Figure 1Immunoblot comparing UFL1 autoUFMylation ( Top ) and UFM1 chain synthesis ( Middle ) in the presence of UFL1 expressed alone and in complex with UFBP1 . ( Bottom ) Assays to monitor UFMylation of substrates using purified TRIP4 . Reaction products were separated on a 4 - 12 % SDS - PAGE gel under reducing conditions and analysed by immunoblotting using indicated antibodies . In vitro reconstitution of 60S ribosome UFMylation . Purified 60S ribosome was incubated with either UFL1 on its own or UFL1 / UFBP1 complex together with UBA5 , UFC1 and UFM1 for indicated time points at 30 ° C . Immunoblot showing formation of free UFM1 chains in the presence ( Lane 3 , 4 ) and absence of UFL1 / UFBP1 ( Lane 1 , 2 ) . ( Bottom ) Graphical representation of the linkage composition of di - UFM1 chains obtained from LC - MS / MS analysis . Immunoblot analysing polyUFMylation ( top ) and UFL1 autoUFMylation ( bottom ) in the presence of indicated Lys to Arg ( K - R ) and Lys - less ( K0 ) mutants of UFM1 . Immunoblot showing in vitro UFMylation assay to check for formation of di - UFM1 chains in the presence of WT , Lys - less ( K0 ) and single lys mutants ( K only ) of UFM1 .

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Figure 1Immunoblot comparing UFL1 autoUFMylation (Top) and UFM1 chain synthesis (Middle) in the presence of UFL1 expressed alone and in complex with UFBP1. (Bottom) Assays to monitor UFMylation of substrates using purified TRIP4. Reaction products were separated on a 4-12% SDS-PAGE gel under reducing conditions and analysed by immunoblotting using indicated antibodies.In vitro reconstitution of 60S ribosome UFMylation. Purified 60S ribosome was incubated with either UFL1 on its own or UFL1/UFBP1 complex together with UBA5, UFC1 and UFM1 for indicated time points at 30°C.Immunoblot showing formation of free UFM1 chains in the presence (Lane 3,4) and absence of UFL1/UFBP1 (Lane 1,2). (Bottom) Graphical representation of the linkage composition of di-UFM1 chains obtained from LC-MS/MS analysis.Immunoblot analysing polyUFMylation (top) and UFL1 autoUFMylation (bottom) in the presence of indicated Lys to Arg (K-R) and Lys-less(K0) mutants of UFM1.Immunoblot showing in vitro UFMylation assay to check for formation of di-UFM1 chains in the presence of WT, Lys-less (K0) and single lys mutants (K only) of UFM1.


Figure 2Coomassie stained gels monitoring discharge of UFM1 from UFC1 in the presence of indicated free amino acids . UBE2D3 is used as a positive control for lysine and cysteine discharge . Immunoblot showing in vitro UFMylation assays in the presence of Cys to Ala ( indicated as C - A ) mutants of UFL1 to check for UFBP1 UFMylation ( top ) , UFL1 autoUFMylation ( middle ) and substrate UFMylation ( bottom ) . Single - turnover lysine discharge assays in the presence and absence of full length UFL1 / UFBP1 complex . The reaction was stopped by addition of non - reducing SDS loading buffer ( 1x final ) and analysed for discharge on a 4 - 12 % SDS PAGE gel under non - reducing conditions . Top gel - LI - COR scan of fluorescently labelled UFM1 ( UFM1 * ) ; bottom gel - Coomassie

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Figure 2Coomassie stained gels monitoring discharge of UFM1 from UFC1 in the presence of indicated free amino acids. UBE2D3 is used as a positive control for lysine and cysteine discharge.Immunoblot showing in vitro UFMylation assays in the presence of Cys to Ala (indicated as C-A) mutants of UFL1 to check for UFBP1 UFMylation (top), UFL1 autoUFMylation (middle) and substrate UFMylation (bottom).Single-turnover lysine discharge assays in the presence and absence of full length UFL1/UFBP1 complex. The reaction was stopped by addition of non-reducing SDS loading buffer (1x final) and analysed for discharge on a 4-12% SDS PAGE gel under non-reducing conditions. Top gel - LI-COR scan of fluorescently labelled UFM1(UFM1*); bottom gel - Coomassie stained


Figure 3Immunoblot from immunoprecipitation ( IP ) assays to validate the predicted model of UFL1 / UFBP1 interaction in cells . UFL1WT - 3xFLAG and UFL1L45R - 3xFLAG were transiently overexpressed along with UFBP1 - SBP in HEK293T - UFL1 KO cells and subjected to separate pulldowns using anti - FLAG antibody or streptavidin . Immunoprecipitated material was run on a 4 - 12 % SDS PAGE gel and analysed by immunoblotting using indicated antibodies . Immunoblot showing in vitro UFMylation assay to monitor formation of free di - UFM1 chains in the presence of UFL1 / UFBP1 complexes bearing different domain boundaries . Immunoblot of in vitro assay monitoring UFMylation of 60S Ribosome in the presence of different UFL1 / UFBP1 truncations as depicted in ( E ) .

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Figure 3Immunoblot from immunoprecipitation (IP) assays to validate the predicted model of UFL1/UFBP1 interaction in cells. UFL1WT-3xFLAG and UFL1L45R-3xFLAG were transiently overexpressed along with UFBP1-SBP in HEK293T-UFL1 KO cells and subjected to separate pulldowns using anti-FLAG antibody or streptavidin. Immunoprecipitated material was run on a 4-12% SDS PAGE gel and analysed by immunoblotting using indicated antibodies.Immunoblot showing in vitro UFMylation assay to monitor formation of free di-UFM1 chains in the presence of UFL1/UFBP1 complexes bearing different domain boundaries.Immunoblot of in vitro assay monitoring UFMylation of 60S Ribosome in the presence of different UFL1/UFBP1 truncations as depicted in (E).


Figure 4In vitro UFMylation assay in the presence of increasing concentrations of CDK5RAP3 to monitor the E3 ligase activity of UFL1 / UFBP1 complex . The reaction products were run on a 4 - 12 % SDS - PAGE gel under reducing conditions and immunoblotting was performed using indicated antibodies . Single turnover lysine discharge assays to check for activation of UFC1 by UFL1 / UFBP1 in the presence and absence of CDK5RAP3 . The reaction products were run on a 4 - 12 % SDS PAGE and visualized by Oriole staining . Quantification of discharge of UFM1 from UFC1 in the presence and absence of CDK5RAP3 as seen in Fig 4D , n = 3 biological replicates , Substrate UFMylation assays using purified 60S Ribosomes in the presence of increasing concentration of CDK5RAP3 . Reaction was performed for 10 min , stopped by addition of SDS Loading dye and analysed on an SDS - PAGE gel under reducing conditions followed by immunoblotting with indicated antibodies . Immunoblot analysis monitoring UFMylation of Histone H4 in the presence of increasing concentrations of CDK5RAP3 ( 1 µM , 2 µM , 3 µM ) . UFL1 / UFBP1 concentrations - 1 µM .

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Figure 4In vitro UFMylation assay in the presence of increasing concentrations of CDK5RAP3 to monitor the E3 ligase activity of UFL1/UFBP1 complex. The reaction products were run on a 4-12% SDS-PAGE gel under reducing conditions and immunoblotting was performed using indicated antibodies.Single turnover lysine discharge assays to check for activation of UFC1 by UFL1/UFBP1 in the presence and absence of CDK5RAP3. The reaction products were run on a 4-12% SDS PAGE and visualized by Oriole staining.   Quantification of discharge of UFM1 from UFC1 in the presence and absence of CDK5RAP3 as seen in Fig 4D, n=3 biological replicates,  Substrate UFMylation assays using purified 60S Ribosomes in the presence of increasing concentration of CDK5RAP3. Reaction was performed for 10 min, stopped by addition of SDS Loading dye and analysed on an SDS-PAGE gel under reducing conditions followed by immunoblotting with indicated antibodies.Immunoblot analysis monitoring UFMylation of Histone H4 in the presence of increasing concentrations of CDK5RAP3 (1 µM, 2 µM, 3 µM). UFL1/UFBP1 concentrations - 1 µM.


Figure 5Immunoblot showing the in vitro UFMylation assay to compare the activity of UFC1 Wild type and mutants in the presence of UFL1 / UFBP1 complex . In vitro UFMylation assay to compare the activity of UFC1 Wild type and UFC1 T106I in the presence of UFL1 / UFBP1 complex . Substrate UFMylation assays using purified 60S Ribosomes in the presence of UFC1 Wild type and different mutants of UFC1 .

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Figure 5Immunoblot showing the in vitro UFMylation assay to compare the activity of UFC1 Wild type and mutants in the presence of UFL1/UFBP1 complex.   In vitro UFMylation assay to compare the activity of UFC1 Wild type and UFC1 T106I in the presence of UFL1/UFBP1 complex.  Substrate UFMylation assays using purified 60S Ribosomes in the presence of UFC1 Wild type and different mutants of UFC1.


Figure 6In vitro UFMylation assays in the presence of Wild type UFC1 ( UFC1WT ) and UFC1 lacking the N - terminal helix ( UFC1ΔN ) . Assay to compare Lys discharge activities of UFC1WT and UFC1ΔN on its own , in the presence of UFL1 / UFBP1 or in the presence of preassembled UFL1 / UFBP1 / CDK5RAP3 complex . Top gel : LI - COR scan of fluorescently labelled UFM1 ( UFM1 * ) ; bottom gel - Coomassie stained ( Representative of three independent experiments ) . H . Effect of deletion of N - term helix of UFC1 in 60S ribosome UFMylation . Purified 60S ribosomes ( 50 nM ) were incubated with 0 . 5 µM UBA5 , 1 µM UFM1 , 100 nM UFL1 / UFBP1 complex in the presence of either 1 µM UFC1WT or 1 µM UFC1ΔN at 30 ° C . The reaction was stopped at indicated time points and run on a 4 - 12 % SDS PAGE gel under reducing conditions followed by immunoblotting using indicated antibodies . ( Right - shorter time duration . Left - Longer time duration ) .

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Figure 6In vitro UFMylation assays in the presence of Wild type UFC1 (UFC1WT) and UFC1 lacking the N-terminal helix (UFC1ΔN).Assay to compare Lys discharge activities of UFC1WT and UFC1ΔN on its own, in the presence of UFL1/UFBP1 or in the presence of preassembled UFL1/UFBP1/CDK5RAP3 complex. Top gel: LI-COR scan of fluorescently labelled UFM1(UFM1*); bottom gel - Coomassie stained (Representative of three independent experiments).H. Effect of deletion of N -term helix of UFC1 in 60S ribosome UFMylation. Purified 60S ribosomes (50 nM) were incubated with 0.5 µM UBA5, 1 µM UFM1, 100 nM UFL1/UFBP1 complex in the presence of either 1 µM UFC1WT or 1 µM UFC1ΔN at 30°C. The reaction was stopped at indicated time points and run on a 4-12% SDS PAGE gel under reducing conditions followed by immunoblotting using indicated antibodies. (Right - shorter time duration. Left - Longer time duration).


Figure 1A Combined IF and DNA FISH analysis of RAD54 ( IF ) and telomeres ( FISH ) in ALT and non - ALT cell lines . White arrows indicate RAD54 foci that colocalize with telomeres . Scale bars = 10 µm B Quantification of data in A . A cell was counted positive if it contained 1 or more colocalization event between RAD54 and the telomere . At least 100 cells were counted per cell line per repeat . For SaOS2 , NOS , SJSA1 , HeLa LT n = 3 . For Cal72 , U2OS , HuO9 n = 4 . Values shown are mean ± sd Combined IF and DNA FISH analysis of RAD54 ( IF ) , PML ( IF ) and telomeres ( FISH ) in ALT cell lines SaOS2 and HuO9 . Scale bars = 10 µmWestern blot showing expression of TRF1 - FOK1 - mCherry fusion protein following doxycycline induction , both WT and D450A ( nuclease dead ) . Cells were treated with 40 ng / mL doxycycline for 16 hrs and then 1µM Shield - 1 and 1µM 4 - OHT for 4 hrsCombined IF and DNA FISH for RAD54 ( IF ) and telomeres ( FISH ) in U2OS - TRF1 - FOK1 - WT or U2OS - TRF1 - FOK1 - D450A ( nuclease dead ) cells . Cells were treated as in E immediately prior to staining . Scale bars = 10 µm Quantification of data from F . RAD54 foci were detected using particle analysis in Fiji . At least a total of 300 cells were counted from 3 repeated experiments . Values shown are mean ± sem . Values were compared using Kruskal - Wallis test followed by Dunn ' s multiple comparison test . Quantification of data from F . RAD54 foci were detected using particle analysis in Fiji . Cells were counted as positive if they counted 8 or more RAD54 foci that colocalized with telomeres . Values shown are mean ± sem of n = 3 biological replicates . Values were compared using standard two - way ANOVA followed by Sidak ' s multiple comparison test .

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Figure 1A Combined IF and DNA FISH analysis of RAD54 (IF) and telomeres (FISH) in ALT and non-ALT cell lines. White arrows indicate RAD54 foci that colocalize with telomeres. Scale bars = 10 µm B Quantification of data in A. A cell was counted positive if it contained 1 or more colocalization event between RAD54 and the telomere. At least 100 cells were counted per cell line per repeat. For SaOS2, NOS, SJSA1, HeLa LT n = 3. For Cal72, U2OS, HuO9 n = 4. Values shown are mean±sd Combined IF and DNA FISH analysis of RAD54 (IF), PML (IF) and telomeres (FISH) in ALT cell lines SaOS2 and HuO9. Scale bars = 10 µmWestern blot showing expression of TRF1-FOK1-mCherry fusion protein following doxycycline induction, both WT and D450A (nuclease dead). Cells were treated with 40 ng/mL doxycycline for 16 hrs and then 1µM Shield-1 and 1µM 4-OHT for 4 hrsCombined IF and DNA FISH for RAD54 (IF) and telomeres (FISH) in U2OS-TRF1-FOK1-WT or U2OS-TRF1-FOK1-D450A (nuclease dead) cells. Cells were treated as in E immediately prior to staining. Scale bars = 10 µm Quantification of data from F. RAD54 foci were detected using particle analysis in Fiji. At least a total of 300 cells were counted from 3 repeated experiments. Values shown are mean±sem. Values were compared using Kruskal-Wallis test followed by Dunn's multiple comparison test. Quantification of data from F. RAD54 foci were detected using particle analysis in Fiji. Cells were counted as positive if they counted 8 or more RAD54 foci that colocalized with telomeres. Values shown are mean±sem of n=3 biological replicates. Values were compared using standard two-way ANOVA followed by Sidak's multiple comparison test.


Figure 2Combined IF and DNA FISH for RAD51 ( IF ) and telomeres ( FISH ) in SaOS2 or HuO9 cells . Cells were transfected with 20nM siRAD54 # 2 for 48 hours immediately prior to staining . Scale bars = 10 µm Quantification of A . A cell was counted as positive it is contained at least 1 colocalization event between RAD51 and telomeres . At least 100 cells were counted per condition per repeat , n = 3 . Values shown are mean ± sd . Data were compared using standard two - way ANOVA followed by Sidak ' s multiple comparison test . ChIP for telomeric DNA associated with RAD51 . DNA dot blot probed with DIG labeled telomere probe or Alu probe . HuO9 cells were transfected with 20nM siRAD54 # 2 , or 15µg myc - TRF2 for 72 hours prior to immunoprecipitation with RAD51 antibody ( Mock , siRAD54 conditions ) or myc antibody ( myc - TRF2 positive control ) . Quantification of ( CCCTAA ) 4 dot blot performed in C . IP values were normalized to loading ( IP / input ) and then normalized to mock control . Values shown are mean ± sd of n = 3 biological replicates . Values were compared using two - tailed unpaired student ' s t - test . Western blot of samples used for ChIP in C - D . Combined IF and DNA FISH for GFP ( IF ) and telomeres ( FISH ) in SaOS2 and HuO9 cells . Cells were forward transfected with Polη - GFP , and then after 24 hours transfected with 20nM siRAD54 # 2 . Cells were stained for GFP and telomeres 48 hours after siRNA transfection . Scale bars = 10 µm Quantification of data in F . GFP negative cells were excluded from analysis . Cells were considered positive if they showed 1 or more colocalization events between GFP ( Polη ) and telomeres . At least 50 GFP positive cells were counted per condition per repeat , n = 3 . Values shown are mean ± sd . Values were compared using standard two - way ANOVA followed by Sidak ' s multiple comparison test .

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Figure 2Combined IF and DNA FISH for RAD51 (IF) and telomeres (FISH) in SaOS2 or HuO9 cells. Cells were transfected with 20nM siRAD54#2 for 48 hours immediately prior to staining. Scale bars = 10 µm Quantification of A. A cell was counted as positive it is contained at least 1 colocalization event between RAD51 and telomeres. At least 100 cells were counted per condition per repeat, n=3. Values shown are mean±sd. Data were compared using standard two-way ANOVA followed by Sidak's multiple comparison test.ChIP for telomeric DNA associated with RAD51. DNA dot blot probed with DIG labeled telomere probe or Alu probe. HuO9 cells were transfected with 20nM siRAD54 #2, or 15µg myc-TRF2 for 72 hours prior to immunoprecipitation with RAD51 antibody (Mock, siRAD54 conditions) or myc antibody (myc-TRF2 positive control). Quantification of (CCCTAA)4 dot blot performed in C. IP values were normalized to loading (IP/input) and then normalized to mock control. Values shown are mean±sd of n=3 biological replicates. Values were compared using two-tailed unpaired student's t-test.Western blot of samples used for ChIP in C-D.Combined IF and DNA FISH for GFP (IF) and telomeres (FISH) in SaOS2 and HuO9 cells. Cells were forward transfected with Polη-GFP, and then after 24 hours transfected with 20nM siRAD54#2. Cells were stained for GFP and telomeres 48 hours after siRNA transfection. Scale bars = 10 µm Quantification of data in F. GFP negative cells were excluded from analysis. Cells were considered positive if they showed 1 or more colocalization events between GFP (Polη) and telomeres. At least 50 GFP positive cells were counted per condition per repeat, n=3. Values shown are mean±sd. Values were compared using standard two-way ANOVA followed by Sidak's multiple comparison test.


Figure 3Representative images of combined EdU staining ( Click - it ) and DNA FISH for telomeres . HuO9 or SaOS2 cells were transfected with 20nM siRAD54 # 2 ( representative images for siRAD54 # 1 not shown ) for 48 hours followed by a 1 . 5 hr pulse of EdU . Pan - nuclear EdU stain represents S - phase cells . White arrows indicate non - S - phase cells containing EdU colocalizing with telomeres . Scale bars = 10 µm Quantification of data shown in A . HuO9 or SaOS2 cells were mock transfected or transfected with 20nM siRAD54 # 1 or 20nM siRAD54 # 2 for 48 hours followed by a 1 . 5 hr pulse of EdU . Cells with pan - nuclear EdU signal or greater than 8 EdU foci were excluded from analysis as S - phase cells . Non - S - phase cells were considered positive if they contained at least one EdU foci colocalizing with telomeres . Data were normalized to mock condition for each repeat . At least 100 non - S - phase cells were counted per condition per repeat , n = 3 . Values shown are mean ± sd . Data were analyzed using two - way ANOVA followed by Dunnett ' s multiple comparison test . Representative DNA dot blot from C - circle assay on HuO9 cells , either mock transfected or transfected with 20nM siRAD54 # 2 for 48 hours . Quantification of C - circle assay in C . Signal was quantified with densitometry , background ( - Φ29 ) was subtracted and signal was normalized to mock . N = 5 biological replicates values shown are mean ± sd . Conditions were analyzed using unpaired two - tailed student ' s t - test . Representative images of combined IF and DNA FISH for PML ( IF ) and telomeres ( FISH ) . HuO9 or SaOS2 cells were mock transfected or transfected with 20nM siRAD54 # 2 for 48 hours . White arrows indicate large colocalization events . Blue arrows indicate small colocalization events . Scale bars = 10 µm Quantification HuO9 or SaOS2 cells were mock transfected or transfected with 20nM siRAD54 # 1 or 20nM siRAD54 # 2 for 48 hours . Cells were considered positive if they contained 1 large or 3 small colocalization events . At least 100 cells were counted per condition per repeat , n = 3 . Values shown are mean ± sd . Data were analyzed using two - way ANOVA followed by Dunnett ' s multiple comparison test . Representative images of combined EdU staining ( Click - it ) and DNA FISH for telomeric DNA . HuO9 cells were forward transfected with 2µg empty vector ( EV ) , RAD54 - WT , RAD54 - K189R , or S49E construct . After 24 hours , cells were transfected with 20 nM siRAD54 # 2 for 48 hours followed by a 1 . 5 hr pulse of EdU . White arrows indicate non - S - phase cells containing EdU colocalizing with telomeres . Scale bars = 10 µm Quantification of data shown in G . Non - S - phase cells were considered positive if they contained at least one EdU foci colocalizing with telomeres . Data were normalized to EV - Mock condition for each repeat . At least 100 non - S - phase cells were counted per condition per repeat , n = 4 for EV , WT , S49E , n = 3 for K189R . Values shown are mean ± sd . Data were analyzed using two - way ANOVA followed by Sidak ' s multiple comparison test .

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Figure 3Representative images of combined EdU staining (Click-it) and DNA FISH for telomeres. HuO9 or SaOS2 cells were transfected with 20nM siRAD54#2 (representative images for siRAD54#1 not shown) for 48 hours followed by a 1.5 hr pulse of EdU. Pan-nuclear EdU stain represents S-phase cells. White arrows indicate non-S-phase cells containing EdU colocalizing with telomeres. Scale bars = 10 µm Quantification of data shown in A. HuO9 or SaOS2 cells were mock transfected or transfected with 20nM siRAD54#1 or 20nM siRAD54#2 for 48 hours followed by a 1.5 hr pulse of EdU. Cells with pan-nuclear EdU signal or greater than 8 EdU foci were excluded from analysis as S-phase cells. Non-S-phase cells were considered positive if they contained at least one EdU foci colocalizing with telomeres. Data were normalized to mock condition for each repeat. At least 100 non-S-phase cells were counted per condition per repeat, n=3. Values shown are mean±sd. Data were analyzed using two-way ANOVA followed by Dunnett's multiple comparison test.Representative DNA dot blot from C-circle assay on HuO9 cells, either mock transfected or transfected with 20nM siRAD54#2 for 48 hours. Quantification of C-circle assay in C. Signal was quantified with densitometry, background (- Φ29) was subtracted and signal was normalized to mock. N=5 biological replicates values shown are mean±sd. Conditions were analyzed using unpaired two-tailed student's t-test.Representative images of combined IF and DNA FISH for PML (IF) and telomeres (FISH). HuO9 or SaOS2 cells were mock transfected or transfected with 20nM siRAD54#2 for 48 hours. White arrows indicate large colocalization events. Blue arrows indicate small colocalization events. Scale bars = 10 µm Quantification HuO9 or SaOS2 cells were mock transfected or transfected with 20nM siRAD54#1 or 20nM siRAD54#2 for 48 hours. Cells were considered positive if they contained 1 large or 3 small colocalization events. At least 100 cells were counted per condition per repeat, n=3. Values shown are mean±sd. Data were analyzed using two-way ANOVA followed by Dunnett's multiple comparison test.Representative images of combined EdU staining (Click-it) and DNA FISH for telomeric DNA. HuO9 cells were forward transfected with 2µg empty vector (EV), RAD54-WT, RAD54-K189R, or S49E construct. After 24 hours, cells were transfected with 20 nM siRAD54#2 for 48 hours followed by a 1.5 hr pulse of EdU. White arrows indicate non-S-phase cells containing EdU colocalizing with telomeres. Scale bars = 10 µm Quantification of data shown in G. Non-S-phase cells were considered positive if they contained at least one EdU foci colocalizing with telomeres. Data were normalized to EV-Mock condition for each repeat. At least 100 non-S-phase cells were counted per condition per repeat, n=4 for EV, WT, S49E, n=3 for K189R. Values shown are mean±sd. Data were analyzed using two-way ANOVA followed by Sidak's multiple comparison test.


Figure 4Representative images of combined IF and DNA FISH for BLM ( IF ) and telomeres ( FISH ) . HuO9 cells were transfected with 20nM siRAD54 # 2 for 48 hours . Scale bars = 10 µm Quantification of data shown in A . Cells were counted positive if they contained at least 1 colocalization event between BLM and telomeres . Data were normalized to mock condition of each repeat . At least 100 cells per condition per repeat were counted , n = 3 . Values shown are mean ± sd . Conditions were analyzed using unpaired two - tailed student ' s t - test . Representative images of IF - DNA FISH for MUS81 and telomeres . HuO9 cells were transfected with 20nM siRAD54 # 2 for 48 hours prior to staining . Scale bars = 10 µm Quantification of data shown in C . Cells were counted positive if they contained at least 1 colocalization event between MUS81 and telomeres . At least 100 cells per condition were counted per repeat . n = 5 , values shown are mean ± sd . Conditions were analyzed using unpaired two - tailed student ' s t - test . Representative images of HuO9 and SaOS2 chromosomes with or without an exchange event . Colocalization of green and red signal indicates an exchange event . Quantification of telomere sister chromatid exchange events . Cells were transfected with 20nM of siRNA for 48 hours prior to the addition of BrdU / BrdC . Metaphases were imaged by confocal microscopy and each data point represents a metaphase . For each condition , there were 3 biological repeats with a total of at least 1300 chromosome ends quantified . Values shown are mean ± sem . Conditions were analyzed using two - way ANOVA followed by Sidak ' s test . Representative images of UFB detection staining for PICH in HuO9 cells . Cells were transfected with 20nM of noted siRNAs for 72 hrs . Scale bars = 10 µmQuantification of data shown in G . Anaphases were scored from 4 independent experiments ( mock ) , or 3 independent experiments ( remaining conditions ) . Anaphases were considered positive for UFB if there was PICH staining between DAPI bodies . Single knockdown conditions had > 50 anaphases scored . Combined knockdown conditions had > 88 anaphases scored . Values shown are mean ± sd . Conditions were compared using one - way ANOVA followed by Dunnett ' s multiple comparison test . Quantification of data shown in G . Number of PICH stained UFBs counted per anaphase . Values shown are total counts over 3 independent experiments , shown mean ± sem . Total number of anaphases per condition were as follows : Mock n = 134 , siBLM / siSLX4 n = 90 , siRAD54 / siSLX4 n = 96 , siRAD54 / siBLM / siSLX4 n = 88 . Conditions were compared using Kruskal - Wallis test followed by Dunn ' s multiple comparison test . Representative images of UFB staining for TRF2 in HuO9 cells . Cells were transfected with 20nM siRAD54 # 2 alone or cotransfected siRAD54 # 2 and siSLX4 for 72 hours prior to staining . Scale bars = 10 µmQuantification of data shown in J . Anaphases were scored from 3 independent experiments . A total of 90 anaphases were scored per condition . Values shown are mean ± sd . Conditions were compared using one - way ANOVA followed by Dunnett ' s multiple comparison test . Quantification of micronuclei from HuO9 cells transfected with siRAD54 # 2 alone or siRAD54 # 2 cotransfected with siSLX4 for 48 hours . Micronuclei containing telomeric DNA were identified using DAPI and telomere FISH . Micronuclei were normalized to the number of nuclei counted . Values shown are mean ± sd of n = 3 biological replicates . Conditions were compared using one - way ANOVA followed by Dunnett ' s multiple comparison test .

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

Figure 4Representative images of combined IF and DNA FISH for BLM (IF) and telomeres (FISH). HuO9 cells were transfected with 20nM siRAD54#2 for 48 hours. Scale bars = 10 µm Quantification of data shown in A. Cells were counted positive if they contained at least 1 colocalization event between BLM and telomeres. Data were normalized to mock condition of each repeat. At least 100 cells per condition per repeat were counted, n=3. Values shown are mean±sd. Conditions were analyzed using unpaired two-tailed student's t-test.Representative images of IF-DNA FISH for MUS81 and telomeres. HuO9 cells were transfected with 20nM siRAD54 #2 for 48 hours prior to staining. Scale bars = 10 µm Quantification of data shown in C. Cells were counted positive if they contained at least 1 colocalization event between MUS81 and telomeres. At least 100 cells per condition were counted per repeat. n=5, values shown are mean±sd. Conditions were analyzed using unpaired two-tailed student's t-test.Representative images of HuO9 and SaOS2 chromosomes with or without an exchange event. Colocalization of green and red signal indicates an exchange event.Quantification of telomere sister chromatid exchange events. Cells were transfected with 20nM of siRNA for 48 hours prior to the addition of BrdU/BrdC. Metaphases were imaged by confocal microscopy and each data point represents a metaphase. For each condition, there were 3 biological repeats with a total of at least 1300 chromosome ends quantified. Values shown are mean±sem. Conditions were analyzed using two-way ANOVA followed by Sidak's test.Representative images of UFB detection staining for PICH in HuO9 cells. Cells were transfected with 20nM of noted siRNAs for 72 hrs. Scale bars = 10 µmQuantification of data shown in G. Anaphases were scored from 4 independent experiments (mock), or 3 independent experiments (remaining conditions). Anaphases were considered positive for UFB if there was PICH staining between DAPI bodies. Single knockdown conditions had >50 anaphases scored. Combined knockdown conditions had >88 anaphases scored. Values shown are mean±sd. Conditions were compared using one-way ANOVA followed by Dunnett's multiple comparison test. Quantification of data shown in G. Number of PICH stained UFBs counted per anaphase. Values shown are total counts over 3 independent experiments, shown mean±sem. Total number of anaphases per condition were as follows: Mock n=134, siBLM/siSLX4 n=90, siRAD54/siSLX4 n=96, siRAD54/siBLM/siSLX4 n=88. Conditions were compared using Kruskal-Wallis test followed by Dunn's multiple comparison test.Representative images of UFB staining for TRF2 in HuO9 cells. Cells were transfected with 20nM siRAD54#2 alone or cotransfected siRAD54#2 and siSLX4 for 72 hours prior to staining. Scale bars = 10 µmQuantification of data shown in J. Anaphases were scored from 3 independent experiments. A total of 90 anaphases were scored per condition. Values shown are mean±sd. Conditions were compared using one-way ANOVA followed by Dunnett's multiple comparison test. Quantification of micronuclei from HuO9 cells transfected with siRAD54#2 alone or siRAD54#2 cotransfected with siSLX4 for 48 hours. Micronuclei containing telomeric DNA were identified using DAPI and telomere FISH. Micronuclei were normalized to the number of nuclei counted. Values shown are mean±sd of n=3 biological replicates. Conditions were compared using one-way ANOVA followed by Dunnett's multiple comparison test.


Figure 1 ( A ) Frequency of CHD1 gene mutation ( green ) deep deletion ( blue ) or amplification ( red ) in prostate cancer patients . ( B ) CHD1 is recruited to an I - SceI - induced DSB site and is co - localized with γH2AX . Immunofluorescence studies using U2OS19 ptight13 GFP - LacR cells containing a stably integrated I - SceI cleavage site flanked by 256 copies of lac operator ( lacO ) on one side and 96 copies of the tetracycline response element on the other side ( tetO ) . The localization of the GFP - lac repressor protein ( GFP - LacR ) at the lac - operator DNA sequences in the nucleus before ( − I - SceI ) and 16 h after I - SceI - induced ( + I - SceI ) DSB . After 16 h of doxy treatment , CHD1 and γH2AX co - localized at I - SceI cleavage site , along with DNA - bound GFP - LacR but not in uninduced cells ( − I - SceI ) . Scale bar 10 µm . ( C ) Quantification of co - localization of CHD1 with the lac array , mean values of three independent experiments counting at least 100 cells are represented in the graph as ± SD . ( D ) PC3 cells were treated with NCS ( 100 ng / ml ) for 2 h and with EdU for 45 min . Cells were stained with EdU first and then proximity ligation assay ( PLA ) was performed using γH2AX and CHD1 antibodies . Scale bar 10 µm . ( E ) Quantification of PLA signal from ( D ) in EdU - positive and - negative cells using ImageJ . For quantification more than 100 cells were analyzed for each condition and represented as mean value ( n = 3 , ± SD ) . ( F ) CHD1 is recruited to the chromatin upon DNA double strand break induction . PC3 cells with stable control ( shCont ) or CHD1 shRNA ( shCHD1 ) expression were treated with NCS for the indicated time points and chromatin fractions were immunoblotted with CHD1 and γH2AX antibodies . H2B was used as a loading control . See also Figure EV1A - EV1F .

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

Figure 1(A) Frequency of CHD1 gene mutation (green) deep deletion (blue) or amplification (red) in prostate cancer patients.(B) CHD1 is recruited to an I-SceI-induced DSB site and is co-localized with γH2AX. Immunofluorescence studies using U2OS19 ptight13 GFP-LacR cells containing a stably integrated I-SceI cleavage site flanked by 256 copies of lac operator (lacO) on one side and 96 copies of the tetracycline response element on the other side (tetO). The localization of the GFP-lac repressor protein (GFP-LacR) at the lac-operator DNA sequences in the nucleus before (− I-SceI) and 16 h after I-SceI-induced (+ I-SceI) DSB. After 16 h of doxy treatment, CHD1 and γH2AX co-localized at I-SceI cleavage site, along with DNA-bound GFP-LacR but not in uninduced cells (−I-SceI). Scale bar 10 µm.(C) Quantification of co-localization of CHD1 with the lac array, mean values of three independent experiments counting at least 100 cells are represented in the graph as ± SD.(D) PC3 cells were treated with NCS (100 ng/ml) for 2 h and with EdU for 45 min. Cells were stained with EdU first and then proximity ligation assay (PLA) was performed using γH2AX and CHD1 antibodies. Scale bar 10 µm.(E) Quantification of PLA signal from (D) in EdU-positive and -negative cells using ImageJ. For quantification more than 100 cells were analyzed for each condition and represented as mean value (n=3, ± SD).(F) CHD1 is recruited to the chromatin upon DNA double strand break induction. PC3 cells with stable control (shCont) or CHD1 shRNA (shCHD1) expression were treated with NCS for the indicated time points and chromatin fractions were immunoblotted with CHD1 and γH2AX antibodies. H2B was used as a loading control. See also Figure EV1A-EV1F.


Figure 2 ( A , B ) BHP1 or PC3 cells with stable expression of either control ( shCont ) or CHD1 shRNA ( shCHD1 ) were treated with γ - radiation ( 3 Gy ) and after 1 h and 24 h cells were immunostained for γH2AX ( A ) and the number of γH2AX foci per cell were determined for each time point , more than 50 cells were counted in each condition ( B ) . Scale bar 5 µm . The mean values of three independent experiments ( ± SD ) are shown . p - values ( 0 . 0008 and 0 . 006 , * * p ≤ 0 . 01 , * * * p ≤ 0 . 001 ) were calculated using ANOVA . ( C ) CHD1 depleted cells show prolonged γH2AX accumulation . PC3 cells with shCont or shCHD1 were treated with γ - radiation as in ( A ) and total protein was analyzed by Western blot . ( D ) CHD1 - depleted cells show increased sensitivity to γ - radiation . For colony formation assay , both BHP1 and PC3 cells with shCont or shCHD1 were treated with the indicated doses of γ - radiation and surviving fractions were measured by counting colonies after 3 weeks , mean values are represented in the plot ( n = 3 , ± SD ) . Data were normalized to the plating efficiency . p - values ( 0 . 0009 and 0 . 002 , * * p ≤ 0 . 01 , * * * p ≤ 0 . 001 ) were calculated using ANOVA . See also Figure EV2A - EV2B .

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 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 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

Figure 2(A, B) BHP1 or PC3 cells with stable expression of either control (shCont) or CHD1 shRNA (shCHD1) were treated with γ-radiation (3 Gy) and after 1 h and 24 h cells were immunostained for γH2AX (A) and the number of γH2AX foci per cell were determined for each time point, more than 50 cells were counted in each condition (B). Scale bar 5 µm. The mean values of three independent experiments (± SD) are shown. p-values (0.0008 and 0.006, **p ≤ 0.01, ***p ≤ 0.001) were calculated using ANOVA.(C) CHD1 depleted cells show prolonged γH2AX accumulation. PC3 cells with shCont or shCHD1 were treated with γ-radiation as in (A) and total protein was analyzed by Western blot.(D) CHD1-depleted cells show increased sensitivity to γ-radiation. For colony formation assay, both BHP1 and PC3 cells with shCont or shCHD1 were treated with the indicated doses of γ-radiation and surviving fractions were measured by counting colonies after 3 weeks, mean values are represented in the plot (n=3, ± SD). Data were normalized to the plating efficiency. p-values (0.0009 and 0.002, **p ≤ 0.01, ***p ≤ 0.001) were calculated using ANOVA. See also Figure EV2A-EV2B.


Figure 3 ( A , B ) HeLa cells harboring single copies of HR ( pGC ) or NHEJ ( pEJ ) repair substrates were transfected with either negative control ( siCont ) or siRNAs targeting CHD1 , RAD51 or XRCC4 . After 24 h of transfection , DSB was induced by transfecting cells with I - Sce - I - expressing vector ( pCMV - I - SceI - 3xNLS ) . After 48 h of transfection , GFP - positive cells were measured by flow cytometry . HR ( A ) or NHEJ ( B ) efficiency was calculated based on the fraction of GFP - positive cells and represented as mean values from three independent experiments as ± SD , 50 , 000 cells were counted for each condition . ( C ) CHD1 - depleted cells show decreased RAD51 foci after DNA damage induction . The shCont or shCHD1 PC3 cells were irradiated and co - immunostained with γH2AX and RAD51 antibodies after the indicated time points . Scale bar 5 µm . ( D ) The number of RAD51 foci co - localized with γH2AX per cell ( from C ) were counted ( n = 50 ) and represented in a graph . The data is represented in mean value of three independent experiments as ± SD , more than 50 cells were counted for each condition . See also Figure EV3A - EV3F .

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

Figure 3(A, B) HeLa cells harboring single copies of HR (pGC) or NHEJ (pEJ) repair substrates were transfected with either negative control (siCont) or siRNAs targeting CHD1, RAD51 or XRCC4. After 24 h of transfection, DSB was induced by transfecting cells with I-Sce-I-expressing vector (pCMV-I-SceI-3xNLS). After 48 h of transfection, GFP-positive cells were measured by flow cytometry. HR (A) or NHEJ (B) efficiency was calculated based on the fraction of GFP-positive cells and represented as mean values from three independent experiments as ± SD, 50,000 cells were counted for each condition.(C) CHD1-depleted cells show decreased RAD51 foci after DNA damage induction. The shCont or shCHD1 PC3 cells were irradiated and co-immunostained with γH2AX and RAD51 antibodies after the indicated time points. Scale bar 5 µm. (D) The number of RAD51 foci co-localized with γH2AX per cell (from C) were counted (n=50) and represented in a graph. The data is represented in mean value of three independent experiments as ± SD, more than 50 cells were counted for each condition. See also Figure EV3A-EV3F.


Figure 4 ( A ) PLA assay with CtIP and CHD1 in PC3 cells after 2 h of NCS treatment . Scale bar 5 µm . ( B ) shCont and shCHD1 PC3 cells were treated with NCS for the indicated times and chromatin fractions were immunoblotted for CtIP , RPA1 and RAD51 . H2B is shown as a loading control . CHD1 depletion leads to decreased ( C ) CtIP and ( D ) RPA1 recruitment to I - SceI - induced DSB in U2OS19 ptight13 GFP - LacR cells . CHD1 was depleted in U2OS19 ptight13 GFP - LacR cells by siRNA and 48 h after transfection cells were treated with doxy for 16 h and co - immunostained for γH2AX and CtIP or RPA1 . The number of cells with CtIP or RPA1 foci from ( C ) and ( D ) were counted and represented the mean in the graph as percentage of foci positive cells as ± SD ( n = 3 ) , more than 50 cells were counted . Scale bar 5 µm . ( E ) PC3 cells were transfected either with mock or siCHD1 followed by empty vector , wt mChd1 ( Wt ) or ATPase - mutant ( Mt ) mChd1 . After 48 h of transfection cells were treated with of 4 - OH tamoxifen ( 4 - OHT ) for 24 h and processed for PLA with γH2AX and CtIP antibodies . Cells demonstrating positive focal interactions indicative of DNA repair hubs ( punctate staining ) are present only in control and ( Wt ) CHD1 - rescued cells but not following knockdown or reconstitution of an ATPase - mutated CHD1 ( Mt ) . Scale bar 20 µm . ( F ) PC3 cells which stably express HA - mChd1 - ERT2 , were transfected with either mock or siCHD1 . After 24 h of transfection cells were treated with 4 - OHT for 24 h to induce HA - mChd1 - ERT2 nuclear translocation . Western blot analysis of chromatin fractions for CHD1 , ERT2 ( mChd1 ) , CtIP and RAD51 shown in Figure EV4O were analyzed by densitometry using ImageJ . The relative quantification for the indicated proteins is shown in the graph . See also Figure EV4A - EV4P .

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

Figure 4(A) PLA assay with CtIP and CHD1 in PC3 cells after 2 h of NCS treatment. Scale bar 5 µm.(B) shCont and shCHD1 PC3 cells were treated with NCS for the indicated times and chromatin fractions were immunoblotted for CtIP, RPA1 and RAD51. H2B is shown as a loading control.CHD1 depletion leads to decreased (C) CtIP and (D) RPA1 recruitment to I-SceI-induced DSB in U2OS19 ptight13 GFP-LacR cells. CHD1 was depleted in U2OS19 ptight13 GFP-LacR cells by siRNA and 48 h after transfection cells were treated with doxy for 16 h and co-immunostained for γH2AX and CtIP or RPA1. The number of cells with CtIP or RPA1 foci from (C) and (D) were counted and represented the mean in the graph as percentage of foci positive cells as ± SD (n=3), more than 50 cells were counted. Scale bar 5 µm.(E) PC3 cells were transfected either with mock or siCHD1 followed by empty vector, wt mChd1 (Wt) or ATPase-mutant (Mt) mChd1. After 48 h of transfection cells were treated with of 4-OH tamoxifen (4-OHT) for 24 h and processed for PLA with γH2AX and CtIP antibodies. Cells demonstrating positive focal interactions indicative of DNA repair hubs (punctate staining) are present only in control and (Wt) CHD1-rescued cells but not following knockdown or reconstitution of an ATPase-mutated CHD1 (Mt). Scale bar 20 µm.(F) PC3 cells which stably express HA-mChd1-ERT2, were transfected with either mock or siCHD1. After 24 h of transfection cells were treated with 4-OHT for 24 h to induce HA-mChd1-ERT2 nuclear translocation. Western blot analysis of chromatin fractions for CHD1, ERT2 (mChd1), CtIP and RAD51 shown in Figure EV4O were analyzed by densitometry using ImageJ. The relative quantification for the indicated proteins is shown in the graph. See also Figure EV4A-EV4P.


Figure 5 ( A ) CHD1 recruitment is dependent on MRE11 activity . PC3 cells were transfected with mock or siMRE11 ( SmartPool ) and 48 h after transfection cells were treated with NCS and chromatin fractions were isolated and analyzed by Western blot . ( B ) CHD1 is upstream of CtIP . PLA with γH2AX and CHD1 antibodies in mock or CtIP depleted cells after 2 h of NCS . Scale bar is 50 µm . Verification of knockdown efficiency is shown by Western blot on the right . HSC70 is shown as a loading control . Scale bar 20 µm . ( C ) Quantification of PLA signal from ( B ) represented as mean values from three independent experiments as ± SD , more than 100 cells were counted per condition . ( D ) qPCR analyses for chromatin accessibility at two HR - repaired sites ( DSB - I and DSB - II ) was analyzed by FAIRE in AsiSI - ER - U20S cells transfected with either mock or CHD1 siRNA . After 48 h of transfection cells were treated with 4 - OHT for the indicated time points and processed for FAIRE . The data are represented as mean ± SD ( n = 3 ) p - values were calculated using ANOVA ( 0 . 02 and 0 . 03 , * p ≤ 0 . 1 ) . ( E ) Native BrdU staining of PC3 cells transfected either with mock or siCHD1 ( SmartPool ) were grown for 48 h and then treated with NCS for 2 h prior to staining with anti - BrdU and γH2AX antibodies . Scale bar 5 µm . ( F ) Quantification of Brdu positive cells from ( E ) . The data are represented as mean ± SD from three independent experiments , more than 50 cells were counted for each condition . p - values ( 0 . 03 , * p ≤ 0 . 1 ) were calculated using ANOVA . See also Figure EV5A - EV4J .

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 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 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 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

Figure 5(A) CHD1 recruitment is dependent on MRE11 activity. PC3 cells were transfected with mock or siMRE11 (SmartPool) and 48 h after transfection cells were treated with NCS and chromatin fractions were isolated and analyzed by Western blot.(B) CHD1 is upstream of CtIP. PLA with γH2AX and CHD1 antibodies in mock or CtIP depleted cells after 2 h of NCS. Scale bar is 50 µm. Verification of knockdown efficiency is shown by Western blot on the right. HSC70 is shown as a loading control. Scale bar 20 µm. (C) Quantification of PLA signal from (B) represented as mean values from three independent experiments as ± SD, more than 100 cells were counted per condition.(D) qPCR analyses for chromatin accessibility at two HR-repaired sites (DSB-I and DSB-II) was analyzed by FAIRE in AsiSI-ER-U20S cells transfected with either mock or CHD1 siRNA. After 48 h of transfection cells were treated with 4-OHT for the indicated time points and processed for FAIRE. The data are represented as mean ± SD (n=3) p-values were calculated using ANOVA (0.02 and 0.03, *p ≤ 0.1).(E) Native BrdU staining of PC3 cells transfected either with mock or siCHD1 (SmartPool) were grown for 48 h and then treated with NCS for 2 h prior to staining with anti-BrdU and γH2AX antibodies. Scale bar 5 µm.(F) Quantification of Brdu positive cells from (E). The data are represented as mean ± SD from three independent experiments, more than 50 cells were counted for each condition. p-values (0.03, *p ≤ 0.1) were calculated using ANOVA. See also Figure EV5A-EV4J.


Figure 6 ( A ) CHD1 - depleted cells show hypersensitivity to Mitomycin ( MMC ) treatment . For colony formation assay shCont and shCHD1 PC3 cells were treated with the indicated MMC concentrations for 4 h and surviving fractions were measured by counting colonies after 3 weeks and mean values were represented ( n = 3 , ± SD , p - value = 0 . 0002 , ANOVA , * * * p ≤ 0 . 001 ) . ( B ) CHD1 - depleted cells show increased hypersensitivity to Irinotecan . For cell proliferation analysis , shCont and shCHD1 cells were treated with 1 µM Irinotecan and proliferation was measured by Celigo and the relative confluency ( mean ) was plotted in the graph ( n = 3 , ± SD , p - value = 0 . 0004 , ANOVA , * * * p ≤ 0 . 001 ) . ( C ) Loss of CHD1 leads to increased sensitivity to PARP inhibition . Control or CHD1 - depleted BPH1 cells were treated with the indicated concentrations of the PARP inhibitor olaparib and surviving fractions were measured by counting colonies after 3 weeks . Data represented as mean values ( n = 3 , ± SD , p - value = 0 . 0003 , ANOVA , * * * p ≤ 0 . 001 ) . ( D ) CHD1 depletion render cells sensitive to PARP inhibition in combination with irradiation . shCont - or shCHD1 - expressing BHP1 cells were treated with 1 µM of PARP inhibitor olaparib for 2 h before irradiation with indicated doses of X - rays and surviving fractions ( normalized to the unirradiated condition ) were measured by counting colonies after 3 weeks . Data is represented as mean ± SD ( n = 3 , p - value = 0 . 07 , ANOVA , * p ≤ 0 . 1 ) . ( E ) Frequency of CHD1 gene alterations in comparison with DSB repair genes in prostate cancer patients . Data obtained from cBioPortal for Cancer Genomics . ( F ) Frequency of CHD1 gene alterations from indicated data sets . See also Figure EV5A - EV5B .

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

Figure 6(A) CHD1-depleted cells show hypersensitivity to Mitomycin (MMC) treatment. For colony formation assay shCont and shCHD1 PC3 cells were treated with the indicated MMC concentrations for 4 h and surviving fractions were measured by counting colonies after 3 weeks and mean values were represented (n=3, ± SD, p-value=0.0002, ANOVA, ***p ≤ 0.001).(B) CHD1-depleted cells show increased hypersensitivity to Irinotecan. For cell proliferation analysis, shCont and shCHD1 cells were treated with 1 µM Irinotecan and proliferation was measured by Celigo and the relative confluency (mean) was plotted in the graph (n=3, ± SD, p-value=0.0004, ANOVA, ***p ≤ 0.001).(C) Loss of CHD1 leads to increased sensitivity to PARP inhibition. Control or CHD1-depleted BPH1 cells were treated with the indicated concentrations of the PARP inhibitor olaparib and surviving fractions were measured by counting colonies after 3 weeks. Data represented as mean values (n=3, ± SD, p-value=0.0003, ANOVA, ***p ≤ 0.001).(D) CHD1 depletion render cells sensitive to PARP inhibition in combination with irradiation. shCont- or shCHD1-expressing BHP1 cells were treated with 1 µM of PARP inhibitor olaparib for 2 h before irradiation with indicated doses of X-rays and surviving fractions (normalized to the unirradiated condition) were measured by counting colonies after 3 weeks. Data is represented as mean ± SD (n=3, p-value=0.07, ANOVA, *p ≤ 0.1).(E) Frequency of CHD1 gene alterations in comparison with DSB repair genes in prostate cancer patients. Data obtained from cBioPortal for Cancer Genomics. (F) Frequency of CHD1 gene alterations from indicated data sets. See also Figure EV5A-EV5B.


Figure 1 ( A ) HOIP , HOIL - 1L , and SHARPIN co - localize with Htt - Q97 aggregates . SH - SY5Y cells co - expressing Htt - Q25 - GFP or Htt - Q97 - GFP ( green ) and HOIP , HOIL - 1L , SHARPIN , or HHARI ( red ) ; DAPI ( blue ) . Scale bar , 10 µm . HOIP co - localizes with Htt inclusion bodies in human HD frontal cortex . Perinuclear Htt aggregates visualized by immunofluorescence ( B ) in human HD frontal cortex . Scale bar , 10 µm ( B ) HOIP co - localizes with Htt inclusion bodies in human HD frontal cortex . Perinuclear Htt aggregates visualized by DAB immunohistochemistry ( C ) in human HD frontal cortex . Scale bar , 15 µm ( 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 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

Figure 1(A) HOIP, HOIL-1L, and SHARPIN co-localize with Htt-Q97 aggregates. SH-SY5Y cells co-expressing Htt-Q25-GFP or Htt-Q97-GFP (green) and HOIP, HOIL-1L, SHARPIN, or HHARI (red); DAPI (blue). Scale bar, 10 µm.HOIP co-localizes with Htt inclusion bodies in human HD frontal cortex. Perinuclear Htt aggregates visualized by immunofluorescence (B) in human HD frontal cortex. Scale bar, 10 µm (B)HOIP co-localizes with Htt inclusion bodies in human HD frontal cortex. Perinuclear Htt aggregates visualized by DAB immunohistochemistry (C) in human HD frontal cortex. Scale bar, 15 µm (C).


Figure 2M1 - linked ubiquitin co - localizes with Htt - polyQ aggregates in cultured SH - SY5Y cells ( A ) Autofluorescent lipofuscin appears in the green channel in human brain . Scale bar , 20 µm ( A ) M1 - linked ubiquitin co - localizes with Htt - polyQ aggregates in cultured R6 / 2 mouse cortex and striatum ( B ) Scale bar 10 µmM1 - linked ubiquitin co - localizes with Htt - polyQ aggregates in cultured human HD frontal cortex ( C ) . Autofluorescent lipofuscin appears in the green channel in human brain . Scale bar , 10 µm ( D ) Htt - Q25 - or Htt - Q97 - expressing HEK293T were analyzed by filter retardation assays . As indicated , the M1 ubiquitin antibody was preincubated with recombinant tetra - ubiquitin linked via M1 ( 4 x M1 ubiquitin ) or K63 ( 4 x K63 ubiquitin ) .

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

Figure 2M1-linked ubiquitin co-localizes with Htt-polyQ aggregates in cultured SH-SY5Y cells (A) Autofluorescent lipofuscin appears in the green channel in human brain. Scale bar, 20 µm (A)M1-linked ubiquitin co-localizes with Htt-polyQ aggregates in cultured R6/2 mouse cortex and striatum (B) Scale bar 10 µmM1-linked ubiquitin co-localizes with Htt-polyQ aggregates in cultured human HD frontal cortex (C). Autofluorescent lipofuscin appears in the green channel in human brain. Scale bar, 10 µm(D) Htt-Q25- or Htt-Q97-expressing HEK293T were analyzed by filter retardation assays. As indicated, the M1 ubiquitin antibody was preincubated with recombinant tetra-ubiquitin linked via M1 (4 x M1 ubiquitin) or K63 (4 x K63 ubiquitin).


Figure 3 ( B ) Quantification of the co - localization of WT HOIP or HOIP mutants and Htt - Q97 aggregates . Data are displayed as mean ± SD and were analyzed by Mann - Whitney U - test , n = 3 . Data information : * p ≤ 0 . 05 , * * * p ≤ 0 . 001 . ( C ) Htt - Q97 - GFP ( green ) was co - expressed with HA - tagged WT or HOIP mutants ( red ) in SH - SY5Y cells and analyzed by immunocytochemistry using an antibody against HA . DAPI ( blue ) . Scale bar , 10 µm . ( D ) Recruitment of HOIP to Htt - Q97 is decreased in p97 / VCP - deficient cells . p97 / VCP - deficient SH - SY5Y cells co - expressing Htt - Q97 and HA - HOIP were reconstituted with either WT VCP or VCP ∆ PIM and analyzed by immunocytochemistry . Data are displayed as mean ± SD and were analyzed by one - way ANOVA followed by Tukey ' s Multiple Comparison Test , n = 9 . Data information : * p ≤ 0 . 05 , * * * p ≤ 0 . 001 . ( E ) Recruitment of p97 / VCP to Htt - Q97 is decreased in HOIP KO cells . WT HAP1 cells ( control ) or HOIP KO HAP1 cells reconstituted with either WT HOIP or HOIP ∆ PUB were analyzed by immunocytochemistry . Data are displayed as mean ± SD and were analyzed by one - way ANOVA followed by Bonferroni Multiple Comparison Test , n = 5 . Data information : * p ≤ 0 . 05 , * * * p ≤ 0 . 001 . ( F ) The interaction of soluble Htt - Q60 with p97 / VCP is dependent on HOIP . WT HAP1 cells or HOIP KO HAP1 cells were transfected with Htt - Q60 - HA . 48 h after transfection cells were lysed with 1 % Triton X - 100 , followed by an immunoprecipitation of Htt - Q60 via the HA tag . Immunopurified proteins were detected by Western blotting using an anti - VCP and anti - HOIP antibody .

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 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 O O O O O O O O O O O O O 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 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 3(B) Quantification of the co-localization of WT HOIP or HOIP mutants and Htt-Q97 aggregates. Data are displayed as mean ± SD and were analyzed by Mann-Whitney U-test, n = 3. Data information: *p ≤ 0.05, ***p ≤ 0.001.(C) Htt-Q97-GFP (green) was co-expressed with HA-tagged WT or HOIP mutants (red) in SH-SY5Y cells and analyzed by immunocytochemistry using an antibody against HA. DAPI (blue). Scale bar, 10 µm.(D) Recruitment of HOIP to Htt-Q97 is decreased in p97/VCP-deficient cells. p97/VCP-deficient SH-SY5Y cells co-expressing Htt-Q97 and HA-HOIP were reconstituted with either WT VCP or VCP∆PIM and analyzed by immunocytochemistry. Data are displayed as mean ± SD and were analyzed by one-way ANOVA followed by Tukey's Multiple Comparison Test, n = 9. Data information: *p ≤ 0.05, ***p ≤ 0.001.(E) Recruitment of p97/VCP to Htt-Q97 is decreased in HOIP KO cells. WT HAP1 cells (control) or HOIP KO HAP1 cells reconstituted with either WT HOIP or HOIP∆PUB were analyzed by immunocytochemistry. Data are displayed as mean ± SD and were analyzed by one-way ANOVA followed by Bonferroni Multiple Comparison Test, n = 5. Data information: *p ≤ 0.05, ***p ≤ 0.001.(F) The interaction of soluble Htt-Q60 with p97/VCP is dependent on HOIP. WT HAP1 cells or HOIP KO HAP1 cells were transfected with Htt-Q60-HA. 48 h after transfection cells were lysed with 1% Triton X-100, followed by an immunoprecipitation of Htt-Q60 via the HA tag. Immunopurified proteins were detected by Western blotting using an anti-VCP and anti-HOIP antibody.


Figure 4 ( A ) Increased expression of HOIP decreases nuclear phospho - c - Jun in primary neurons . Primary mouse hippocampal neurons expressing Htt - Q25 or Htt - Q97 + / - HOIP were analyzed by immunocytochemistry using an antibody against phospho - c - Jun . Data are displayed as mean ± SD and were analyzed by 2 independent Mann - Whitney U - tests , n = 3 . * p ≤ 0 . 05 , * * * p ≤ 0 . 001 . ( B ) Htt - Q97 - induced toxicity is increased in cells silenced for HOIP . SH - SY5Y cells transiently transfected with Htt - Q25 or Htt - Q97 and either control siRNA ( CO ) or HOIP siRNA were analyzed by immunocytochemistry using an antibody against phospho - c - Jun . For rescue experiments , HOIP was co - expressed 24 h after silencing . Quantification of Htt - expressing cells with nuclear phospho - c - Jun is based on Student ' s t test from 3 independent experiments . Data are displayed as mean ± SD unless otherwise stated . * p ≤ 0 . 05 , * * * p ≤ 0 . 001 . OTULIN silencing decreases the toxicity of Htt - Q97 . SH - SY5Y cells transiently transfected with either control siRNA ( CO ) or OTULIN siRNA and Htt - Q25 or Htt - Q97 were analyzed by immunocytochemistry using antibodies against phospho - c - Jun ( C ) Data are displayed as mean ± SD and were analyzed by one - way ANOVA followed by Tukey ' s Multiple Comparison Test , n = 9 - 11 . * p ≤ 0 . 05 , * * * p ≤ 0 . 001 . OTULIN silencing decreases the toxicity of Htt - Q97 . SH - SY5Y cells transiently transfected with either control siRNA ( CO ) or OTULIN siRNA and Htt - Q25 or Htt - Q97 were analyzed by immunocytochemistry using antibodies against cleaved caspase - 3 ( D ) . Data are displayed as mean ± SD and were analyzed by one - way ANOVA followed by Tukey ' s Multiple Comparison Test , n = 9 - 11 . * p ≤ 0 . 05 , * * * p ≤ 0 . 001 . ( E ) The protective effect of HOIP is independent of NF - κB signaling . SH - SY5Y cells transiently transfected with Htt - Q97 - GFP and either control vector ( CO ) or HOIP ± the NF - κB super - repressor IκB - 2S / A were analyzed by immunocytochemistry using an antibody against phospho - c - Jun . Data are displayed as mean ± SD and were analyzed by one - way ANOVA followed by Tukey ' s Multiple Comparison Test , n = 9 . * p ≤ 0 . 05 , * * * p ≤ 0 . 001 . ( F ) SDS - soluble Htt - polyQ species are modified by linear ubiquitin chains . HEK293T cells were co - transfected with Htt - Q60 - GFP ( as a control for specific immunoprecipitation of HA - tagged Htt - Q60 ) , Htt - Q60 - HA or Htt - Q60 - 3R ( 3 lysine residues replaced by arginines ) and the plasmids indicated . Cells were lysed under denaturing conditions ( 1 % SDS ) , followed by immunoprecipitation of Htt via the HA tag ( 1 % Triton X - 100 , 0 . 1 % SDS ) . Immunopurified proteins were detected by Western blotting using the M1 ubiquitin - specific antibody 1E3 and anti - HA . ( G ) Silencing of OTULIN increases linear ubiquitination of Htt - Q97 . A linear ubiquitination assay was performed as described in ( F ) with HEK293T cells transfected with Htt - Q97 - HA and control siRNA or OTULIN siRNA . ( H ) SDS - insoluble Htt - polyQ species are modified by linear ubiquitin chains . HEK293T cells expressing Htt - Q97 - HA or Htt - Q97 - GFP were lysed under denaturing conditions in 1 . 5 % SDS . After centrifugation , the pellets containing the SDS - insoluble aggregates ( SDS - insoluble fraction ) were dissolved in formic acid . Formic acid - dissolved aggregates were analyzed by immunoblotting using the M1 ubiquitin - specific 1F11 / 3F5 / Y102L antibody . ( I ) Endogenous NEMO co - localizes with Htt - Q97 aggregates . NEMO ( red ) and Htt - Q97 - GFP ( green ) were analyzed by immunocytochemistry in SH - SY5Y cells . DAPI ( blue ) . Scale bar , 10 µm . ( J ) Endogenous Optineurin co - localizes with Htt - Q97 aggregates . Optineurin ( red ) and Htt - Q97 - GFP ( green ) were analyzed by immunocytochemistry in SH - SY5Y cells . DAPI ( blue ) . Scale bar , 10 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 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 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 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 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 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 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

Figure 4(A) Increased expression of HOIP decreases nuclear phospho-c-Jun in primary neurons. Primary mouse hippocampal neurons expressing Htt-Q25 or Htt-Q97 +/- HOIP were analyzed by immunocytochemistry using an antibody against phospho-c-Jun. Data are displayed as mean ± SD and were analyzed by 2 independent Mann-Whitney U-tests, n = 3. *p ≤ 0.05, ***p ≤ 0.001.(B) Htt-Q97-induced toxicity is increased in cells silenced for HOIP. SH-SY5Y cells transiently transfected with Htt-Q25 or Htt-Q97 and either control siRNA (CO) or HOIP siRNA were analyzed by immunocytochemistry using an antibody against phospho-c-Jun. For rescue experiments, HOIP was co-expressed 24 h after silencing. Quantification of Htt-expressing cells with nuclear phospho-c-Jun is based on Student's t test from 3 independent experiments. Data are displayed as mean ± SD unless otherwise stated. *p ≤ 0.05, ***p ≤ 0.001.OTULIN silencing decreases the toxicity of Htt-Q97. SH-SY5Y cells transiently transfected with either control siRNA (CO) or OTULIN siRNA and Htt-Q25 or Htt-Q97 were analyzed by immunocytochemistry using antibodies against phospho-c-Jun (C) Data are displayed as mean ± SD and were analyzed by one-way ANOVA followed by Tukey's Multiple Comparison Test, n = 9-11. *p ≤ 0.05, ***p ≤ 0.001.OTULIN silencing decreases the toxicity of Htt-Q97. SH-SY5Y cells transiently transfected with either control siRNA (CO) or OTULIN siRNA and Htt-Q25 or Htt-Q97 were analyzed by immunocytochemistry using antibodies against cleaved caspase-3 (D). Data are displayed as mean ± SD and were analyzed by one-way ANOVA followed by Tukey's Multiple Comparison Test, n = 9-11. *p ≤ 0.05, ***p ≤ 0.001.(E) The protective effect of HOIP is independent of NF-κB signaling. SH-SY5Y cells transiently transfected with Htt-Q97-GFP and either control vector (CO) or HOIP ± the NF-κB super-repressor IκB-2S/A were analyzed by immunocytochemistry using an antibody against phospho-c-Jun. Data are displayed as mean ± SD and were analyzed by one-way ANOVA followed by Tukey's Multiple Comparison Test, n = 9. *p ≤ 0.05, ***p ≤ 0.001.(F) SDS-soluble Htt-polyQ species are modified by linear ubiquitin chains. HEK293T cells were co-transfected with Htt-Q60-GFP (as a control for specific immunoprecipitation of HA-tagged Htt-Q60), Htt-Q60-HA or Htt-Q60-3R (3 lysine residues replaced by arginines) and the plasmids indicated. Cells were lysed under denaturing conditions (1% SDS), followed by immunoprecipitation of Htt via the HA tag (1% Triton X-100, 0.1% SDS). Immunopurified proteins were detected by Western blotting using the M1 ubiquitin-specific antibody 1E3 and anti-HA.(G) Silencing of OTULIN increases linear ubiquitination of Htt-Q97. A linear ubiquitination assay was performed as described in (F) with HEK293T cells transfected with Htt-Q97-HA and control siRNA or OTULIN siRNA.(H) SDS-insoluble Htt-polyQ species are modified by linear ubiquitin chains. HEK293T cells expressing Htt-Q97-HA or Htt-Q97-GFP were lysed under denaturing conditions in 1.5% SDS. After centrifugation, the pellets containing the SDS-insoluble aggregates (SDS-insoluble fraction) were dissolved in formic acid. Formic acid-dissolved aggregates were analyzed by immunoblotting using the M1 ubiquitin-specific 1F11/3F5/Y102L antibody.(I) Endogenous NEMO co-localizes with Htt-Q97 aggregates. NEMO (red) and Htt-Q97-GFP (green) were analyzed by immunocytochemistry in SH-SY5Y cells. DAPI (blue). Scale bar, 10 µm. (J) Endogenous Optineurin co-localizes with Htt-Q97 aggregates. Optineurin (red) and Htt-Q97-GFP (green) were analyzed by immunocytochemistry in SH-SY5Y cells. DAPI (blue). Scale bar, 10 µm.


Figure 5 ( A ) The interaction between Htt - Q97 and Sp1 is decreased by linear ubiquitination . HEK293T cells were co - transfected with Htt - Q97 - HA and the siRNAs indicated . Htt - Q97 - GFP was transfected as a control for specific immunoprecipitation of HA - Htt - Q97 . Nuclear ( left panel ) and cytosolic fractions ( middle panel ) were subjected to an anti - HA immunoprecipitation followed by Western blotting using an anti - Sp1 antibody . Right panel : Input controls ( whole cell lysates ) . ( B ) Quantification of the interaction between Htt - Q97 and Sp1 in nuclear fractions . Shown is the relative amount of Sp1 co - purifying with Htt - Q97 . Signal intensities were normalized to nuclear Sp1 . Data are displayed as mean ± SD and were analyzed by 2 independent Mann - Whitney U - tests , n = 4 . * p ≤ 0 . 05 , * * * p ≤ 0 . 001 . ( C , D ) Impairment of Sp1 - mediated transcription by Htt - Q97 is modified by HOIP . ( C ) Shown is the relative luciferase activity in HEK293T cells expressing Htt - Q97 - HA , a Sp1 luciferase reporter construct and HOIP cDNA . Data are displayed as mean ± SD and were analyzed by Kruskal - Wallis test followed by Dunn ' s Multiple Comparison Test , n = 9 - 11 . ( D ) Shown is the relative luciferase activity in HEK293T cells expressing Htt - Q97 - HA , a Sp1 luciferase reporter construct and HOIP siRNA . Data are displayed as mean ± SD and were analyzed by one - way ANOVA followed by Tukey ' s Multiple Comparison Test , n = 9 . * p ≤ 0 . 05 , * * * p ≤ 0 . 001 . ( E ) Sp1 silencing decreases HOIP and SHARPIN protein expression . SH - SY5Y cells were transfected with control or Sp1 - specific siRNAs and analyzed for protein expression 2 days after transfection by immunoblotting ( left panel ) . Quantification of normalized Sp1 , HOIP and SHARPIN protein expression levels ( right panel ) . Data are displayed as mean ± SEM with n = 5 from unpaired two - tailed Student ' s t - tests . * p ≤ 0 . 05 , * * * p ≤ 0 . 001 . ( F ) HOIP - , HOIL - 1L - and SHARPIN - specific mRNA is decreased in R6 / 2 striatum . Quantification of mRNA from the striatum of 9 - 12 weeks old R6 / 2 mice and non - transgenic littermates . Data are displayed as mean ± SD with n = 6 - 9 biological replicates from unpaired two - tailed Student ' s t - tests . * p ≤ 0 . 05 , * * * p ≤ 0 . 001 . ( G ) HOIP protein levels are decreased in human HD brain . Total protein lysates from human brain ( anterior cingulate cortex ) analyzed by immunoblotting using antibodies against HOIP and βIII - tubulin . HOIP - positive signals were normalized to βIII - tubulin . To verify the specificity of the HOIP antibody , WT and HOIP KO HAP1 cells were analyzed in parallel on a different gel . Data are displayed as mean ± SEM with n = 5 for control and n = 7 for HD patients from unpaired two - tailed Student ' s t - tests . * p ≤ 0 . 05 , * * * p ≤ 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 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 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 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 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 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 5(A) The interaction between Htt-Q97 and Sp1 is decreased by linear ubiquitination. HEK293T cells were co-transfected with Htt-Q97-HA and the siRNAs indicated. Htt-Q97-GFP was transfected as a control for specific immunoprecipitation of HA-Htt-Q97. Nuclear (left panel) and cytosolic fractions (middle panel) were subjected to an anti-HA immunoprecipitation followed by Western blotting using an anti-Sp1 antibody. Right panel: Input controls (whole cell lysates).(B) Quantification of the interaction between Htt-Q97 and Sp1 in nuclear fractions. Shown is the relative amount of Sp1 co-purifying with Htt-Q97. Signal intensities were normalized to nuclear Sp1. Data are displayed as mean ± SD and were analyzed by 2 independent Mann-Whitney U-tests, n = 4. *p ≤ 0.05, ***p ≤ 0.001.(C, D) Impairment of Sp1-mediated transcription by Htt-Q97 is modified by HOIP. (C) Shown is the relative luciferase activity in HEK293T cells expressing Htt-Q97-HA, a Sp1 luciferase reporter construct and HOIP cDNA. Data are displayed as mean ± SD and were analyzed by Kruskal-Wallis test followed by Dunn's Multiple Comparison Test, n = 9-11. (D) Shown is the relative luciferase activity in HEK293T cells expressing Htt-Q97-HA, a Sp1 luciferase reporter construct and HOIP siRNA. Data are displayed as mean ± SD and were analyzed by one-way ANOVA followed by Tukey's Multiple Comparison Test, n = 9. *p ≤ 0.05, ***p ≤ 0.001.(E) Sp1 silencing decreases HOIP and SHARPIN protein expression. SH-SY5Y cells were transfected with control or Sp1-specific siRNAs and analyzed for protein expression 2 days after transfection by immunoblotting (left panel). Quantification of normalized Sp1, HOIP and SHARPIN protein expression levels (right panel). Data are displayed as mean ± SEM with n=5 from unpaired two-tailed Student's t-tests. *p ≤ 0.05, ***p ≤ 0.001.(F) HOIP-, HOIL-1L- and SHARPIN-specific mRNA is decreased in R6/2 striatum. Quantification of mRNA from the striatum of 9-12 weeks old R6/2 mice and non-transgenic littermates. Data are displayed as mean ± SD with n = 6-9 biological replicates from unpaired two-tailed Student's t-tests. *p ≤ 0.05, ***p ≤ 0.001.(G) HOIP protein levels are decreased in human HD brain. Total protein lysates from human brain (anterior cingulate cortex) analyzed by immunoblotting using antibodies against HOIP and βIII-tubulin. HOIP-positive signals were normalized to βIII-tubulin. To verify the specificity of the HOIP antibody, WT and HOIP KO HAP1 cells were analyzed in parallel on a different gel. Data are displayed as mean ± SEM with n=5 for control and n=7 for HD patients from unpaired two-tailed Student's t-tests. *p ≤ 0.05, ***p ≤ 0.001.


Figure 6 ( A ) HOIP is recruited to disease - associated protein aggregates formed by Ataxin - 3 - Q84 , SOD1 G85R , TDP - 43 Q331K or Optineurin R96L . SH - SY5Y cells co - expressing disease - associated proteins ( green ) and HOIP ( red ) were analyzed by immunocytochemistry ; DAPI ( blue ) . Scale bar , 20 µLinear ubiquitin accumulates at disease - associated protein aggregates under endogenous LUBAC expression . M1 - linked ubiquitin ( red ) and disease - associated proteins ( green ) were analyzed by immunocytochemistry in SH - SY5Y cells using the 1E3 antibody ( Millipore ) ( B ) DAPI ( blue ) . Scale bar , 20 µm . Linear ubiquitin accumulates at disease - associated protein aggregates under endogenous LUBAC expression . M1 - linked ubiquitin ( red ) and disease - associated proteins ( green ) were analyzed by immunocytochemistry in primary striatal neurons using the 1F11 / 3F5 / Y102L antibody ( Genentech ) ( C ) . DAPI ( blue ) . Scale bar , 20 µm . ( D ) SDS - soluble SOD G85R and TDP - 43 Q331K species are modified by linear ubiquitin chains . HEK293T cells were co - transfected with disease - associated proteins and either the plasmids indicated or with control siRNA or OTULIN siRNA . 48 h after transfection cells were lysed under denaturing conditions ( 1 % SDS ) , followed by immunoprecipitation of SOD G85R and TDP - 43 Q331K via the HA tag ( 1 % Triton X - 100 , 0 . 1 % SDS ) . Immunopurified proteins were detected by Western blotting using the M1 ubiquitin - specific antibody 1E3 .

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 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 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

Figure 6(A) HOIP is recruited to disease-associated protein aggregates formed by Ataxin-3-Q84, SOD1 G85R, TDP-43 Q331K or Optineurin R96L. SH-SY5Y cells co-expressing disease-associated proteins (green) and HOIP (red) were analyzed by immunocytochemistry; DAPI (blue). Scale bar, 20 µLinear ubiquitin accumulates at disease-associated protein aggregates under endogenous LUBAC expression. M1-linked ubiquitin (red) and disease-associated proteins (green) were analyzed by immunocytochemistry in SH-SY5Y cells using the 1E3 antibody (Millipore) (B) DAPI (blue). Scale bar, 20 µm.Linear ubiquitin accumulates at disease-associated protein aggregates under endogenous LUBAC expression. M1-linked ubiquitin (red) and disease-associated proteins (green) were analyzed by immunocytochemistry in primary striatal neurons using the 1F11/3F5/Y102L antibody (Genentech) (C). DAPI (blue). Scale bar, 20 µm.(D) SDS-soluble SOD G85R and TDP-43 Q331K species are modified by linear ubiquitin chains. HEK293T cells were co-transfected with disease-associated proteins and either the plasmids indicated or with control siRNA or OTULIN siRNA. 48 h after transfection cells were lysed under denaturing conditions (1% SDS), followed by immunoprecipitation of SOD G85R and TDP-43 Q331K via the HA tag (1% Triton X-100, 0.1% SDS). Immunopurified proteins were detected by Western blotting using the M1 ubiquitin-specific antibody 1E3.


Figure 7 ( A ) Linear ubiquitination reduces the fraction of cells with Htt - Q97 aggregates . SH - SY5Y cells were transfected with Htt - Q97 and either control siRNA , HOIP siRNA or OTULIN siRNA . Data are displayed as mean ± SD and were analyzed by one - way ANOVA followed by Tukey ' s Multiple Comparison Test , n = 9 . Expression levels of HOIP and OTULIN were analyzed by Western blotting . Data information : * p ≤ 0 . 05 , * * p ≤ 0 . 01 , * * * p ≤ 0 . 001 . ( B ) Linear ubiquitination reduces the fraction of cells with Htt - Q97 aggregates . SH - SY5Y cells were transfected with Htt - Q97 and either WT HOIP or the catalytically inactive HOIP mutant C885A . Data are displayed as mean ± SD and were analyzed by one - way ANOVA followed by Tukey s Multiple Comparison Test , n = 5 . Expression levels of WT and mutant HOIP and OTULIN were analyzed by Western blotting . Data information : * p ≤ 0 . 05 , * * p ≤ 0 . 01 , * * * p ≤ 0 . 001 . ( C ) The effect of HOIP on Htt - Q97 aggregation is independent of NF - κB signaling . SH - SY5Y cells transiently transfected with Htt - Q97 and either control vector ( CO ) or WT HOIP plus the NF - κB super - repressor IκB - 2S / A were analyzed by immunocytochemistry . Data are mean ± SD with n = 5 from an unpaired two - tailed Student ' s t - test . Expression levels of WT HOIP and IκB - 2S / A were analyzed by Western blotting . Data information : * p ≤ 0 . 05 , * * p ≤ 0 . 01 , * * * p ≤ 0 . 001 . ( D ) Catalytically active HOIP decreases the number of cells with Htt - Q97 aggregates independently of autophagy . WT and ATG5 KO MEFs were transiently transfected with Htt - Q97 - GFP and either control vector ( CO ) , HOIP or HOIP C885A . Cells were treated 24 h after transfection with the proteasomal inhibitor MG132 ( 1 µM , 16 h ) . Data are displayed as mean ± SD and were analyzed by one - way ANOVA followed by Tukey ' s Multiple Comparison Test , n = 7 . Data information : * p ≤ 0 . 05 , * * p ≤ 0 . 01 , * * * p ≤ 0 . 001 . ( E ) Linear ubiquitination reduces the fraction of cells with TDP - 43 Q331K aggregates . SH - SY5Y cells were transfected with TDP - 43 Q331K and either control siRNA , HOIP siRNA or OTULIN siRNA . Data are displayed as mean ± SD and were analyzed by Kruskal - Wallis test , followed by Dunn ' s Multiple Comparison Test , n = 7 . Expression levels of HOIP and OTULIN were analyzed by Western blotting . Data information : * p ≤ 0 . 05 , * * p ≤ 0 . 01 , * * * p ≤ 0 . 001 . ( F ) Catalytically active HOIP decreases the number of cells with TDP - 43 aggregates dependent on proteasomal degradation . SH - SY5Y cells were transfected with TDP - 43 Q331K and either control vector ( CO ) , HOIP or HOIP C885A . Cells were treated 48 h after transfection with the proteasomal inhibitor MG132 ( 1 µM ) or the p97 / VCP inhibitor NSM - 873 ( 1 µM ) for 3 h . Data are displayed as mean ± SD and were analyzed by one - way ANOVA followed by Dunnett ' s Multiple Comparison Test , n = 5 . Data information : * p ≤ 0 . 05 , * * p ≤ 0 . 01 , * * * p ≤ 0 . 001 . ( G ) Inhibition of the proetasome and p97 / VCP do not have additive effects on the degradation of misfolded TDP - 43 species . SH - SY5Y cells expressing TDP - 43 Q331K were treated 48 h after transfection with MG132 ( 1 µM ) and / or NSM - 873 ( 1 µM ) for 3 h . All data are mean ± SD , data were analyzed by one - way ANOVA followed by Bonferroni ' s Multiple Comparison Test , n = 5 . Data information : * p ≤ 0 . 05 , * * p ≤ 0 . 01 , * * * p ≤ 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 O O 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 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 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 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 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 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

Figure 7(A) Linear ubiquitination reduces the fraction of cells with Htt-Q97 aggregates. SH-SY5Y cells were transfected with Htt-Q97 and either control siRNA, HOIP siRNA or OTULIN siRNA. Data are displayed as mean ± SD and were analyzed by one-way ANOVA followed by Tukey's Multiple Comparison Test, n = 9. Expression levels of HOIP and OTULIN were analyzed by Western blotting. Data information: *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001.(B) Linear ubiquitination reduces the fraction of cells with Htt-Q97 aggregates. SH-SY5Y cells were transfected with Htt-Q97 and either WT HOIP or the catalytically inactive HOIP mutant C885A. Data are displayed as mean ± SD and were analyzed by one-way ANOVA followed by Tukey s Multiple Comparison Test, n = 5. Expression levels of WT and mutant HOIP and OTULIN were analyzed by Western blotting. Data information: *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001.(C) The effect of HOIP on Htt-Q97 aggregation is independent of NF-κB signaling. SH-SY5Y cells transiently transfected with Htt-Q97 and either control vector (CO) or WT HOIP plus the NF-κB super-repressor IκB-2S/A were analyzed by immunocytochemistry. Data are mean ± SD with n=5 from an unpaired two-tailed Student's t-test. Expression levels of WT HOIP and IκB-2S/A were analyzed by Western blotting. Data information: *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001.(D) Catalytically active HOIP decreases the number of cells with Htt-Q97 aggregates independently of autophagy. WT and ATG5 KO MEFs were transiently transfected with Htt-Q97-GFP and either control vector (CO), HOIP or HOIP C885A. Cells were treated 24 h after transfection with the proteasomal inhibitor MG132 (1 µM, 16 h). Data are displayed as mean ± SD and were analyzed by one-way ANOVA followed by Tukey's Multiple Comparison Test, n = 7. Data information: *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001.(E) Linear ubiquitination reduces the fraction of cells with TDP-43 Q331K aggregates. SH-SY5Y cells were transfected with TDP-43 Q331K and either control siRNA, HOIP siRNA or OTULIN siRNA. Data are displayed as mean ± SD and were analyzed by Kruskal-Wallis test, followed by Dunn's Multiple Comparison Test, n = 7. Expression levels of HOIP and OTULIN were analyzed by Western blotting. Data information: *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001.(F) Catalytically active HOIP decreases the number of cells with TDP-43 aggregates dependent on proteasomal degradation. SH-SY5Y cells were transfected with TDP-43 Q331K and either control vector (CO), HOIP or HOIP C885A. Cells were treated 48 h after transfection with the proteasomal inhibitor MG132 (1 µM) or the p97/VCP inhibitor NSM-873 (1 µM) for 3 h. Data are displayed as mean ± SD and were analyzed by one-way ANOVA followed by Dunnett's Multiple Comparison Test, n = 5. Data information: *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001.(G) Inhibition of the proetasome and p97/VCP do not have additive effects on the degradation of misfolded TDP-43 species. SH-SY5Y cells expressing TDP-43 Q331K were treated 48 h after transfection with MG132 (1 µM) and/or NSM-873 (1 µM) for 3 h. All data are mean ± SD, data were analyzed by one-way ANOVA followed by Bonferroni's Multiple Comparison Test, n = 5. Data information: *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001.


Figure 1Representative TEM images of normal and injured acinar cells . Scale bar , 2μm . Right ( insets ) - higher magnification images of boxed regions from left show emergence of double - membraned autophagic vesicles engulfing mature granules , mitochondria , and rough ER 24h after cerulein ( CER ) injury . Scale bar , 500nm . Arrowheads mark degradation events without double membranes . Graph of the number of acinar cell granules per cell in vehicle vs . 24h CER treatment . Each data point = total granules in a cell in a single cell counted from one mouse . Red line indicates mean . Graph of the number of degradation events scored in vehicle vs . 24h CER treatment . Each data point = total events in a cell in a single cell counted from one mouse . Red line indicates mean . mRNA expression by qRT - PCR of Rab5 , Rab7a , Rab7b , and Rab9 . Significance determined by one - way ANOVA , Tukey post hoc . Each data point represents the mean of triplicate technical replicates from a single mouse , n = 3 - 4 mice in independent experiments . Error bars denote standard deviation .

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 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

Figure 1Representative TEM images of normal and injured acinar cells. Scale bar, 2μm. Right (insets)- higher magnification images of boxed regions from left show emergence of double-membraned autophagic vesicles engulfing mature granules, mitochondria, and rough ER 24h after cerulein (CER) injury. Scale bar, 500nm. Arrowheads mark degradation events without double membranes. Graph of the number of acinar cell granules per cell in vehicle vs. 24h CER treatment. Each data point=total granules in a cell in a single cell counted from one mouse. Red line indicates mean. Graph of the number of degradation events scored in vehicle vs. 24h CER treatment. Each data point=total events in a cell in a single cell counted from one mouse. Red line indicates mean.mRNA expression by qRT-PCR of Rab5, Rab7a, Rab7b, and Rab9. Significance determined by one-way ANOVA, Tukey post hoc. Each data point represents the mean of triplicate technical replicates from a single mouse, n=3-4 mice in independent experiments. Error bars denote standard deviation.