Figure 1 ( B ) Chlamydomonas reinhardtii ( Cr ) UBA5 and UFC1 are active E1 and E2 enzymes . SDS - PAGE analysis showing transfer of UFM1 to CrUBA5 and CrUFC1 . The gels are run in non - reducing conditions except where otherwise specified . The presented gel is representative of two independent experiments . BME : β - mercaptoethanol ; ATP : Adenosine triphosphate . ( C ) RPL26 mono - and di - UFMylation is lost in Chlamydomonas reinhardtii ( Cr ) uba5 and ufl1 mutants . Liquid TAP cultures were either left untreated ( control ) or treated for 24 hours with 200 ng / mL tunicamycin . Protein extracts were analyzed by immunoblotting with anti - UFM1 antibodies . Total proteins were analyzed by Ponceau S staining . 12 hours and 24 hours treatment replicates are shown in Fig . S3C . Right Panel , Quantification of UFMylated RPL26 . RPL26 mono - UFMylated ; RPL26 - ( UFM1 ) 2 : RPL26 di - UFMylated . ( D ) Chlamydomonas reinhardtii ( Cr ) UFMylation pathway mutants are sensitive to ER stress triggered by tunicamycin . Liquid TAP cultures of wild type ( wt ) , uba5 , ufl1 and ire1 mutants were either left untreated ( control ) or treated for 3 days with 200 ng / mL of tunicamycin . Left panel , representative images of control and treated liquid cultures taken 3 - days after incubation . Middle Panel , optical density ( OD ) 600 ( OD600 ) quantification of each genetic background under control conditions . Bars represent the mean ( ± SD ) of 5 biological replicates . Two - tailed unpaired t - tests were performed to analyze the differences between wild type and mutants . Right Panel , normalized OD600 quantification of each genetic background under tunicamycin treatment conditions . Bars represent the mean ( ± SD ) of 5 biological replicates . Two - tailed unpaired t - tests were performed to analyze the differences between wild type and mutants . ns , p - value > 0 . 05 ; * * * , p - value < 0 . 001 . BR : Biological Replicate .

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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 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 1(B) Chlamydomonas reinhardtii (Cr) UBA5 and UFC1 are active E1 and E2 enzymes. SDS-PAGE analysis showing transfer of UFM1 to CrUBA5 and CrUFC1. The gels are run in non-reducing conditions except where otherwise specified. The presented gel is representative of two independent experiments. BME: β-mercaptoethanol; ATP: Adenosine triphosphate.(C) RPL26 mono- and di-UFMylation is lost in Chlamydomonas reinhardtii (Cr) uba5 and ufl1 mutants. Liquid TAP cultures were either left untreated (control) or treated for 24 hours with 200 ng/mL tunicamycin. Protein extracts were analyzed by immunoblotting with anti-UFM1 antibodies. Total proteins were analyzed by Ponceau S staining. 12 hours and 24 hours treatment replicates are shown in Fig. S3C. Right Panel, Quantification of UFMylated RPL26. RPL26 mono-UFMylated; RPL26-(UFM1)2: RPL26 di-UFMylated.(D) Chlamydomonas reinhardtii (Cr) UFMylation pathway mutants are sensitive to ER stress triggered by tunicamycin. Liquid TAP cultures of wild type (wt), uba5, ufl1 and ire1 mutants were either left untreated (control) or treated for 3 days with 200 ng/mL of tunicamycin. Left panel, representative images of control and treated liquid cultures taken 3-days after incubation. Middle Panel, optical density (OD) 600 (OD600) quantification of each genetic background under control conditions. Bars represent the mean (± SD) of 5 biological replicates. Two-tailed unpaired t-tests were performed to analyze the differences between wild type and mutants. Right Panel, normalized OD600 quantification of each genetic background under tunicamycin treatment conditions. Bars represent the mean (± SD) of 5 biological replicates. Two-tailed unpaired t-tests were performed to analyze the differences between wild type and mutants. ns, p-value > 0.05; ***, p-value < 0.001. BR: Biological Replicate.


Figure 2 ( F , G ) C53 IDR is essential for UFM1 interaction . HsC53 ( B ) and AtC53 ( C ) IDRs are necessary to mediate the interaction with AtUFM1 and HsUFM1 respectively . MBP - AtC53ΔIDR : MBP - AtC53 ( 1 - 239 , ( KGSGSTSGSG ) 2 , 373 - 549 ) ; MBP - HsC53ΔIDR : HsC53 ( 1 - 262 , ( KGSGSTSGSG ) , 317 - 506 ) . F , G ) Bacterial lysates containing recombinant protein were mixed and pulled down with glutathione magnetic agarose beads . Input and bound proteins were visualized by immunoblotting with anti - GST and anti - MBP antibodies . ( H ) AtC53sAIM cannot interact with AtUFM1 . Individual or combinatorial mutations in sAIM1 ( 1A : W276A ) , sAIM2 ( 2A : W287A ) and sAIM3 ( 3A : W335A ) suggest sAIM1 is crucial for UFM1 interaction .

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 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

Figure 2(F, G) C53 IDR is essential for UFM1 interaction. HsC53 (B) and AtC53 (C) IDRs are necessary to mediate the interaction with AtUFM1 and HsUFM1 respectively. MBP-AtC53ΔIDR: MBP-AtC53(1-239, (KGSGSTSGSG)2, 373-549); MBP-HsC53ΔIDR: HsC53(1-262, (KGSGSTSGSG), 317-506). F, G) Bacterial lysates containing recombinant protein were mixed and pulled down with glutathione magnetic agarose beads. Input and bound proteins were visualized by immunoblotting with anti-GST and anti-MBP antibodies.(H) AtC53sAIM cannot interact with AtUFM1. Individual or combinatorial mutations in sAIM1 (1A: W276A), sAIM2 (2A: W287A) and sAIM3 (3A: W335A) suggest sAIM1 is crucial for UFM1 interaction.


Figure 3 ( B ) The cAIM peptide cannot outcompete HsUFM1 - HsC53 interaction . Bacterial lysates containing recombinant protein were mixed and pulled down with glutathione magnetic agarose beads . Input and bound proteins were visualized by immunoblotting with anti - GST and anti - MBP antibodies . cAIM peptide was used to a final concentration of 200 µM . HsC53sAIM : HsC53W269A , W294A , W312A . ( C , D ) Microscopy - based protein - protein interaction assays showing unlike GABARAP - C53 interaction , UFM1 - C53 interaction is insensitive to cAIM peptide competition . Glutathione - sepharose beads were prepared by incubating them with GST - GABARAP ( C ) or GST - HsUFM1 ( D ) . The pre - assembled beads were then washed and mixed with 1 µM of HsC53 containing increasing concentrations of cAIM peptide ( 0 - 100 µM ) . The beads were then imaged using a confocal microscope . Left Panel , representative confocal images ( inverted grayscale ) for each condition are shown . Right panel , normalized fluorescence is shown for each condition with the mean ( ± SD ) of 4 replicates . Unpaired two - samples Wilcoxon test with continuity correction was performed to analyze the differences between wild type and wild type with 100 µM AIM peptide . ns , not significant , p - value > 0 . 05 , * * * , p - value < 0 . 001 .

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 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

Figure 3(B) The cAIM peptide cannot outcompete HsUFM1-HsC53 interaction. Bacterial lysates containing recombinant protein were mixed and pulled down with glutathione magnetic agarose beads. Input and bound proteins were visualized by immunoblotting with anti-GST and anti-MBP antibodies. cAIM peptide was used to a final concentration of 200 µM. HsC53sAIM: HsC53W269A, W294A, W312A.(C, D) Microscopy-based protein-protein interaction assays showing unlike GABARAP-C53 interaction, UFM1-C53 interaction is insensitive to cAIM peptide competition. Glutathione-sepharose beads were prepared by incubating them with GST-GABARAP (C) or GST-HsUFM1 (D). The pre-assembled beads were then washed and mixed with 1 µM of HsC53 containing increasing concentrations of cAIM peptide (0-100 µM). The beads were then imaged using a confocal microscope. Left Panel, representative confocal images (inverted grayscale) for each condition are shown. Right panel, normalized fluorescence is shown for each condition with the mean (± SD) of 4 replicates. Unpaired two-samples Wilcoxon test with continuity correction was performed to analyze the differences between wild type and wild type with 100 µM AIM peptide. ns, not significant, p-value > 0.05, ***, p-value < 0.001.


Figure 4 ( B ) Binding of AtUFM1 to AtC53 IDR leads to a general drop in signal intensity . NMR signal intensity of AtC53 IDR ( 100 µM ) in the presence of 300 µM AtUFM1 normalized to signal intensity of AtC53 IDR alone . Bars corresponding to residues in the AIMs are highlighted . ( D ) The backbone amide signals of AtC53 IDR shift upon AtUFM1 addition in a concentration - dependent manner . Insets of overlaid 1H - 15N HSQC spectra of isotope - labelled AtC53 IDR ( 100 µM ) showing chemical shift perturbations of individual peaks from backbone amides of AIM residues in their free ( gray ) or bound state to unlabelled AtUFM1 . Chemical shifts are indicated with arrows .

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

Figure 4(B) Binding of AtUFM1 to AtC53 IDR leads to a general drop in signal intensity. NMR signal intensity of AtC53 IDR (100 µM) in the presence of 300 µM AtUFM1 normalized to signal intensity of AtC53 IDR alone. Bars corresponding to residues in the AIMs are highlighted.(D) The backbone amide signals of AtC53 IDR shift upon AtUFM1 addition in a concentration-dependent manner. Insets of overlaid 1H-15N HSQC spectra of isotope-labelled AtC53 IDR (100 µM) showing chemical shift perturbations of individual peaks from backbone amides of AIM residues in their free (gray) or bound state to unlabelled AtUFM1. Chemical shifts are indicated with arrows.


Figure 5 ( B , Conversion of sAIM into cAIM leads to reduced UFM1 binding and stronger ATG8 interaction . Bacterial lysates containing recombinant proteins were mixed and pulled down with glutathione magnetic agarose beads . Input and bound proteins were visualized by immunoblotting with anti - GST and anti - MBP antibodies . AtC53sAIM : AtC53 ( W276A , W287A , W335A ) ; AtC53cAIM : AtC53 ( IDWD274WDDI , IDWD285WDDI , IDWD333WDDI ) ; ATG8LDS : ATG8YL50AA . ( G ) AtC53cAIM forms more GFP - ATG8A colocalizing puncta upon ER stress . Upper Panel , representative confocal images of transgenic Arabidopsis seedlings co - expressing C53 - mCherry ( magenta ) , C53sAIM - mCherry and C53cAIM - mCherry with GFP - ATG8a in c53 mutant background under normal condition and after tunicamycin stress . 6 - day old seedlings were incubated in liquid 1 / 2 MS medium with 1 % sucrose supplemented with DMSO as control or tunicamycin ( 10 μg / ml ) for 6 hours before imaging . Scale bars , 30 μm . Inset scale bars , 10 μm . Right Panel , Quantification of the C53 - autophagosomes ( C53 - APG ) per normalized Z - stacks . Bars represent the mean ( ± SD ) of at least twenty roots from 3 biological replicates for each genotype and treatment . Unpaired two - samples Wilcoxon test with continuity correction was performed to analyze the differences between wild type and mutants . * * * , p - value < 0 . 001 .

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 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

Figure 5(B, Conversion of sAIM into cAIM leads to reduced UFM1 binding and stronger ATG8 interaction. Bacterial lysates containing recombinant proteins were mixed and pulled down with glutathione magnetic agarose beads. Input and bound proteins were visualized by immunoblotting with anti-GST and anti-MBP antibodies. AtC53sAIM: AtC53 (W276A, W287A, W335A); AtC53cAIM: AtC53(IDWD274WDDI, IDWD285WDDI, IDWD333WDDI); ATG8LDS: ATG8YL50AA.(G) AtC53cAIM forms more GFP-ATG8A colocalizing puncta upon ER stress. Upper Panel, representative confocal images of transgenic Arabidopsis seedlings co-expressing C53-mCherry (magenta), C53sAIM-mCherry and C53cAIM-mCherry with GFP-ATG8a in c53 mutant background under normal condition and after tunicamycin stress. 6-day old seedlings were incubated in liquid 1/2 MS medium with 1% sucrose supplemented with DMSO as control or tunicamycin (10 μg/ml) for 6 hours before imaging. Scale bars, 30 μm. Inset scale bars, 10 μm. Right Panel, Quantification of the C53-autophagosomes (C53-APG) per normalized Z-stacks. Bars represent the mean (± SD) of at least twenty roots from 3 biological replicates for each genotype and treatment. Unpaired two-samples Wilcoxon test with continuity correction was performed to analyze the differences between wild type and mutants. ***, p-value < 0.001.


Figure 1B . Genotype distribution in litters from heterozygous matings ( Fbxl4 + / - x Fbxl4 + / - ) at embryonic stage 13 . 5 ( E13 . 5 ) and of live animals at weaning . Data is presented as a percentage . Embryos n = 79 , weaned mice n = 339 . Dashed lines indicate the expected Mendelian ratios . Chi2 test vs expected Mendelian ratios ; * * * , p < 0 . 001 . C . Representative images of wild - type ( + / + ) , heterozygous knockout ( + / - ) and homozygous Fbxl4 knockout embryos ( - / - ) at E13 . 5 . D . Relative levels of mtDNA of Fbxl4 knockout ( - / - ) embryos and the corresponding wild type ( + / + ) embryos at E13 . 5 . Data represent mean ± SEM , n = 5 . Student ' s t - test ; * , p < 0 . 05 . E . Representative images of male Fbxl4 knockout ( - / - ) and matched wild - type ( + / + ) animals at one year of age ( left ) . A hunch - back phenotype was observed both in male and female knockout animals ( right , indicated by arrow ) . F - I . Whole body ( F ) , heart ( G ) , liver ( H ) and kidney ( I ) weights of 1 - year old animals . Data represent mean ± SEM , n ≥ 5 animals . Student ' s t - test ; * , p < 0 . 05 ; * * , p < 0 . 01 ; * * * , p < 0 . 001 ; n . s . , not significant ( p ≥ 0 . 05 ) .

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 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 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 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

Figure 1B. Genotype distribution in litters from heterozygous matings (Fbxl4+/- x Fbxl4+/-) at embryonic stage 13.5 (E13.5) and of live animals at weaning. Data is presented as a percentage. Embryos n=79, weaned mice n=339. Dashed lines indicate the expected Mendelian ratios. Chi2 test vs expected Mendelian ratios; ***, p<0.001.C. Representative images of wild-type (+/+), heterozygous knockout (+/-) and homozygous Fbxl4 knockout embryos (-/-) at E13.5.D. Relative levels of mtDNA of Fbxl4 knockout (-/-) embryos and the corresponding wild type (+/+) embryos at E13.5. Data represent mean ± SEM, n=5. Student's t-test; *, p<0.05.E. Representative images of male Fbxl4 knockout (-/-) and matched wild-type (+/+) animals at one year of age (left). A hunch-back phenotype was observed both in male and female knockout animals (right, indicated by arrow).F-I. Whole body (F), heart (G), liver (H) and kidney (I) weights of 1-year old animals. Data represent mean ± SEM, n ≥ 5 animals. Student's t-test; *, p<0.05; **, p<0.01; ***, p<0.001; n.s., not significant (p ≥ 0.05).


Figure 2A . Relative levels of mtDNA in tissues of 1 - year - old Fbxl4 knockout mice determined by quantitative real - time PCR ( normalized to controls ) . Data are presented as mean ± SEM , n = 6 for controls and n = 8 for knockout animals . Student ' s t - test ; * * , p < 0 . 01 ; * * * , p < 0 . 001 . B . Transcript levels in indicated tissues of 1 - year - old Fbxl4 knockout mice determined by quantitative real - time PCR . Relative mRNA levels in control animals were set as 1 ( dashed line ) . Data are presented as mean ± SEM , n = 6 for controls and n = 8 for knockout animals . Student ' s t - test ; * , p < 0 . 05 ; * * , p < 0 . 01 ; * * * , p < 0 . 001 . C . Western blot analysis of protein steady - state levels in liver protein extracts from wild type ( Fbxl4 + / + ) and Fbxl4 knock - out ( Fbxl4 - / - ) animals . D . Quantification of the western blot in ( C ) . Signal normalized to the average of controls and presented as mean ± SEM , n = 4 for both controls and knockout animals . Student ' s t - test ; * * , p < 0 . 01 ; * * * , p < 0 , 001 . E . Quantitative TMT - based proteomic analysis of wild - type and Fbxl4 knock - out liver tissue protein extracts . Proteins from three control and three Fbxl4 knock - out liver protein extracts were TMT - labelled , quantified and analyzed separately as described in Materials and Methods ; the data from control and knock - out animals were averaged after the analysis and presented as - log10 of the adjusted p - value vs log2 - fold change ( logFC ) . Mitochondrial proteins were selected according to mouse MitoCarta 2 . 0 ( Calvo et al , 2016 ) and highlighted in red ; lysosomal proteins were selected according to hLGDB database ( Brozzi et al , 2013 ) and highlighted in yellow . F . Enrichment analysis of lysosomal and mitochondrial proteins according to the logFC median of each group of proteins . Data is presented as 25 to 75 percentile box with the indicated median and whiskers representing the ± 1 . 5x inter - quartile range . Proteins included in each group : total = 1523 , mitochondrial = 324 , lysosomal = 50 .

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 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 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 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

Figure 2A. Relative levels of mtDNA in tissues of 1-year-old Fbxl4 knockout mice determined by quantitative real-time PCR (normalized to controls). Data are presented as mean ± SEM, n=6 for controls and n=8 for knockout animals. Student's t-test; **, p<0.01; ***, p<0.001.B. Transcript levels in indicated tissues of 1-year-old Fbxl4 knockout mice determined by quantitative real-time PCR. Relative mRNA levels in control animals were set as 1 (dashed line). Data are presented as mean ± SEM, n=6 for controls and n=8 for knockout animals. Student's t-test; *, p<0.05; **, p<0.01; ***, p<0.001.C. Western blot analysis of protein steady-state levels in liver protein extracts from wild type (Fbxl4+/+) and Fbxl4 knock-out (Fbxl4-/-) animals. D. Quantification of the western blot in (C). Signal normalized to the average of controls and presented as mean ± SEM, n=4 for both controls and knockout animals. Student's t-test; **, p<0.01; ***, p<0,001.E. Quantitative TMT-based proteomic analysis of wild-type and Fbxl4 knock-out liver tissue protein extracts. Proteins from three control and three Fbxl4 knock-out liver protein extracts were TMT-labelled, quantified and analyzed separately as described in Materials and Methods; the data from control and knock-out animals were averaged after the analysis and presented as -log10 of the adjusted p-value vs log2-fold change (logFC). Mitochondrial proteins were selected according to mouse MitoCarta 2.0(Calvo et al, 2016) and highlighted in red; lysosomal proteins were selected according to hLGDB database(Brozzi et al, 2013) and highlighted in yellow.F. Enrichment analysis of lysosomal and mitochondrial proteins according to the logFC median of each group of proteins. Data is presented as 25 to 75 percentile box with the indicated median and whiskers representing the ±1.5x inter-quartile range. Proteins included in each group: total=1523, mitochondrial=324, lysosomal=50.


Figure 3B . Relative mtDNA content in fibroblasts from patients and controls as determined by qRT - PCR using an ND6 probe and normalized to nuclear genomic DNA content using an 18S probe . Individual values are presented for each sample , as well as their mean ± SEM values . C . Steady - state protein levels in three control and three FBXL4 patient - derived fibroblasts analyzed by western blotting . D . Quantification of the western blot in ( C ) . Signals from the control and FBXL4 - mutated cells were considered as biological replicates , normalized to the average of controls and displayed as mean ± SEM ( n = 3 for each ) . Student ' s t - test ; * , p < 0 . 05 ; * * , p < 0 . 01 ; * * * , p < 0 . 001 . E . Quantification of nuclear and mitochondrial transcript levels in control and patient fibroblasts . The results from control and patient cells were averaged , normalized to controls and displayed as mean ± SEM ( n = 3 biological replicates for each condition ) . F . Quantitative TMT - based proteomic analysis of control and patient fibroblasts . Proteins from three control and three patient fibroblast cell lines were TMT - labelled , quantified and analyzed separately as described in Materials and Methods ; the data from control and patients were averaged after the analysis and presented as - log10 of the adjusted p - value vs log2 - fold change ( logFC ) . Mitochondrial proteins were selected according to human MitoCarta 2 . 0 ( Calvo et al , 2016 ) and highlighted in blue ; lysosomal proteins were selected according to hLGDB database ( Brozzi et al , 2013 ) and highlighted in yellow . G . Log2FC correlation analysis of Mouse Liver proteomic dataset and Fibroblast Proteomic dataset . Proteins with a log2FC > 0 . 5 are highlighted in black and lysosomal proteins are highlighted in yellow .

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 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 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 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 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

Figure 3B. Relative mtDNA content in fibroblasts from patients and controls as determined by qRT-PCR using an ND6 probe and normalized to nuclear genomic DNA content using an 18S probe. Individual values are presented for each sample, as well as their mean ± SEM values.C. Steady-state protein levels in three control and three FBXL4 patient-derived fibroblasts analyzed by western blotting.D. Quantification of the western blot in (C). Signals from the control and FBXL4-mutated cells were considered as biological replicates, normalized to the average of controls and displayed as mean ± SEM (n=3 for each). Student's t-test; *, p<0.05; **, p<0.01; ***, p<0.001.E. Quantification of nuclear and mitochondrial transcript levels in control and patient fibroblasts. The results from control and patient cells were averaged, normalized to controls and displayed as mean ± SEM (n=3 biological replicates for each condition).F. Quantitative TMT-based proteomic analysis of control and patient fibroblasts. Proteins from three control and three patient fibroblast cell lines were TMT-labelled, quantified and analyzed separately as described in Materials and Methods; the data from control and patients were averaged after the analysis and presented as -log10 of the adjusted p-value vs log2-fold change (logFC). Mitochondrial proteins were selected according to human MitoCarta 2.0 (Calvo et al, 2016) and highlighted in blue; lysosomal proteins were selected according to hLGDB database (Brozzi et al, 2013) and highlighted in yellow.G. Log2FC correlation analysis of Mouse Liver proteomic dataset and Fibroblast Proteomic dataset. Proteins with a log2FC > 0.5 are highlighted in black and lysosomal proteins are highlighted in yellow.


Figure 4B . Steady - state protein levels in wild - type ( WT ) and FBXL4 knockout ( KO ) RKO cells . C . ( Upper panel ) Pulse - chase labeling of mitochondrial translation products in wild type ( WT ) and FBXL4 knockout ( KO ) cells . The cells were incubated for 1 hour with a 35S methionine - cysteine labeling mix in the presence of anisomycin to inhibit cytosolic translation ( pulse ) . After removal of the medium , the cells were incubated in complete growth medium without inhibitors and labeled amino acids for 16 or 24 hours ( chase ) . A representative experiment of 4 biological replicates is shown . A Coomassie - stained part of the gel is shown as loading control . ( Lower panel ) Quantification of the MTCO1 - ND4 signal presented as mean ± SEM ; n = 4 biological replicates ; Student ' s t - test ; * , p < 0 . 05 . D . ( Upper panel ) Mitochondrial translation products were labeled in wild type ( WT ) and FBXL4 knock - out ( KO ) RKO cells for 60 minutes as in C , followed by 24 h chase in the presence of 5 μM epoxomicin ( epoxo ) or 20 mM ammonium chloride ( NH4Cl ) . A representative experiment of n ≥ 3 biological replicates is shown . A fragment of the Coomassie - stained gel is shown as loading control . ( Lower panel ) Quantification of the ND4 - MTCO1 signal . n ≥ 3 biological replicates , data are presented as mean ± SEM . Student ' s t - test ; * , p < 0 . 05 . E - F . Steady - state levels of indicated proteins in wild type and FBXL4 knockout RKO cells treated with NH4ClE - F . Steady - state levels of indicated proteins in wild type and FBXL4 knockout RKO cells treated with epoxomicin ( Epoxo , F ) .

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

Figure 4B. Steady-state protein levels in wild-type (WT) and FBXL4 knockout (KO) RKO cells.C. (Upper panel) Pulse-chase labeling of mitochondrial translation products in wild type (WT) and FBXL4 knockout (KO) cells. The cells were incubated for 1 hour with a 35S methionine-cysteine labeling mix in the presence of anisomycin to inhibit cytosolic translation (pulse). After removal of the medium, the cells were incubated in complete growth medium without inhibitors and labeled amino acids for 16 or 24 hours (chase). A representative experiment of 4 biological replicates is shown. A Coomassie-stained part of the gel is shown as loading control. (Lower panel) Quantification of the MTCO1-ND4 signal presented as mean ± SEM; n=4 biological replicates; Student's t-test; *, p<0.05.D. (Upper panel) Mitochondrial translation products were labeled in wild type (WT) and FBXL4 knock-out (KO) RKO cells for 60 minutes as in C, followed by 24 h chase in the presence of 5 μM epoxomicin (epoxo) or 20 mM ammonium chloride (NH4Cl). A representative experiment of n≥3 biological replicates is shown. A fragment of the Coomassie-stained gel is shown as loading control. (Lower panel) Quantification of the ND4-MTCO1 signal. n≥3 biological replicates, data are presented as mean ± SEM. Student's t-test; *, p<0.05.E-F. Steady-state levels of indicated proteins in wild type and FBXL4 knockout RKO cells treated with NH4ClE-F. Steady-state levels of indicated proteins in wild type and FBXL4 knockout RKO cells treated with epoxomicin (Epoxo, F).


Figure 5A . Confocal images from the mito - QC - expressing fibroblast lines . Scale bar 25 μm . B . Quantification of the mitolysosomes / cell area ( μm2 ) using the mito - QC counter plugin for ImageJ ( Montava - Garriga et al , 2020 ) . At least 3 technical replicates for each line and n > 15 cells analyzed for each technical replicate . Data is presented as 25 to 75 percentile box with the indicated median and whiskers representing 5 - 95 percentile interval . Non - parametric Mann - Whitney test ; * * * , p < 0 . 001 .

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

Figure 5A. Confocal images from the mito-QC-expressing fibroblast lines. Scale bar 25 μm. B. Quantification of the mitolysosomes/cell area (μm2) using the mito-QC counter plugin for ImageJ (Montava-Garriga et al, 2020). At least 3 technical replicates for each line and n>15 cells analyzed for each technical replicate. Data is presented as 25 to 75 percentile box with the indicated median and whiskers representing 5-95 percentile interval. Non-parametric Mann-Whitney test; ***, p<0.001.


Figure 1 ( 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 . ( 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 ) ] .

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 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 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

Figure 1(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.(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)].


Figure 2 ( 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 ) . ( 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 . ( 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 ) .

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

Figure 2(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).(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.(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 3Figure 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 ) .

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

Figure 3Figure 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).


Figure 4 ( 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 . ( 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 . ( 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 . ( 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 .

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 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 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 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 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 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 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 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

Figure 4(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.(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.(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.(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.


Figure 5 ( 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 . ( c ) U2 - Prp5 binding assay . FLAG - Prp5 and a radiolabeled short ( nts 1 - 76 ) ( lanes 1 and 2 ) or long ( nts 1 - 120 ) ( lanes 5 and 6 ) U2 fragment , or FLAG - Prp5 and a radiolabeled fully pseudouridylated short ( nts 1 - 76 ) ( lanes 3 and 4 ) or long ( nts 1 - 120 ) ( lanes 7 and 8 ) U2 fragment , were incubated under the conditions used for ATPase activity assay . Anti - FLAG IP was then carried out . Co - precipitated ( lanes 2 , 4 , 6 and 8 ) and un - precipitated ( lanes 1 , 3 , 5 and 7 ) U2 RNAs were analyzed by electrophoresis . The U2 band is indicated . ( d ) Quantification of Prp5 - U2 binding . Relative intensities of U2 bands shown in ( c ) were calculated [ setting the un - precipitated uridine - containing U2 ( lane 1 and 5 ) to 1 ] . The numbers at the bottom of ( d ) correspond to the lane numbers of ( c ) . Quantification was based on three independent experiments . ( e ) Prp5 ' s ATPase activity assay using cellular U2 . U2 RNA isolated from the wild - type strain ( lanes 2 and 4 ) or from the snR81Δ pus1Δ strain ( lanes 6 and 8 ) was used for the ATPase activity assay . In odd - numbered lanes , no RNA was added . Both the wild - type Prp5 ( lanes 1 - 4 ) and the mutant Prp5 ( GAR ) ( lanes 5 - 8 ) were tested . ( f ) Quantification of Prp5 ' s ATPase activity activated by U2 from cells . Prp5 ' s ATPase activity shown in ( e ) was calculated using the formula ( ADP ) / ( ADT + ATP ) . The numbers at the bottom of ( f ) correspond to the lane numbers of ( e ) . Quantification was based on four independent experiments . ( g ) U2 level in the ATPase activity reactions . RNAs from lanes 2 and 4 in ( c ) were recovered , and U2 level was measured using primer - extension . Lanes 1 - 5 , a titration of U2 snRNA . Lane 6 , U2 from the wild - type strain [ lane 2 of ( c ) ] . Lane 7 , U2 from the snR81Δ pus1Δ strain [ lane 4 of ( c ) ] .

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 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 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 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 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 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 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

Figure 5(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.(c) U2-Prp5 binding assay. FLAG-Prp5 and a radiolabeled short (nts 1-76) (lanes 1 and 2) or long (nts 1-120) (lanes 5 and 6) U2 fragment, or FLAG-Prp5 and a radiolabeled fully pseudouridylated short (nts 1-76) (lanes 3 and 4) or long (nts 1-120) (lanes 7 and 8) U2 fragment, were incubated under the conditions used for ATPase activity assay. Anti-FLAG IP was then carried out. Co-precipitated (lanes 2, 4, 6 and 8) and un-precipitated (lanes 1, 3, 5 and 7) U2 RNAs were analyzed by electrophoresis. The U2 band is indicated.(d) Quantification of Prp5-U2 binding. Relative intensities of U2 bands shown in (c) were calculated [setting the un-precipitated uridine-containing U2 (lane 1 and 5) to 1]. The numbers at the bottom of (d) correspond to the lane numbers of (c). Quantification was based on three independent experiments.(e) Prp5's ATPase activity assay using cellular U2. U2 RNA isolated from the wild-type strain (lanes 2 and 4) or from the snR81Δ pus1Δ strain (lanes 6 and 8) was used for the ATPase activity assay. In odd-numbered lanes, no RNA was added. Both the wild-type Prp5 (lanes 1-4) and the mutant Prp5 (GAR) (lanes 5-8) were tested.(f) Quantification of Prp5's ATPase activity activated by U2 from cells. Prp5's ATPase activity shown in (e) was calculated using the formula (ADP) / (ADT+ATP). The numbers at the bottom of (f) correspond to the lane numbers of (e). Quantification was based on four independent experiments.(g) U2 level in the ATPase activity reactions. RNAs from lanes 2 and 4 in (c) were recovered, and U2 level was measured using primer-extension. Lanes 1-5, a titration of U2 snRNA. Lane 6, U2 from the wild-type strain [lane 2 of (c)]. Lane 7, U2 from the snR81Δ pus1Δ strain [lane 4 of (c)].


Figure 6 ( a ) Native gel analysis of pre - splicing complexes . Pre - splicing complex assembly was carried out in the test tube with Prp5 - reconstituted cell extracts and labeled pre - mRNA . Lane 1 , Prp5 - depleted wild - type cell extract . Lane 2 , Prp5 - depleted wild - type cell extract , plus wild - type Prp5 . Lane 3 , Prp5 - depleted snR81Δ pus1Δ cell extract , plus wild - type Prp5 . Lane 4 , Prp5 - depleted wild - type cell extract , plus mutant Prp5 ( GAR ) . Lane 5 , Prp5 - depleted snR81Δ pus1Δ cell extract , plus mutant Prp5 ( GAR ) . The pre - splicing complexes A and B are indicated . ( b ) DMS in vivo probing . After being exposed to DMS , yeast cells were lysed , total RNA collected , and primer - extension analysis carried out ( left panel ) . Lane 1 , cells that were not exposed to DMS . Lane 2 , wild - type cells exposed to DMS . Lane 3 , snR81Δ pus1Δ cells exposed to DMS . Lane 4 , snR81Δ pus1Δ cells , transformed with a plasmid carrying an artificial guide RNA gene targeting positions 42 and 44 , and exposed to DMS . Lane 5 , snR81Δ pus1Δ cells , transformed with a plasmid carrying a control guide RNA gene with random guide sequences , and exposed to DMS . The nucleotides in parentheses are DMS - modified C and A residues that are one nucleotide after the actual primer - extension stops ( indicated by the arrows ) . A partial U2 sequence ( along with the BSL structure ) is shown ( right panel ) . DMS - modified adenosine and cytosine in the BSL are also indicated . ( c ) Residue accessibility quantification . Relative accessibility ( relative band intensity ) was quantified based on three independent experiments . The intensity of every band in each lane was normalized against the C46 band ( the least changed band ; compare lanes 2 - 5 in ( b ) ) in its respective lane [ ( intensity of a band ) / ( intensity of C46 in the same lane ) ] . Each C46 - normailzed band in lanes 3 - 5 ( refer to ( b ) ) was then further normalized against its respective counterpart in lane 2 [ ( the value of a C46 - normalized band in lanes 3 - 5 ) / ( the value of its counterpart in lane 2 ) ] . The error bars represent the standard deviations of the measurements .

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 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 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

Figure 6(a) Native gel analysis of pre-splicing complexes. Pre-splicing complex assembly was carried out in the test tube with Prp5-reconstituted cell extracts and labeled pre-mRNA. Lane 1, Prp5-depleted wild-type cell extract. Lane 2, Prp5-depleted wild-type cell extract, plus wild-type Prp5. Lane 3, Prp5-depleted snR81Δ pus1Δ cell extract, plus wild-type Prp5. Lane 4, Prp5-depleted wild-type cell extract, plus mutant Prp5 (GAR). Lane 5, Prp5-depleted snR81Δ pus1Δ cell extract, plus mutant Prp5 (GAR). The pre-splicing complexes A and B are indicated.(b) DMS in vivo probing. After being exposed to DMS, yeast cells were lysed, total RNA collected, and primer-extension analysis carried out (left panel). Lane 1, cells that were not exposed to DMS. Lane 2, wild-type cells exposed to DMS. Lane 3, snR81Δ pus1Δ cells exposed to DMS. Lane 4, snR81Δ pus1Δ cells, transformed with a plasmid carrying an artificial guide RNA gene targeting positions 42 and 44, and exposed to DMS. Lane 5, snR81Δ pus1Δ cells, transformed with a plasmid carrying a control guide RNA gene with random guide sequences, and exposed to DMS. The nucleotides in parentheses are DMS-modified C and A residues that are one nucleotide after the actual primer-extension stops (indicated by the arrows). A partial U2 sequence (along with the BSL structure) is shown (right panel). DMS-modified adenosine and cytosine in the BSL are also indicated.(c) Residue accessibility quantification. Relative accessibility (relative band intensity) was quantified based on three independent experiments. The intensity of every band in each lane was normalized against the C46 band (the least changed band; compare lanes 2-5 in (b)) in its respective lane [(intensity of a band) / (intensity of C46 in the same lane)]. Each C46-normailzed band in lanes 3-5 (refer to (b)) was then further normalized against its respective counterpart in lane 2 [(the value of a C46-normalized band in lanes 3-5) / (the value of its counterpart in lane 2)]. The error bars represent the standard deviations of the measurements.


Figure 3 ( A ) Representative western blot of proteins pulled down by IP against Pum1 compared to IgG from wild - type mice brain without ( left ) and with ( right ) RNase treatment . In this panel , after IP - Pum1 , we immunoblotted for Pum1 ( positive control ) , Pum2 , Fmrp , Ago2 , Rbfox3 , Cnot1 , and Mov10 ( B ) Representative western blots with and without RNase . Molecular weights at the right are in kilodaltons ( kDa ) . IP against IgG was used as a negative control , and Input ( 1 % from the initial protein lysate as a loading control .

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 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

Figure 3(A) Representative western blot of proteins pulled down by IP against Pum1 compared to IgG from wild-type mice brain without (left) and with (right) RNase treatment. In this panel, after IP-Pum1, we immunoblotted for Pum1 (positive control), Pum2, Fmrp, Ago2, Rbfox3, Cnot1, and Mov10(B) Representative western blots with and without RNase. Molecular weights at the right are in kilodaltons (kDa). IP against IgG was used as a negative control, and Input (1% from the initial protein lysate as a loading control.


Figure 4 ( A - C ) Representative western blots of Pum1 , Pum2 , Fmrp , Ago2 , Rbfox3 , Cnot1 , and Mov10 in ( A ) cerebellum , ( B ) hippocampus , and ( C ) cortex in both male ( left panel ) and female ( right panel ) WT , Pum1 + / - , and Pum1 - / - mice . All the experiments were conducted with equal numbers of 10 - week - old male and female mice per genotype , for a total of at least 6 mice per genotype Graphs below show quantification for each protein by brain region , sex , and genotype . All data were normalized to Gapdh protein levels . P values were calculated by two - tailed Student ' s t test . * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 . P1 indicates Pum1 . ( D ) Heatmap showing 166 microRNAs from cerebella of Pum1 - / - male and female mice that were dysregulated ( fold change - 3 to + 3 ) relative to wild - type cerebellum . ( E ) Heatmap showing mRNA quantification by qPCR for 49 targets co - bound by a minimum of eight dysregulated miRNAs ( > 25 % change ) from panel D . Statistical significance and magnitude of dysregulation are illustrated for both male and

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 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

Figure 4(A-C) Representative western blots of Pum1, Pum2, Fmrp, Ago2, Rbfox3, Cnot1, and Mov10 in (A) cerebellum, (B) hippocampus, and (C) cortex in both male (left panel) and female (right panel) WT, Pum1+/-, and Pum1-/- mice. All the experiments were conducted with equal numbers of 10-week-old male and female mice per genotype, for a total of at least 6 mice per genotype Graphs below show quantification for each protein by brain region, sex, and genotype. All data were normalized to Gapdh protein levels. P values were calculated by two-tailed Student's t test. *p < 0.05, **p < 0.01, ***p < 0.001. P1 indicates Pum1.(D) Heatmap showing 166 microRNAs from cerebella of Pum1-/- male and female mice that were dysregulated (fold change -3 to +3) relative to wild-type cerebellum. (E) Heatmap showing mRNA quantification by qPCR for 49 targets co-bound by a minimum of eight dysregulated miRNAs (>25% change) from panel D. Statistical significance and magnitude of dysregulation are illustrated for both male and female


Figure 5 ( C ) GSEA analysis scores of Pum1 , Pum2 , Rbfox3 , and Ago2 HITS - CLIP data plotted against Fmrp HITS - CLIP data divided into 10 - percentile ranked bins shows the shared targets are among the top percentiles of targets for each protein .

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

Figure 5(C) GSEA analysis scores of Pum1, Pum2, Rbfox3, and Ago2 HITS-CLIP data plotted against Fmrp HITS-CLIP data divided into 10-percentile ranked bins shows the shared targets are among the top percentiles of targets for each protein.


Figure 6 ( A , B ) IP against PUM1 from Subject 1 ( S1 , R1147W ) ( A ) and Subject 2 ( S2 , T1035S ) ( B ) compared to their respective age - , sex - , and cell type - matched healthy controls confirm the interactions between PUM1 ( used here as a positive control ) , and PUM2 , FMRP , AGO2 , CNOT1 , and MOV10 . Input ( 1 % ) was used as a loading control and IP against IgG was used as a negative control . GAPDH was used here as loading control , see Methods for quantification . ( C , D ) Representative western blots ( C ) and relative quantification ( D ) of protein levels for PUM1 , PUM2 , FMRP , AGO2 , CNOT1 , and MOV10 in PADDAS patient - derived and PRCA patient - derived cells compared to their respective controls . Data were normalized to GAPDH protein levels .

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 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

Figure 6(A, B) IP against PUM1 from Subject 1 (S1, R1147W) (A) and Subject 2 (S2, T1035S) (B) compared to their respective age-, sex-, and cell type- matched healthy controls confirm the interactions between PUM1 (used here as a positive control), and PUM2, FMRP, AGO2, CNOT1, and MOV10. Input (1%) was used as a loading control and IP against IgG was used as a negative control. GAPDH was used here as loading control, see Methods for quantification.(C, D) Representative western blots (C) and relative quantification (D) of protein levels for PUM1, PUM2, FMRP, AGO2, CNOT1, and MOV10 in PADDAS patient-derived and PRCA patient-derived cells compared to their respective controls. Data were normalized to GAPDH protein levels.


Figure 7 ( B ) Representative western blots ( left panel ) and relative quantifications ( right panel ) of PUM1 and its interactors ( AGO2 , CNOT1 , FMRP , and MOV10 ) in R1147W patient - derived cell lines after Myc - PUM1 - WT expression . ( C ) mRNA quantification of the top 15 shared target genes from panel A in R1147W patient - derived cell lines after Myc - PUM1 - WT expression .

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

Figure 7(B) Representative western blots (left panel) and relative quantifications (right panel) of PUM1 and its interactors (AGO2, CNOT1, FMRP, and MOV10) in R1147W patient-derived cell lines after Myc-PUM1-WT expression.(C) mRNA quantification of the top 15 shared target genes from panel A in R1147W patient-derived cell lines after Myc-PUM1-WT expression.


Figure 1 ( C ) Heat map demonstration of genes that were mostly enriched in the pathway of pluripotency of stem cells . The color bar showed the relative RNA - seq signal ( Z - score of normalized FPKM ) . Histological examinations of tumor types in ( F ) K versus KrasG12D ; NanogL / L ( KN ) Tumor tissues collected form indicated mice analyzed using Haematoxylin Eosin ( H & E ) staining . Representative H & E images are shown . Scale bar , 500 μm ( top ) , 50 μm ( bottom ) .

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

Figure 1(C) Heat map demonstration of genes that were mostly enriched in the pathway of pluripotency of stem cells. The color bar showed the relative RNA-seq signal (Z-score of normalized FPKM).Histological examinations of tumor types in (F) K versus KrasG12D; NanogL/L (KN) Tumor tissues collected form indicated mice analyzed using Haematoxylin Eosin (H&E) staining. Representative H&E images are shown. Scale bar, 500 μm (top), 50 μm (bottom).


Figure 2 ( G ) qPCR analysis of stomach - restricted gene set between KL ADC and KLN mucinous tumor sections ( n = 3 or 4 per group ) . Results were shown in a box and whisker plot with the minimum value , first quartile , median , third quartile and maximum value . The box represented the 50 % of the central data , with a line inside represented the median . The significance was calculated by two - tailed unpaired Student ' s t - test with Welch ' s correction . * p = 0 . 0260 ( Hnf4α ) , * p = 0 . 0275 ( Vsig1 ) , * p = 0 . 0122 ( Gkn3 ) , * p = 0 . 0324 ( Ctse ) , * * p = 0 . 0084 ( Onecut2 ) .

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

Figure 2(G) qPCR analysis of stomach-restricted gene set between KL ADC and KLN mucinous tumor sections (n=3 or 4 per group). Results were shown in a box and whisker plot with the minimum value, first quartile, median, third quartile and maximum value. The box represented the 50% of the central data, with a line inside represented the median. The significance was calculated by two-tailed unpaired Student's t-test with Welch's correction. *p=0.0260 (Hnf4α), *p=0.0275 (Vsig1), *p=0.0122 (Gkn3), *p=0.0324 (Ctse), **p=0.0084 (Onecut2).


Figure 3 ( D ) Western blot for NANOG and TUBULIN in 7 paired human normal lungs ( N ) and ADC samples ( T ) and human ESCs . Mr , relative molecular mass . kDa , killoDalton . ( H ) Statistic analysis of LKB1 expression in ADC and IMA . The tumor tissues were analyzed by IHC staining and graded as low , medium , high expression . Significance was calculated by χ2 test for trend . ( I ) The expression grade of IHC staining of NANOG , NKX2 - 1 , LKB1 in 76 human IMA ( 1 sample / row ) .

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 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

Figure 3(D) Western blot for NANOG and TUBULIN in 7 paired human normal lungs (N) and ADC samples (T) and human ESCs. Mr, relative molecular mass. kDa, killoDalton.(H) Statistic analysis of LKB1 expression in ADC and IMA. The tumor tissues were analyzed by IHC staining and graded as low, medium, high expression. Significance was calculated by χ2 test for trend.(I) The expression grade of IHC staining of NANOG, NKX2-1, LKB1 in 76 human IMA (1 sample/row).


Figure 4 ( B - D ) H & E staining of tumors , IHC staining ( B ) and quantification of NICD ( C ) and HES1 ( D ) in KL ADC and KLN IMA . Representative images were shown . Scale bar , 50 μm . Results were shown as mean ± SD . The numbers of tumors analyzed over KL ADC and KLN IMA were 15 , 18 for NICD , 16 , 15 for HES1 . Significance was calculated by two - tailed unpaired Student ' s t - test with Welch ' s correction . * p = 0 . 0188 ( C ) , * * p = 0 . 0040 ( D ) . ( G , H ) Statistical analysis of NICD ( G ) and HES1 ( H ) expression in lung sections of KLN mice following vehicle or LY - 411575 treatment . The numbers of tumors analyzed over vehicle and LY - 411575 groups were 22 , 19 for NICD , 22 , 22 for HES1 . Results were shown as mean ± SD . Significance was calculated by two - tailed unpaired Student ' s t - test with Welch ' s correction . * * * * p < 0 . 0001 . ( I ) H & E and IHC staining of NICD and HES1 in lung tumor tissues of vehicle and LY - 411575 groups . Scale bar , 50 μm . ( J ) H & E , AB and PAS staining in lung tumors of vehicle and LY - 411575 groups . Scale bar , 50 μm .

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 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 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

Figure 4(B-D) H&E staining of tumors, IHC staining (B) and quantification of NICD (C) and HES1 (D) in KL ADC and KLN IMA. Representative images were shown. Scale bar, 50 μm. Results were shown as mean ± SD. The numbers of tumors analyzed over KL ADC and KLN IMA were 15, 18 for NICD, 16, 15 for HES1. Significance was calculated by two-tailed unpaired Student's t-test with Welch's correction. *p=0.0188 (C), **p=0.0040 (D).(G, H) Statistical analysis of NICD (G) and HES1 (H) expression in lung sections of KLN mice following vehicle or LY-411575 treatment. The numbers of tumors analyzed over vehicle and LY-411575 groups were 22, 19 for NICD, 22, 22 for HES1. Results were shown as mean ± SD. Significance was calculated by two-tailed unpaired Student's t-test with Welch's correction. ****p<0.0001.(I) H&E and IHC staining of NICD and HES1 in lung tumor tissues of vehicle and LY-411575 groups. Scale bar, 50 μm. (J) H&E, AB and PAS staining in lung tumors of vehicle and LY-411575 groups. Scale bar, 50 μm.


Figure 5 ( I , J ) Statistical analysis of Ki67 ( I ) and CC3 ( J ) activity in ADC and IMA lung sections of vehicle and phenformin ( Phen ) groups . The numbers of tumors analyzed over vehicle group and phenformin groups for ADC and IMA were 17 , 8 , 9 for Ki67 , 10 , 10 , 7 for CC3 . Results were shown as mean ± SEM . Significance was calculated by one - way ANOVA with Dunnett ' s multiple comparisons test . * * p = 0 . 0010 ( I ) , * * * p = 0 . 0009 ( I ) , * p = 0 . 0427 ( J ) .

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

Figure 5(I, J) Statistical analysis of Ki67 (I) and CC3 (J) activity in ADC and IMA lung sections of vehicle and phenformin (Phen) groups. The numbers of tumors analyzed over vehicle group and phenformin groups for ADC and IMA were 17, 8, 9 for Ki67, 10, 10, 7 for CC3. Results were shown as mean ± SEM. Significance was calculated by one-way ANOVA with Dunnett's multiple comparisons test. **p=0.0010 (I), ***p=0.0009 (I), *p=0.0427(J).


Figure 6 ( E , F ) Statistical analysis of Ki67 ( E ) and CC3 ( F ) reactivity in lung sections of KLN mice following vehicle , LY - 411575 treatment or a combinational therapy of phenformin and LY - 411575 . The numbers of tumors analyzed over vehicle , phenformin and combinational therapy groups were 20 , 30 , 13 for Ki67 , 14 , 11 , 9 for CC3 . Results were shown as mean ± SEM . Significance was calculated by one - way ANOVA with Dunnett ' s multiple comparisons test . * * p = 0 . 0014 ( E ) , * * * p = 0 . 0001 ( F ) .

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

Figure 6(E, F) Statistical analysis of Ki67 (E) and CC3 (F) reactivity in lung sections of KLN mice following vehicle, LY-411575 treatment or a combinational therapy of phenformin and LY-411575. The numbers of tumors analyzed over vehicle, phenformin and combinational therapy groups were 20, 30, 13 for Ki67, 14, 11, 9 for CC3. Results were shown as mean ± SEM. Significance was calculated by one-way ANOVA with Dunnett's multiple comparisons test. **p=0.0014 (E), ***p=0.0001 (F).


Figure 1 ( A ) BI ‐ 1 WT and KO MEFs cells were stably transduced with retroviruses expressing cytochrome b5 - GFP to visualize the ER ( green ) . Then cells were stained with lysotracker ( red ) and observed with a confocal microscope . Nucleus was stained with Hoechst ( blue ) . Scale bar : 30 μm . ( B ) BI ‐ 1 WT and KO MEFs cells stimulated with EBSS for 3 h to induce autophagy . Acidic vesicles were visualized with a confocal microscope after lysotracker staining . Data represent the results of three independent experiments . Scale bar : 50 μm . ( C ) ( a ) Epifluorescence imaging of BI ‐ 1 KO cells stained with lysotracker ; ( b ) electron micrograph of the same field of cells shown in ( a ) ; ( c - e ) , magnification of lysotracker ‐ positive vesicles in BI ‐ 1 KO cells exposed to EBSS for 3 h and visualized with a fluorescent microscope ( c ) and the same field subsequently imaged by EM ( d ) . Overlapping images are presented ( e ) . Left panel : analysis of vesicular structures by EM with morphologies resembling early ( f ) , intermediate ( g ) and late ( h ) autophagy vesicles . ( D ) Left panel : the distribution of endogenous LC3 was monitored by immunofluorescence and confocal microscopy in BI ‐ 1 WT and KO MEFs cells at basal conditions ( NT ) or after exposure to EBSS for 3 h . Scale bar : left 15 μm and right 10 μm . Right panel : quantification of the number cells containing three or more LC3 ‐ positive vesicles ( N = 160 cells ) . Mean and standard deviation are presented ( N = 4 ) . Student ' s t ‐ test was used to analyse statistical significance , * * P0 . 001 and * P0 . 01 . ( E ) BI ‐ 1 WT and KO cells were transiently transfected with an expression vector for a monomeric ‐ tandem LC3 - RFP - GFP construct . After 24 h , cells were exposed to EBSS for 3 h . Autophagy fluxes were monitored in living cells by visualizing the distribution of LC3 ‐ positive dots in the red and green channels using a confocal microscope . Scale bar : 10 μm . Right panel : quantification of the ratio between red and yellow dots per cell is presented . Mean and standard error of the analysis of 15 cells are shown .

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 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 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 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 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

Figure 1(A) BI‐1 WT and KO MEFs cells were stably transduced with retroviruses expressing cytochrome b5-GFP to visualize the ER (green). Then cells were stained with lysotracker (red) and observed with a confocal microscope. Nucleus was stained with Hoechst (blue). Scale bar: 30 μm.(B) BI‐1 WT and KO MEFs cells stimulated with EBSS for 3 h to induce autophagy. Acidic vesicles were visualized with a confocal microscope after lysotracker staining. Data represent the results of three independent experiments. Scale bar: 50 μm.(C) (a) Epifluorescence imaging of BI‐1 KO cells stained with lysotracker; (b) electron micrograph of the same field of cells shown in (a); (c-e), magnification of lysotracker‐positive vesicles in BI‐1 KO cells exposed to EBSS for 3 h and visualized with a fluorescent microscope (c) and the same field subsequently imaged by EM (d). Overlapping images are presented (e). Left panel: analysis of vesicular structures by EM with morphologies resembling early (f), intermediate (g) and late (h) autophagy vesicles.(D) Left panel: the distribution of endogenous LC3 was monitored by immunofluorescence and confocal microscopy in BI‐1 WT and KO MEFs cells at basal conditions (NT) or after exposure to EBSS for 3 h. Scale bar: left 15 μm and right 10 μm. Right panel: quantification of the number cells containing three or more LC3‐positive vesicles (N=160 cells). Mean and standard deviation are presented (N=4). Student's t‐test was used to analyse statistical significance, **P0.001 and *P0.01.(E) BI‐1 WT and KO cells were transiently transfected with an expression vector for a monomeric‐tandem LC3-RFP-GFP construct. After 24 h, cells were exposed to EBSS for 3 h. Autophagy fluxes were monitored in living cells by visualizing the distribution of LC3‐positive dots in the red and green channels using a confocal microscope. Scale bar: 10 μm. Right panel: quantification of the ratio between red and yellow dots per cell is presented. Mean and standard error of the analysis of 15 cells are shown.


Figure 2 ( A ) BI ‐ 1 WT and KO MEFs were treated with EBSS ( left panel ) or glucose / serum ‐ free RPMI media ( right panel ) for the indicated time points . Then , levels of LC3 were determined by western blot analysis . LC3 ‐ I and LC3 ‐ II forms are indicated . Hsp90 levels were assessed as loading control . ( B ) Quantification of LC3 ‐ II levels relative to Hsp90 expression was performed in several experiments performed as presented in ( A ) . ( C ) Cells were pre ‐ treated with a cocktail of lysosomal inhibitors ( 200 nM bafilomycin A1 , 10 μg / ml pepstatin , and E64d ; left panel ) or 10 mM 3 ‐ methyladenine ( 3 ‐ MA ; right panel ) for 12 h and then exposed to starvation . LC3 levels monitored by western blot ( D ) and quantification of LC3 ‐ II levels relative to Hsp90 were performed in the experimental conditions described in ( C ) . ( E ) Basal autophagy flux was monitored in cells treated with a cocktail of lysosomal inhibitors ( Lys . Inh . ) in the presence of normal cell culture media . Right panel : quantification of independent experiments is presented . ( F ) BI ‐ 1 KO cells were stably transduced with retroviruses expressing BI ‐ 1 - GFP or empty vector , and then levels of LC3 ‐ II were assessed over time by western blot after exposure to EBSS . Right panel : as control , the levels of BI ‐ 1 - GFP were monitored by western blot . Hsp90 levels were used as loading control . In ( B , D and E ) mean and standard deviation are presented . Two ‐ way ANOVA was applied to analyse statistical significance . In parenthesis , the number of independent experiments for each time point is indicated . Student ' s t ‐ test was also used in ( E ) to analyse the statistical significance between each time point ( * P0 . 001 ) . In ( B , D and E ) , normalization was performed as a ratio with the LC3 ‐ II / Hsp90 normalized levels from non ‐ treated BI ‐ 1 WT cells .

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 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 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 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

Figure 2(A) BI‐1 WT and KO MEFs were treated with EBSS (left panel) or glucose/serum‐free RPMI media (right panel) for the indicated time points. Then, levels of LC3 were determined by western blot analysis. LC3‐I and LC3‐II forms are indicated. Hsp90 levels were assessed as loading control.(B) Quantification of LC3‐II levels relative to Hsp90 expression was performed in several experiments performed as presented in (A).(C)Cells were pre‐treated with a cocktail of lysosomal inhibitors (200 nM bafilomycin A1, 10 μg/ml pepstatin, and E64d; left panel) or 10 mM 3‐methyladenine (3‐MA; right panel) for 12 h and then exposed to starvation. LC3 levels monitored by western blot (D) and quantification of LC3‐II levels relative to Hsp90 were performed in the experimental conditions described in (C).(E) Basal autophagy flux was monitored in cells treated with a cocktail of lysosomal inhibitors (Lys. Inh.) in the presence of normal cell culture media. Right panel: quantification of independent experiments is presented.(F) BI‐1 KO cells were stably transduced with retroviruses expressing BI‐1-GFP or empty vector, and then levels of LC3‐II were assessed over time by western blot after exposure to EBSS. Right panel: as control, the levels of BI‐1-GFP were monitored by western blot. Hsp90 levels were used as loading control. In (B, D and E) mean and standard deviation are presented. Two‐way ANOVA was applied to analyse statistical significance. In parenthesis, the number of independent experiments for each time point is indicated. Student's t‐test was also used in (E) to analyse the statistical significance between each time point (*P0.001). In (B, D and E), normalization was performed as a ratio with the LC3‐II/Hsp90 normalized levels from non‐treated BI‐1 WT cells.


Figure 3 ( A ) Left panel : BI ‐ 1 WT and KO MEFs cells were incubated in EBSS , and then cell viability was monitored using the MTS assay . Right panel : a similar experiment was performed after treating cells with the indicated concentration of tunicamycin for 24 h . Mean and standard deviation are presented of triplicates representative of three independent experiments . ( B ) BI ‐ 1 WT and KO cells were treated with three different starvation stimuli for 6 and 24 h . Cell death was determined after propidium iodide ( PI ) staining and FACS analysis . In addition , cells were treated with 100 ng / ml tunicamycin ( Tm ) for 24 h . Mean and standard deviation are presented of one experiment performed in triplicates . ( C ) Cells were exposed to EBSS for 6 h or 1 μg / ml Tm for 2 h , and then replated in normal cell culture media . After 5 days , cell viability was monitored by staining with crystal violet . Data are representative of three independent experiments . ( D ) BI ‐ 1 WT MEFs were stably transduced with lentiviral expression vectors to deliver an shRNA against bi ‐ 1 mRNA or control mRNA ( luciferase shRNA ) . Cell survival was measured after treatment of cells as described in ( B ) . Mean and standard deviation of an experiment made by triplicate , representative of two independent experiments . ( E ) BI ‐ 1 KO cells were stably transduced with retroviruses expressing BI ‐ 1 - GFP or empty vector , and then exposed to EBSS for indicated time points . Cell viability was monitored after PI staining by FACS . Mean and standard deviation are presented of triplicates representative of two independent experiments .

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 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 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 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 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

Figure 3(A) Left panel: BI‐1 WT and KO MEFs cells were incubated in EBSS, and then cell viability was monitored using the MTS assay. Right panel: a similar experiment was performed after treating cells with the indicated concentration of tunicamycin for 24 h. Mean and standard deviation are presented of triplicates representative of three independent experiments.(B) BI‐1 WT and KO cells were treated with three different starvation stimuli for 6 and 24 h. Cell death was determined after propidium iodide (PI) staining and FACS analysis. In addition, cells were treated with 100 ng/ml tunicamycin (Tm) for 24 h. Mean and standard deviation are presented of one experiment performed in triplicates.(C) Cells were exposed to EBSS for 6 h or 1 μg/ml Tm for 2 h, and then replated in normal cell culture media. After 5 days, cell viability was monitored by staining with crystal violet. Data are representative of three independent experiments.(D) BI‐1 WT MEFs were stably transduced with lentiviral expression vectors to deliver an shRNA against bi‐1 mRNA or control mRNA (luciferase shRNA). Cell survival was measured after treatment of cells as described in (B). Mean and standard deviation of an experiment made by triplicate, representative of two independent experiments.(E) BI‐1 KO cells were stably transduced with retroviruses expressing BI‐1-GFP or empty vector, and then exposed to EBSS for indicated time points. Cell viability was monitored after PI staining by FACS. Mean and standard deviation are presented of triplicates representative of two independent experiments.