type and mutant ACT1-CUP1 reporters. RNAs isolated from the wild-type and the snr81Δ pusΔ strains (indicated on the top), which carry the wild-type ACT1-CUP1 reporter pre-mRNA or any of the mutant ACT1-CUP1 reporter pre-mRNAs [indicated, also see (c)], were used for splicing assay (primer-extension analysis). The un-spliced pre-mRNA, lariat intermediate, and spliced mRNA are indicated. A primer complementary to U6 was also used (as an internal control) in the assay, and the U6 band is indicated as well. In addition, the growth phenotype of each strain (in various concentrations of [Cu2+]) is also shown. Lanes 1-12, lanes 13 and 14, and lanes 15 and 16 are from separate gels.


( e ) Quantification of ACT1 - CUP1 mRNA levels . Spliced ACT1 - CUP1 mRNA levels were calculated relative to U6 in each lane . The quantification was based on three independent experiments .

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(e) Quantification of ACT1-CUP1 mRNA levels. Spliced ACT1-CUP1 mRNA levels were calculated relative to U6 in each lane. The quantification was based on three independent experiments.


( f ) Relative improvement / reduction for the mutant reporters . Relative improvement or reduction in splicing was calculated by normalizing the splicing efficiency ( mRNA / U6 ) of each mutant reporter to that of WT reporter ( set at 1 ) .

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(f) Relative improvement/reduction for the mutant reporters. Relative improvement or reduction in splicing was calculated by normalizing the splicing efficiency (mRNA/U6) of each mutant reporter to that of WT reporter (set at 1).


Figure 3 . Synthetic lethality assay . The Prp5 domain structure , and the mutations in some of the domains ( indicated ) are schematized at the top . Each of these PRP5 genes ( wild - type or mutants , plasmid - borne ) was used to replace the chromosomal PRP5 . The numbers in parentheses are the relative ATPase activities of the wild - type ( SAT ) Prp5 and some of the Prp5 mutants tested before ( Xu Query , 2007 ) . The U2 status , either containing Ψ42 and Ψ44 ( WT - U2 and snR81Δ pus1Δ with pΨ42 or Ψ44 ) or lacking Ψ42 and Ψ44 ( snR81Δ pus1Δ ) , is indicated ( on the right ) . Upon transformation with the plasmids carrying PRP5 genes ( wild - type or any of the mutants ) , cells were grown on the regular medium ( - FOA ) or on medium containing 5 - FOA ( + FOA ) .

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Figure 3. Synthetic lethality assay. The Prp5 domain structure, and the mutations in some of the domains (indicated) are schematized at the top. Each of these PRP5 genes (wild-type or mutants, plasmid-borne) was used to replace the chromosomal PRP5. The numbers in parentheses are the relative ATPase activities of the wild-type (SAT) Prp5 and some of the Prp5 mutants tested before (Xu Query, 2007). The U2 status, either containing Ψ42 and Ψ44 (WT-U2 and snR81Δ pus1Δ with pΨ42 or Ψ44) or lacking Ψ42 and Ψ44 (snR81Δ pus1Δ), is indicated (on the right). Upon transformation with the plasmids carrying PRP5 genes (wild-type or any of the mutants), cells were grown on the regular medium (-FOA) or on medium containing 5-FOA (+FOA).


( a ) Prp5 - U2 co - immunoprecipitation ( IP ) . Wild - type ( lanes 1 and 2 ) and snR81Δ pus1Δ ( lane 3 ) strains , each containing a FLAG - tagged PRP5 gene ( indicated on the top ) , were used for anti - FLAG IP in the absence ( lane 1 ) or presence ( lanes 2 and 3 ) of ATP . The precipitated Prp5 ( WT Prp5 - FLAG ) as well as co - precipitated U1 and U2 , detected by primer - extension , are indicated .

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(a) Prp5-U2 co-immunoprecipitation (IP). Wild-type (lanes 1 and 2) and snR81Δ pus1Δ (lane 3) strains, each containing a FLAG-tagged PRP5 gene (indicated on the top), were used for anti-FLAG IP in the absence (lane 1) or presence (lanes 2 and 3) of ATP. The precipitated Prp5 (WT Prp5-FLAG) as well as co-precipitated U1 and U2, detected by primer-extension, are indicated.


( b ) Quantification of co - precipitated U2 snRNA . For each lane [ the WT lane and the snr81Δ pus1Δ lane , corresponding to lane 2 and lane 3 of ( a ) , respectively ] , Prp5 - co - precipitated U2 was normalized against co - precipitated U1 [ ( intensity of the U2 band ) / ( intensity of the U1 band ) ] . The snr81Δ pus1Δ lane was then further normalized against the WT lane [ ( the value of normalized U2 from the snr81Δ pus1Δ lane ) / ( the value of normalized U2 from the WT lane ) ] . Quantification was based on three independent experiments .

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(b) Quantification of co-precipitated U2 snRNA. For each lane [the WT lane and the snr81Δ pus1Δ lane, corresponding to lane 2 and lane 3 of (a), respectively], Prp5-co-precipitated U2 was normalized against co-precipitated U1 [(intensity of the U2 band) / (intensity of the U1 band)]. The snr81Δ pus1Δ lane was then further normalized against the WT lane [(the value of normalized U2 from the snr81Δ pus1Δ lane) / (the value of normalized U2 from the WT lane)]. Quantification was based on three independent experiments.


( c ) U2 expression level in wild - type and snR81Δ pus1Δ strains . Total RNA , isolated from the wild - type ( lane 1 ) or snR81Δ pus1Δ ( lane 2 ) strains , was used for primer - extension to measure the levels of U2 and U6 . The U2 and U6 bands are indicated .

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(c) U2 expression level in wild-type and snR81Δ pus1Δ strains. Total RNA, isolated from the wild-type (lane 1) or snR81Δ pus1Δ (lane 2) strains, was used for primer-extension to measure the levels of U2 and U6. The U2 and U6 bands are indicated.


d ) Depletion of Prp5 . Wild - type ( lanes 1 , 3 and 4 ) and snR81Δ pus1Δ ( lanes 2 and 5 ) cells containing a FLAG - tagged PRP5 were lysed , and anti - FLAG IP was performed at a high concentration of salt . Cell extracts , before ( lanes 1 and 2 ) , and after [ lanes 3 ( mock ) , 4 and 5 ] anti - FLAG IP , were used for western analysis using anti - FLAG . The Prp5 band is indicated .

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d) Depletion of Prp5. Wild-type (lanes 1, 3 and 4) and snR81Δ pus1Δ (lanes 2 and 5) cells containing a FLAG-tagged PRP5 were lysed, and anti-FLAG IP was performed at a high concentration of salt. Cell extracts, before (lanes 1 and 2), and after [lanes 3 (mock), 4 and 5] anti-FLAG IP, were used for western analysis using anti-FLAG. The Prp5 band is indicated.


( e ) IP of reconstituted Prp5 ( GAR ) - U2 . An equal amount of FLAG - tagged Prp5 ( GAR ) was added to the wild - type ( lane 1 ) and snR81Δ pus1Δ ( lane 2 ) cell extracts depleted of endogenous Prp5 [ see ( c ) ] . After the salt concentration was brought back to its original level , anti - FLAG IP was carried out . The Prp5 ( GAR ) band , detected by western , is indicated . The co - precipitated U1 and U2 bands , detected by primer - extension , are also indicated .

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(e) IP of reconstituted Prp5 (GAR)-U2. An equal amount of FLAG-tagged Prp5 (GAR) was added to the wild-type (lane 1) and snR81Δ pus1Δ (lane 2) cell extracts depleted of endogenous Prp5 [see (c)]. After the salt concentration was brought back to its original level, anti-FLAG IP was carried out. The Prp5 (GAR) band, detected by western, is indicated. The co-precipitated U1 and U2 bands, detected by primer-extension, are also indicated.


f ) Quantification of U2 that is co - precipitated with mutant Prp5 ( GAR ) . For each lane [ including the WT lane and the snr81Δ pus1Δ lane , corresponding to lane 1 and lane 2 of ( e ) , respectively ] , U2 co - precipitated with Prp5 ( GAR ) was normalized against co - precipitated U1 [ ( intensity of the U2 band ) / ( intensity of the U1 band ) ] . The snr81Δ pus1Δ lane was then further normalized against the WT lane [ ( the value of normalized U2 from the snr81Δ pus1Δ lane ) / ( the value of normalized U2 from the WT lane ) ] . Quantification was based on three independent experiments .

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f) Quantification of U2 that is co-precipitated with mutant Prp5 (GAR). For each lane [including the WT lane and the snr81Δ pus1Δ lane, corresponding to lane 1 and lane 2 of (e), respectively], U2 co-precipitated with Prp5 (GAR) was normalized against co-precipitated U1 [(intensity of the U2 band) / (intensity of the U1 band)]. The snr81Δ pus1Δ lane was then further normalized against the WT lane [(the value of normalized U2 from the snr81Δ pus1Δ lane) / (the value of normalized U2 from the WT lane)]. Quantification was based on three independent experiments.


( g ) Prp5 - U2 - pre - mRNA co - IP . Reconstitution was carried out as described in ( d ) . Prior to IP , an equal amount of radiolabeled pre - mRNA was also added to each reaction . Anti - FLAG IP was then performed in the presence of ATP . The precipitated Prp5 ( WT and GAR ) band , detected by western , is indicated . The co - precipitated pre - mRNA , directly visualized after electrophoresis , is also indicated . The pre - mRNA input is also shown . In lanes 1 and 2 , wild - type and snR81Δ pus1Δ cell extracts , depleted of Prp5 , were reconstituted with the wild - type FLAG - Prp5 . In lanes 3 and 4 , wild - type and snR81Δ pus1Δ cell extracts , depleted of Prp5 , were reconstituted with mutant FLAG - Prp5 ( GAR ) .

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(g) Prp5-U2-pre-mRNA co-IP. Reconstitution was carried out as described in (d). Prior to IP, an equal amount of radiolabeled pre-mRNA was also added to each reaction. Anti-FLAG IP was then performed in the presence of ATP. The precipitated Prp5 (WT and GAR) band, detected by western, is indicated. The co-precipitated pre-mRNA, directly visualized after electrophoresis, is also indicated. The pre-mRNA input is also shown. In lanes 1 and 2, wild-type and snR81Δ pus1Δ cell extracts, depleted of Prp5, were reconstituted with the wild-type FLAG-Prp5. In lanes 3 and 4, wild-type and snR81Δ pus1Δ cell extracts, depleted of Prp5, were reconstituted with mutant FLAG-Prp5 (GAR).


( h ) Quantification of pre - mRNA co - precipitated with Prp5 and U2 . For each lane in ( g ) , pre - mRNA co - precipitated with Prp5 ( Wild - type or GAR ) was normalized against the Prp5 ( WT / GAR ) band in the same lane ( detected by Anti - FLAG ) [ ( intensity of pre - mRNA ) / ( intensity of WT / GAR Prp5 ) ] . Lanes 2 , 3 , and 4 ( see ( g ) ) were then further normalized against lane 1 ( see ( g ) ) [ ( the value of normalized pre - mRNA from lane 2 , 3 or 4 in ( g ) ) / ( the value of normalized pre - mRNA from lane 1 in ( g ) ) ] . The numbers at the bottom of ( h ) correspond to the lane numbers of ( g ) . Quantification was based on three independent experiments .

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(h) Quantification of pre-mRNA co-precipitated with Prp5 and U2. For each lane in (g), pre-mRNA co-precipitated with Prp5 (Wild-type or GAR) was normalized against the Prp5 (WT/GAR) band in the same lane (detected by Anti-FLAG) [(intensity of pre-mRNA) / (intensity of WT/GAR Prp5)]. Lanes 2,3,and 4 (see (g)) were then further normalized against lane 1 (see (g)) [(the value of normalized pre-mRNA from lane 2, 3 or 4 in (g)) / (the value of normalized pre-mRNA from lane 1 in (g))]. The numbers at the bottom of (h) correspond to the lane numbers of (g). Quantification was based on three independent experiments.


( a ) Prp5 ' s ATPase activity assay using synthetic U2 . The 5 ' sequence of S . cerevisiae U2 is shown ( top ) . The three ψs ( 35 , 42 and 44 ) as well as the branch site recognition sequence ( underlined ) are indicated . In the ATPase assay ( bottom ) , no RNA ( lane 3 ) , or an equal amount of LiCl - precipitated RNA ( lane 1 ) or tRNA ( lane 2 ) , a short U2 fragment ( nts 1 - 76 ) ( lane 4 ) , a fully pseudouridylated short U2 fragment ( nts 1 - 76 ) ( lane 5 ) , a long U2 fragment ( nts 1 - 120 ) ( lane 6 ) , or a fully pseudouridylated long U2 fragment ( nts 1 - 120 ) ( lane 7 ) , was used . The un - hydrolyzed ATP and hydrolyzed product ADP are indicated . Lanes 1 and 2 , lanes 3 - 5 , and lanes 6 and 7 are from separate gels .

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

(a) Prp5's ATPase activity assay using synthetic U2. The 5' sequence of S. cerevisiae U2 is shown (top). The three ψs (35, 42 and 44) as well as the branch site recognition sequence (underlined) are indicated. In the ATPase assay (bottom), no RNA (lane 3), or an equal amount of LiCl-precipitated RNA (lane 1) or tRNA (lane 2), a short U2 fragment (nts 1-76) (lane 4), a fully pseudouridylated short U2 fragment (nts 1-76) (lane 5), a long U2 fragment (nts 1-120) (lane 6), or a fully pseudouridylated long U2 fragment (nts 1-120) (lane 7), was used. The un-hydrolyzed ATP and hydrolyzed product ADP are indicated. Lanes 1 and 2, lanes 3-5, and lanes 6 and 7 are from separate gels.


( b ) Quantification of Prp5 ' s ATPase activity activated by in vitro transcribed U2 . Prp5 ' s ATPase activity shown in ( a ) was calculated using the formula ( ADP ) / ( ADT + ATP ) . The numbers at the bottom of ( b ) correspond to the lane numbers of ( a ) . Quantification was based on three independent experiments .

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

(b) Quantification of Prp5's ATPase activity activated by in vitro transcribed U2. Prp5's ATPase activity shown in (a) was calculated using the formula (ADP) / (ADT+ATP). The numbers at the bottom of (b) correspond to the lane numbers of (a). Quantification was based on three independent experiments.