Figure 6A . Cumulative distribution plots showing the difference in distribution of transcript optimality between upregulated and downregulated transcripts in K562 cells subject to ILF2 CRISPR interference targeting ILF2 . Transcript quantities are indicated in the figure legend . Data information : Wilcoxon signed rank tests were performed on the upregulated and downregulated groups against the control group . P - values are denoted ( right ) . B , C . HEK293 Tet - off experiments showing the degradation of REL - OPT ( B ) and REL - WT ( C ) transcripts with ILF2 and ILF3 siRNA and Control ( CTR ) siRNA treatment , post - doxycycline addition . Data information : data is representative of 3 independent experiments in which the data represents the mean ± SD for 3 biological replicates . A two - way ANOVA with Holm - Sidak multiple comparisons was performed . P - values are denoted as follows : p < 0 . 05 ( * ) , p < 0 . 01 ( * * ) . D . Representative immunoblot of FLAG - tagged REL - OPT and REL - WT expressed in HEK293T cells under ILF2 and ILF3 siRNA treatment . The immunoblot is representative of 3 independent experiments . ACTB is shown as loading controls . E . Representative immunoblot of FLAG - tagged REL - OPT and REL - WT in HEK293T cells co - expressed with two different isoforms of ILF2 . The immunoblot is representative of 3 independent experiments . ACTB is shown as loading controls .

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

Figure 6A. Cumulative distribution plots showing the difference in distribution of transcript optimality between upregulated and downregulated transcripts in K562 cells subject to ILF2 CRISPR interference targeting ILF2. Transcript quantities are indicated in the figure legend. Data information: Wilcoxon signed rank tests were performed on the upregulated and downregulated groups against the control group. P-values are denoted (right).B, C. HEK293 Tet-off experiments showing the degradation of REL-OPT (B) and REL-WT (C) transcripts with ILF2 and ILF3 siRNA and Control (CTR) siRNA treatment, post-doxycycline addition. Data information: data is representative of 3 independent experiments in which the data represents the mean ± SD for 3 biological replicates. A two-way ANOVA with Holm-Sidak multiple comparisons was performed. P-values are denoted as follows: p < 0.05 (*), p<0.01 (**).D. Representative immunoblot of FLAG-tagged REL-OPT and REL-WT expressed in HEK293T cells under ILF2 and ILF3 siRNA treatment. The immunoblot is representative of 3 independent experiments. ACTB is shown as loading controls.E. Representative immunoblot of FLAG-tagged REL-OPT and REL-WT in HEK293T cells co-expressed with two different isoforms of ILF2. The immunoblot is representative of 3 independent experiments. ACTB is shown as loading controls.


Figure 1A . KD efficiency of three different Syt11 shRNAs in DRG neurons . Cultured DRG neurons were infected with control or shRNA - expressing lentiviruses at DIV 1 , and Western blotting for Syt11 , Syt1 , Syt4 , synaptobrevin 2 ( Syb2 ) , SNAP25 , complexin 1 / 2 ( Cpx1 , Cpx2 ) , clathrin , adaptor protein 2 ( AP - 2 ) , and β - actin were performed at DIV 6 - 7 . N = 4 independent experiments . B , C . Representative Cm traces induced by 200 - ms pulse depolarization ( arrows ) ib DRG neurons . DRG neurons were transfected with plasmids expressing shSyt11 - 2 ( Syt11 KD , KD ) or scrambled shRNA ( Sc ) and Cm recording was performed 5 days after transfection . Endo - 5s represents the Cm decay 5 s after stimulation . Insets show Ca2 + currents recorded in the same neurons . D . Representative Cm overshoot recorded from an Syt11 KD neuron . About 45 % of the KD neurons showed excessive membrane retrieval after a 200 - ms depolarizing pulse . E . Averaged Cm traces recorded from Control ( Ctrl ) , Sc , KD ( from all three shRNAs , n = 39 for shSyt11 - 2 , n = 7 for shSyt11 - 1 , n = 14 for shSyt11 - 3 ) , and rescued ( with the shSyt11 - 2 - resistant form of Syt11 ) DRG neurons . F - H . Statistics of Cm jumps , Endo - 5s , and Ca2 + current recorded from DRGneurons as in ( B - E ) . Data were collected from 4 ( Sc , Rescue ) , 8 ( Ctrl ) , and 16 ( KD ) independent experiments . I . Normalized Cm changes induced by two pulses of 200 - ms depolarization ( arrows ) at a 1 - s interval . Data were normalized to the capacitance jump induced by the first depolarization . J . Paired - pulse ratios of ΔCm with different interstimulus intervals as in ( I ) . DRG neurons were stimulated with two 200 - ms pulses at different intervals as indicated , and the paired - pulse ratio was calculated by normalizing the ΔCm in response to the second stimulation to that induced by the first one .

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Figure 1A. KD efficiency of three different Syt11 shRNAs in DRG neurons. Cultured DRG neurons were infected with control or shRNA-expressing lentiviruses at DIV 1, and Western blotting for Syt11, Syt1, Syt4, synaptobrevin 2 (Syb2), SNAP25, complexin 1/2 (Cpx1, Cpx2), clathrin, adaptor protein 2 (AP-2), and β-actin were performed at DIV 6-7. N = 4 independent experiments.B, C. Representative Cm traces induced by 200-ms pulse depolarization (arrows) ib DRG neurons. DRG neurons were transfected with plasmids expressing shSyt11-2 (Syt11 KD, KD) or scrambled shRNA (Sc) and Cm recording was performed 5 days after transfection. Endo-5s represents the Cm decay 5 s after stimulation. Insets show Ca2+ currents recorded in the same neurons.D. Representative Cm overshoot recorded from an Syt11 KD neuron. About 45% of the KD neurons showed excessive membrane retrieval after a 200-ms depolarizing pulse.E. Averaged Cm traces recorded from Control (Ctrl), Sc, KD (from all three shRNAs, n = 39 for shSyt11-2, n = 7 for shSyt11-1, n = 14 for shSyt11-3), and rescued (with the shSyt11-2-resistant form of Syt11) DRG neurons.F-H. Statistics of Cm jumps, Endo-5s, and Ca2+ current recorded from DRGneurons as in (B-E). Data were collected from 4 (Sc, Rescue), 8 (Ctrl), and 16 (KD) independent experiments.I. Normalized Cm changes induced by two pulses of 200-ms depolarization (arrows) at a 1-s interval. Data were normalized to the capacitance jump induced by the first depolarization.J. Paired-pulse ratios of ΔCm with different interstimulus intervals as in (I). DRG neurons were stimulated with two 200-ms pulses at different intervals as indicated, and the paired-pulse ratio was calculated by normalizing the ΔCm in response to the second stimulation to that induced by the first one.


Figure 2A , B . FM 1 - 43 uptake into DRG neurons stimulated by 100 mM K + for 30 s . The right - shifted cumulative frequency in ( B ) indicates the increased uptake level in KD neurons . Scale bars , 5 μm . C . Quantification of KCl - evoked FM 1 - 43 uptake with different stimulation times . D . Representative micrographs showing the increased constitutive FM 1 - 43 uptake ( for 5 min ) in KD neurons . Scale bars , 5 μm . E . Quantification of constitutive FM 1 - 43 uptake with different loading times . F . Basal [ Ca2 + ] i in DRG neurons measured with Fura - 2 . G . FM1 - 43 uptake by Syt11 KD ( RFP - positive ) and control ( RFP - negative ) hippocampal neurons stimulated by 100 mM K + for 2 min . Quantitative data are shown in the lower - right panel . Scale bars , 20 μm . H . Representative images showing the preloaded FM4 - 64 fluorescence in presynaptic boutons of hippocampal neurons before and after 800 stimuli at 40 Hz . Scale bars , 5 μm . I . Time - course of FM4 - 64 unloading from control , Syt11 KD , and rescued nerve terminals within the same field of view ( 6 coverslips from at least 3 biological repeats each ) .

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

Figure 2A, B. FM 1-43 uptake into DRG neurons stimulated by 100 mM K+ for 30 s. The right-shifted cumulative frequency in (B) indicates the increased uptake level in KD neurons. Scale bars, 5 μm.C. Quantification of KCl-evoked FM 1-43 uptake with different stimulation times.D. Representative micrographs showing the increased constitutive FM 1-43 uptake (for 5 min) in KD neurons. Scale bars, 5 μm.E. Quantification of constitutive FM 1-43 uptake with different loading times.F. Basal [Ca2+]i in DRG neurons measured with Fura-2.G. FM1-43 uptake by Syt11 KD (RFP-positive) and control (RFP-negative) hippocampal neurons stimulated by 100 mM K+ for 2 min. Quantitative data are shown in the lower-right panel. Scale bars, 20 μm.H. Representative images showing the preloaded FM4-64 fluorescence in presynaptic boutons of hippocampal neurons before and after 800 stimuli at 40 Hz. Scale bars, 5 μm.I. Time-course of FM4-64 unloading from control, Syt11 KD, and rescued nerve terminals within the same field of view (6 coverslips from at least 3 biological repeats each).


Figure 3A . Averaged ΔCm traces induced by 200 - ms depolarization in the presence and absence of dynasore . Endocytic inhibition by dynasore ( Dyna ) was estimated by comparison of Cm traces recorded during 1 - 3 min ( KD ) and 5 - 10 min ( KD + Dyna and Ctrl + Dyna ) after whole - cell dialysis . B , C . Endo - 5s and Cm jumps recorded as in ( A ) . D . Averaged ΔCm traces induced by 200 - ms depolarization in the presence and absence of dynole - 34 - 2TM ( Dynole ) . Cm traces and endocytic rate were measured as in ( A ) except that 10 μM dynole - 34 - 2TM with 0 . 1 % DMSO was dialyzed into patched cells . E , F . Endo - 5s and Cm jumps as in ( D ) . G , H . Averaged ∆ Cm traces and Endo - 5s recorded from KD neurons under control conditions ( 0 . 1 % DMSO ) . I . KCl - evoked FM 1 - 43 uptake in DRG neurons after 10 - min pre - incubation with 0 . 1 % DMSO ( control ) or with 100 μM dynasore .

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Figure 3A. Averaged ΔCm traces induced by 200-ms depolarization in the presence and absence of dynasore. Endocytic inhibition by dynasore (Dyna) was estimated by comparison of Cm traces recorded during 1-3 min (KD) and 5-10 min (KD + Dyna and Ctrl + Dyna) after whole-cell dialysis.B, C. Endo-5s and Cm jumps recorded as in (A).D. Averaged ΔCm traces induced by 200-ms depolarization in the presence and absence of dynole-34-2TM (Dynole). Cm traces and endocytic rate were measured as in (A) except that 10 μM dynole-34-2TM with 0.1% DMSO was dialyzed into patched cells.E, F. Endo-5s and Cm jumps as in (D).G, H. Averaged ∆Cm traces and Endo-5s recorded from KD neurons under control conditions (0.1 % DMSO).I. KCl-evoked FM 1-43 uptake in DRG neurons after 10-min pre-incubation with 0.1% DMSO (control) or with 100 μM dynasore.


Figure 4A . DRG neurons expressing Myc - Syt11 were immunostained for Myc - Syt11 , dynamin ( Dyn ) , AP - 2 , clathrin ( CL ) , TGN46 , synaptobrevin 2 ( Syb2 ) , and LAMP1 . For dynamin , the box along the plasma membrane was straightened , enlarged , and is shown in the upper right panels with arrows indicating co - localized puncta . An enlarged inset of the inside of the cell ( box ) is shown in the lower panel . For localization of the transferrin receptor , EGFP - transferrin receptor ( TfR ) was expressed in DRG neurons . Scale bars , 10 μm . B . Analysis of co - localization of Myc - Syt11 with dynamin , AP - 2 , clathrin , TGN46 , synaptobrevin 2 , transferrin receptor , and LAMP1 . N = 3 independent experiments .

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Figure 4A. DRG neurons expressing Myc-Syt11 were immunostained for Myc-Syt11, dynamin (Dyn), AP-2, clathrin (CL), TGN46, synaptobrevin 2 (Syb2), and LAMP1. For dynamin, the box along the plasma membrane was straightened, enlarged, and is shown in the upper right panels with arrows indicating co-localized puncta. An enlarged inset of the inside of the cell (box) is shown in the lower panel. For localization of the transferrin receptor, EGFP-transferrin receptor (TfR) was expressed in DRG neurons. Scale bars, 10 μm.B. Analysis of co-localization of Myc-Syt11 with dynamin, AP-2, clathrin, TGN46, synaptobrevin 2, transferrin receptor, and LAMP1. N = 3 independent experiments.


Figure 5A . Transferrin ( Tf , red ) uptake in Syt11 KD ( GFP - positive ) and control neurons . Quantitative data are on the right . Scale bars , 20 μm . B . Tf uptake in Syt11 over - expressing and control neurons . Quantitative data are on the right . Scale bars , 20 μm . C . Normalized ∆ Cm induced by 200 - ms depolarization of a Syt11 KD neuron in the presence of 100 μM MDC was fitted to a double - exponential decay function ( solid black and red , fitted curves ) . D . Fast and slow time - constants of endocytosis in Syt11 KD neurons recorded as in ( C ) . E . KCl - evoked FM 1 - 43 uptake in DRG neurons after 1 - h pre - incubation with 0 . 1 % DMSO or 100 μM MDC in the bath solution .

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Figure 5A. Transferrin (Tf, red) uptake in Syt11 KD (GFP-positive) and control neurons. Quantitative data are on the right. Scale bars, 20 μm.B. Tf uptake in Syt11 over-expressing and control neurons. Quantitative data are on the right. Scale bars, 20 μm.C. Normalized ∆Cm induced by 200-ms depolarization of a Syt11 KD neuron in the presence of 100 μM MDC was fitted to a double-exponential decay function (solid black and red, fitted curves).D. Fast and slow time-constants of endocytosis in Syt11 KD neurons recorded as in (C).E. KCl-evoked FM 1-43 uptake in DRG neurons after 1-h pre-incubation with 0.1% DMSO or 100 μM MDC in the bath solution.


Figure 6A , B . Cm traces with bulk endocytosis ( arrowheads ) in control ( A ) and KD ( B ) neurons induced by 200 - ms depolarization . Expanded bulk events are shown on the right . C , D . Frequency distribution and bulk endocytic events / cell within 16 s after stimulation . E , F . Size and duration of Cm decay during bulk endocytosis . G , H . Representative z - projected fluorescence images and statistics showing the large dextran uptake in Syt11 KD and control DRG neurons . Neurons were loaded with 50 μM tetramethylrhodamine - dextran ( 40 kD ) by 2 - min exposure to 100 mM KCl . Scale bars , 10 μm .

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

Figure 6A, B. Cm traces with bulk endocytosis (arrowheads) in control (A) and KD (B) neurons induced by 200-ms depolarization. Expanded bulk events are shown on the right.C, D. Frequency distribution and bulk endocytic events/cell within 16 s after stimulation.E, F. Size and duration of Cm decay during bulk endocytosis.G, H. Representative z-projected fluorescence images and statistics showing the large dextran uptake in Syt11 KD and control DRG neurons. Neurons were loaded with 50 μM tetramethylrhodamine-dextran (40 kD) by 2-min exposure to 100 mM KCl. Scale bars, 10 μm.


Figure 7A . Representative electron micrographs showing a small / clear vesicle ( SV , arrow ) , dense - core vesicles ( DCVs , arrowheads ) and a clathrin - coated pit ( hatched arrow ) in DRG neurons . B , C . Diameter distributions of SVs and DCVs ( statistics from 6 control cells and 9 KD cells ) in DRG neurons . The red and green lines are curves fitted to a Gaussian function . D , E . Diameters of SVs and DCVs in Syt11 KD and control neurons with ( 100K , statistics from 6 control cells and 9 KD cells ) or without 100 mM K + stimulation ( SE , statistics from 6 cells for each group ) .

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Figure 7A. Representative electron micrographs showing a small/clear vesicle (SV, arrow), dense-core vesicles (DCVs, arrowheads) and a clathrin-coated pit (hatched arrow) in DRG neurons.B, C. Diameter distributions of SVs and DCVs (statistics from 6 control cells and 9 KD cells) in DRG neurons. The red and green lines are curves fitted to a Gaussian function.D, E. Diameters of SVs and DCVs in Syt11 KD and control neurons with (100K, statistics from 6 control cells and 9 KD cells) or without 100 mM K+ stimulation (SE, statistics from 6 cells for each group).


Figure 8A . Representative electron micrographs of HRP uptake from standard ( SE ) or 100 mM K + ( 100K ) bath solution . Scale bar , 2 μm . B - D . HRP - labeled small vesicles ( filled arrows ) and endosomes ( arrowheads ) in DRG neurons . Quantitative data are shown in ( C ) and ( D ) . Scale bar , 200 nm . B - D . HRP - labeled small vesicles ( filled arrows ) and endosomes ( arrowheads ) in DRG neurons . Quantitative data are shown in ( C ) and ( D ) . Scale bar , 200 nm . E . Representative docked vesicles ( arrows ) in a DRG neuron . Quantitative data are on the right . Scale bar , 200 nm . F - H . Clathrin - coated pits ( CCPs , hatched arrows ) and bulk - endocytosis - related structures of invaginating membrane ( open arrowhead ) in DRGneurons . Quantitative data are shown in ( G ) and ( H ) . Scale bars , 200 nm . F - H . Clathrin - coated pits ( CCPs , hatched arrows ) and bulk - endocytosis - related structures of invaginating membrane ( open arrowhead ) in DRGneurons . Quantitative data are shown in ( G ) and ( H ) . Scale bars , 200 nm . I , J . Cumulative frequencies of diameter distributions of small vesicles and endosomes ( statistics from 6 control cells and 9 KD cells ) . Kolmogorov - Smirnov test , P = 0 . 304 for ( I ) and P = 0 . 553 for ( J ) .

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

Figure 8A. Representative electron micrographs of HRP uptake from standard (SE) or 100 mM K+ (100K) bath solution. Scale bar, 2 μm.B-D. HRP-labeled small vesicles (filled arrows) and endosomes (arrowheads) in DRG neurons. Quantitative data are shown in (C) and (D). Scale bar, 200 nm.B-D. HRP-labeled small vesicles (filled arrows) and endosomes (arrowheads) in DRG neurons. Quantitative data are shown in (C) and (D). Scale bar, 200 nm.E. Representative docked vesicles (arrows) in a DRG neuron. Quantitative data are on the right. Scale bar, 200 nm.F-H. Clathrin-coated pits (CCPs, hatched arrows) and bulk-endocytosis-related structures of invaginating membrane (open arrowhead) in DRGneurons. Quantitative data are shown in (G) and (H). Scale bars, 200 nm.F-H. Clathrin-coated pits (CCPs, hatched arrows) and bulk-endocytosis-related structures of invaginating membrane (open arrowhead) in DRGneurons. Quantitative data are shown in (G) and (H). Scale bars, 200 nm.I, J. Cumulative frequencies of diameter distributions of small vesicles and endosomes (statistics from 6 control cells and 9 KD cells). Kolmogorov-Smirnov test, P = 0.304 for (I) and P = 0.553 for (J).


Figure 9Figure 9 . Functional domains of Syt11 in endocytosisA . Diagram of the mutant forms of Syt11 used for rescue experiments . B - D . Statistics of FM4 - 64 ( 5 min ) , Tf , and large dextran ( 40 - KD ) uptake into KD neurons with or without rescue by indicated forms of Syt11 . All data are presented as mean ± s . e . m . of 3 independent experiments . One - way ANOVA , values labeled with different letters ( a , b , and c ) are significantly different from each other , * * * P < 0 . 001 . Student ' s t - test for the comparison between KD and control cells in ( B - D ) , * * P < 0 . 01 , * * * P < 0 . 001 .

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Figure 9Figure 9. Functional domains of Syt11 in endocytosisA. Diagram of the mutant forms of Syt11 used for rescue experiments.B-D. Statistics of FM4-64 (5 min), Tf, and large dextran (40-KD) uptake into KD neurons with or without rescue by indicated forms of Syt11.All data are presented as mean ± s.e.m. of 3 independent experiments. One-way ANOVA, values labeled with different letters (a, b, and c) are significantly different from each other, ***P < 0.001. Student's t-test for the comparison between KD and control cells in (B-D), **P < 0.01, ***P < 0.001.


Figure 1SUD binds to Paip1 and Paip1M with the same binding intensity . n . s . : not significant . The Mac2 subdomain is crucial for interacting with Paip1M .

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Figure 1SUD binds to Paip1 and Paip1M with the same binding intensity. n. s.: not significant.The Mac2 subdomain is crucial for interacting with Paip1M.


Figure 3Δ16 - Mac2 cannot bind Paip1M , Mac1 − 2 binds to Paip1M in the split - YFP assay . Scale bars represent 50 µM .

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Figure 3Δ16-Mac2 cannot bind Paip1M, Mac1−2 binds to Paip1M in the split-YFP assay. Scale bars represent 50 µM.


Figure 4SUD and PABP do not compete with each other for interacting with Paip1 . ( A ) Excess expression of SUD - RFP does not impair the split - YFP signal between PABP and Paip1 ( lower images ) , compared to the control ( upper images ) . ( B ) Co - expression of excess RFP - PABP does not result in an impaired interaction between Paip1 and SUD either . RFP : red fluorescent protein . Scale bars represent 50 µM . GFP - trap - based co - immunoprecipitation ( CoIP ) assay demonstrating a complex formed by PABP , Paip1 , and SUD . GFP : green fluorescent protein .

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Figure 4SUD and PABP do not compete with each other for interacting with Paip1. (A) Excess expression of SUD-RFP does not impair the split-YFP signal between PABP and Paip1 (lower images), compared to the control (upper images). (B) Co-expression of excess RFP-PABP does not result in an impaired interaction between Paip1 and SUD either. RFP: red fluorescent protein. Scale bars represent 50 µM.GFP-trap-based co-immunoprecipitation (CoIP) assay demonstrating a complex formed by PABP, Paip1, and SUD. GFP: green fluorescent protein.


Figure 5Left image : SUD generally stimulates protein translation level detected by the luciferase - pcDNA3 reporter ( n = 6 ) . Middle image : SUD does not increase the amounts of total protein synthesis ( host and viral proteins ) in the replicon - transfected cells . Right image : SUD augments viral protein synthesis ( n = 8 ) . HEK - 293 cells growing in 12 - well plates were transfected with the indicated plasmids and replicon DNA . Twenty - four hours post - transfection , cells were harvested for Renilla luciferase activity measurement ( left and right image ) . For the ribopuromycylation assay , 24 hours post - transfection cells were pulsed with 3 µM puromycin for 1 hour at 37 ° C before harvesting for western blot analysis ( middle image ) .

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Figure 5Left image: SUD generally stimulates protein translation level detected by the luciferase-pcDNA3 reporter (n=6). Middle image: SUD does not increase the amounts of total protein synthesis (host and viral proteins) in the replicon-transfected cells. Right image: SUD augments viral protein synthesis (n=8). HEK-293 cells growing in 12-well plates were transfected with the indicated plasmids and replicon DNA. Twenty-four hours post-transfection, cells were harvested for Renilla luciferase activity measurement (left and right image). For the ribopuromycylation assay, 24 hours post-transfection cells were pulsed with 3 µM puromycin for 1 hour at 37°C before harvesting for western blot analysis (middle image).


Figure 6SARS - CoV - 2 SUD ( SARS2 - SUD ) interacts with Paip1 in HEK - 293 cells as demonstrated by the split - YFP assay . SARS2 - Mac2 binds to Paip1 . SARS2 - Mac2 but not Δ16 - SARS2 - Mac2 interacts with Paip1M . MERS - CoV SUD - MC ( Mac3 − DPUP ) or XMC ( Mac1 − Mac3 − DPUP ) do not bind to Paip1 . Scale bars represent 50 µM .

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Figure 6SARS-CoV-2 SUD (SARS2-SUD) interacts with Paip1 in HEK-293 cells as demonstrated by the split-YFP assay.SARS2-Mac2 binds to Paip1. SARS2-Mac2 but not Δ16-SARS2-Mac2 interacts with Paip1M.MERS-CoV SUD-MC (Mac3−DPUP) or XMC (Mac1−Mac3−DPUP) do not bind to Paip1. Scale bars represent 50 µM.


Figure 1 ( A ) Venn diagram of STK38 partners identified in both starvation and suspension conditions by proximity biotinylation assay coupled to mass spectrometry identification ( B ) Heatmap representation of common STK38 interactors identified in both starvation and suspension conditions according to their dynamic of association with STK38 ( with a minimal fold increase of 1 . 38 ± 0 . 06 ) . Unsupervised hierarchical clustering was generated based on their association fold using Pearson correlation . ( C ) Representation of the 10 most cellular component enriched terms of the two clusters ( assessed by using the Gene Ontology ( GO )

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Figure 1(A) Venn diagram of STK38 partners identified in both starvation and suspension conditions by proximity biotinylation assay coupled to mass spectrometry identification (B) Heatmap representation of common STK38 interactors identified in both starvation and suspension conditions according to their dynamic of association with STK38 (with a minimal fold increase of 1.38 ± 0.06). Unsupervised hierarchical clustering was generated based on their association fold using Pearson correlation. (C) Representation of the 10 most cellular component enriched terms of the two clusters (assessed by using the Gene Ontology (GO) database


Figure 2 ( A ) STK38 interacts with XPO1 . HekRasV12 cells were transiently transfected with myc - STK38 ( wt ) together with either Flag - XPO1 ( wt ) plasmid , Flag - control ( ctrl = Sirt3 ) plasmid or without DNA . 24h later , cells were incubated with Okadaic Acid ( OA ) , ( final concentration = 1µM ) for 1 hour or with DMSO . Flag fusions were pulled - down and co - immunoprecipited proteins were analyzed by western blotting ( WB ) . Upper panel displays whole cell lysates ( WCL ) and lower panel represents immunoprecipitated proteins . STK38 accumulates in the cytoplasm upon nutrient starvation in a XPO1 - dependant manner . ( B ) HeLa cells were transfected with myc - STK38 ( wt ) plasmid . The next day , cells were incubated with DMEM or EBSS in the presence of XPO1 inhibitors KPT - 185 or KPT - 330 as indicated ( final concentration = 1 µM ) or DMSO for 4 hours . Cells were then fixed and stained for myc - tag . Representative images are shown and scale bars are 40 µm . STK38 accumulates in the cytoplasm upon nutrient starvation in a XPO1 - dependant manner . ( C ) Quantification of myc - STK38 ( wt ) nuclear / cytoplasmic staining ( n > 30 cells from 3 independent experiments , mean ± SEM ; * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 , Mann - Whitney test ) . XPO1 activity is required for nutrient starvation - induced autophagy . ( D ) Immunoblotting with indicated antibodies of whole cell lysates of cells and its graphical representation ( n = 3 independent experiments , mean ± SEM ; * * * p < 0 . 001 , ns , not significant , Student ' s t test ) . As expected , p62 degradation in starvation conditions was inhibited in presence of XPO1 inhibitors . XPO1 activity is required for nutrient starvation - induced autophagy . ( E ) HeLa cells stably expressing the GFP - LC3 - RFP - LC3ΔG reporter autophagic probe [ 26 ] were incubated with DMEM or EBSS in the presence of XPO1 inhibitors KPT - 185 or KPT - 330 ( final concentration = 1 µM ) or DMSO for 4 hours . The GFP - LC3 ( degraded upon autophagy ) and RFP - LC3ΔG ( non - degraded upon autophagy ) signals were recorded by FACS analysis and then shown as a ratio recapitulating overall LC3 level ( n = 3 independent experiments , mean ± SEM ; * p < 0 . 05 , * * p < 0 . 01 , ns , not significant , Mann - Whitney test ) . Here again , incubation with XPO1 inhibitors significantly impaired the autophagy

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 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 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 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

Figure 2(A) STK38 interacts with XPO1. HekRasV12 cells were transiently transfected with myc-STK38(wt) together with either Flag-XPO1(wt) plasmid, Flag-control (ctrl = Sirt3) plasmid or without DNA. 24h later, cells were incubated with Okadaic Acid (OA), (final concentration = 1µM) for 1 hour or with DMSO. Flag fusions were pulled-down and co-immunoprecipited proteins were analyzed by western blotting (WB). Upper panel displays whole cell lysates (WCL) and lower panel represents immunoprecipitated proteins.STK38 accumulates in the cytoplasm upon nutrient starvation in a XPO1-dependant manner. (B) HeLa cells were transfected with myc-STK38(wt) plasmid. The next day, cells were incubated with DMEM or EBSS in the presence of XPO1 inhibitors KPT-185 or KPT-330 as indicated (final concentration = 1 µM) or DMSO for 4 hours. Cells were then fixed and stained for myc-tag. Representative images are shown and scale bars are 40 µm.STK38 accumulates in the cytoplasm upon nutrient starvation in a XPO1-dependant manner. (C) Quantification of myc-STK38(wt) nuclear/cytoplasmic staining (n > 30 cells from 3 independent experiments, mean ± SEM; *p<0.05, **p<0.01, ***p<0.001, Mann-Whitney test).XPO1 activity is required for nutrient starvation-induced autophagy. (D) Immunoblotting with indicated antibodies of whole cell lysates of cells and its graphical representation (n = 3 independent experiments, mean ± SEM; ***p<0.001, ns, not significant, Student's t test). As expected, p62 degradation in starvation conditions was inhibited in presence of XPO1 inhibitors.XPO1 activity is required for nutrient starvation-induced autophagy. (E) HeLa cells stably expressing the GFP-LC3-RFP-LC3ΔG reporter autophagic probe [26] were incubated with DMEM or EBSS in the presence of XPO1 inhibitors KPT-185 or KPT-330 (final concentration = 1 µM) or DMSO for 4 hours. The GFP-LC3 (degraded upon autophagy) and RFP-LC3ΔG (non-degraded upon autophagy) signals were recorded by FACS analysis and then shown as a ratio recapitulating overall LC3 level (n=3 independent experiments, mean ± SEM; *p<0.05, **p<0.01, ns, not significant, Mann-Whitney test). Here again, incubation with XPO1 inhibitors significantly impaired the autophagy process


Figure 3STK38 kinase activity is required and sufficient for its cytoplasmic accumulation upon nutrient starvation . ( A ) HeLa cells silenced for endogenous STK38 were transfected with myc - STK38 ( wt ) expressing plasmid , myc - STK38 ( K118R ) ( STK38 kinase - dead version ) plasmid or with HA - STK38 ( PIF ) ( STK38 constitutively active version ) plasmid . 24 hours later , cells were incubated with DMEM or EBSS for 4 hours , fixed and stained for myc - tag or HA - tag . Representative images are shown and scale bars are 40 µm . STK38 kinase activity is required and sufficient for its cytoplasmic accumulation upon nutrient starvation . ( B ) Graphical representation of tag - STK38 variants nuclear staining / cytoplasmic staining ( n > 30 cells from 3 independent experiments , mean ± SEM ; * * p < 0 . 01 , * * * p < 0 . 001 , ns , not significant , Mann - Whitney test ) . STK38 kinase activity is required and sufficient for nutrient - starvation induced autophagy . ( C ) HeLa cells stably expressing the GFP - LC3 - RFP - LC3ΔG autophagic probe [ 26 ] were transiently transfected with siRNA targeting the 3 ' UTR region of endogenous STK38 ( or with non - targeting siRNA ( siNT ) ) . The next day , cells were transiently transfected with the indicated STK38 mutants expressing plasmids 24 hours after plasmid transfection , cells were incubated with DMEM or EBSS for 4 hours . The GFP - LC3 ( degraded upon autophagy ) and RFP - LC3ΔG ( non - degraded upon autophagy ) signals were recorded by FACS analysis and then shown as a ratio recapitulating overall LC3 level ( n = 4 independent experiments , mean ± SEM ; * p < 0 . 05 , * * * p < 0 . 001 , ns , not significant , Mann - Whitney test ) . depleting STK38 prevents autophagy to take place . This effect was partially reversed by expressing the wt ORF as well as constitutively active STK38 whereas kinase - dead version failed to reproduce endogenous STK38 effect . Expression of constitutively active STK38 is sufficient to induce a substantial change in the autophagic flux upon nutrient rich conditionsSTK38 kinase activity is required and sufficient for nutrient - starvation induced autophagy . ( D ) HeLa cells were transiently transfected with siRNA targeting the 3 ' UTR region of endogenous STK38 ( or with non - targeting siRNA ( siNT ) ) and then transiently transfected with the indicated STK38 mutants expressing plasmids 48h after . The next day , the cells were incubated with DMEM or EBSS for 4 hours and overall p62 level was quantified for autophagy estimation ( n = 3 independent experiments , mean ± SEM ; * * * p < 0 . 001 , ns , not significant , Student ' s t test ) .

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Figure 3STK38 kinase activity is required and sufficient for its cytoplasmic accumulation upon nutrient starvation. (A) HeLa cells silenced for endogenous STK38 were transfected with myc-STK38(wt) expressing plasmid, myc-STK38(K118R) (STK38 kinase-dead version) plasmid or with HA-STK38(PIF) (STK38 constitutively active version) plasmid. 24 hours later, cells were incubated with DMEM or EBSS for 4 hours, fixed and stained for myc-tag or HA-tag. Representative images are shown and scale bars are 40 µm.STK38 kinase activity is required and sufficient for its cytoplasmic accumulation upon nutrient starvation. (B) Graphical representation of tag-STK38 variants nuclear staining/cytoplasmic staining (n > 30 cells from 3 independent experiments, mean ± SEM; **p<0.01, ***p<0.001, ns, not significant, Mann-Whitney test).STK38 kinase activity is required and sufficient for nutrient-starvation induced autophagy. (C) HeLa cells stably expressing the GFP-LC3-RFP-LC3ΔG autophagic probe [26] were transiently transfected with siRNA targeting the 3'UTR region of endogenous STK38 (or with non-targeting siRNA (siNT)). The next day, cells were transiently transfected with the indicated STK38 mutants expressing plasmids 24 hours after plasmid transfection, cells were incubated with DMEM or EBSS for 4 hours. The GFP-LC3 (degraded upon autophagy) and RFP-LC3ΔG (non-degraded upon autophagy) signals were recorded by FACS analysis and then shown as a ratio recapitulating overall LC3 level (n=4 independent experiments, mean ± SEM; *p<0.05, ***p<0.001, ns, not significant, Mann-Whitney test). depleting STK38 prevents autophagy to take place. This effect was partially reversed by expressing the wt ORF as well as constitutively active STK38 whereas kinase-dead version failed to reproduce endogenous STK38 effect. Expression of constitutively active STK38 is sufficient to induce a substantial change in the autophagic flux upon nutrient rich conditionsSTK38 kinase activity is required and sufficient for nutrient-starvation induced autophagy. (D) HeLa cells were transiently transfected with siRNA targeting the 3'UTR region of endogenous STK38 (or with non-targeting siRNA (siNT)) and then transiently transfected with the indicated STK38 mutants expressing plasmids 48h after. The next day, the cells were incubated with DMEM or EBSS for 4 hours and overall p62 level was quantified for autophagy estimation (n = 3 independent experiments, mean ± SEM; ***p<0.001, ns, not significant, Student's t test).


Figure 4 ( A - B ) STK38 is required for XPO1 _ S1055 phosphorylation . ( A ) HeLa cells were transiently transfected with the indicated siRNA and subjected to Flag - XPO1 ( wt ) transient transfection the following day . 48 hours later , cells were incubated with Okadaic Acid ( OA , final concentration = 1 µM ) for 1 hour or with DMSO . Immunoblotting was performed on whole cell lysates with indicated antibodies . ( B ) Graphical representation of the phospho S1055 - XPO1 signal on total XPO1 ( n = 3 independent experiments , mean ± SEM ; * p < 0 . 05 , ns , not significant , Mann - Whitney test ) . As expected , XPO1 is phosphorylated on its Ser1055 upon OA treatment but not when STK38 is silenced ( C ) STK38 kinase activity is required for XPO1 _ S1055 phosphorylation . HekRasV12 cells silenced ( or not ) for endogenous STK38 were transfected with Flag - XPO1 ( wt ) expression plasmid in addition to myc - STK38 ( wt ) or myc - STK38 ( K118R ) ( STK38 kinase - dead version ) plasmids . The next day , cells were treated with 1 µM OA or with vehicle ( DMSO ) for 1 hour . Flag fusions were immunoprecipited and pulled - down proteins were analyzed by western blotting . Upper panel displays immunoprecipited proteins and lower panel represents whole cell lysates .

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Figure 4(A-B) STK38 is required for XPO1_S1055 phosphorylation. (A) HeLa cells were transiently transfected with the indicated siRNA and subjected to Flag-XPO1(wt) transient transfection the following day. 48 hours later, cells were incubated with Okadaic Acid (OA, final concentration = 1 µM) for 1 hour or with DMSO. Immunoblotting was performed on whole cell lysates with indicated antibodies. (B) Graphical representation of the phospho S1055-XPO1 signal on total XPO1 (n = 3 independent experiments, mean ± SEM; *p<0.05, ns, not significant, Mann-Whitney test). As expected, XPO1 is phosphorylated on its Ser1055 upon OA treatment but not when STK38 is silenced(C) STK38 kinase activity is required for XPO1_S1055 phosphorylation. HekRasV12 cells silenced (or not) for endogenous STK38 were transfected with Flag-XPO1(wt) expression plasmid in addition to myc-STK38(wt) or myc-STK38(K118R) (STK38 kinase-dead version) plasmids. The next day, cells were treated with 1 µM OA or with vehicle (DMSO) for 1 hour. Flag fusions were immunoprecipited and pulled-down proteins were analyzed by western blotting. Upper panel displays immunoprecipited proteins and lower panel represents whole cell lysates.


Figure 5 ( A ) Graphical representation of XPO1 protein . C528 to S amino acid mutation confers XPO1 resistance to both KPT - 185 and KPT - 330 chemical inhibitors [ 24 ] . S1055 amino acid correspond to the phosphorylation target of the STK38 . Finally , the ΔCter region is also highlighted corresponding to a XPO1 construct lacking the 39 C - terminal residues ( B - C ) XPO1 _ S1055 phosphorylation is required and sufficient for STK38 nuclear exit upon nutrient starvation . ( B ) HeLa cells were transiently transfected with myc - STK38 ( wt ) in addition to indicated Flag - XPO1 mutant plasmids . 24 hours later , cells were incubated with DMEM or EBSS for 4 hours both supplemented with KPT - 185 ( final concentration = 1 µM ) in order to inhibit endogenous XPO1 activity . Cells were then fixed and stained for Flag and myc tags . Only cells positives for both Flag - XPO1 and myc - STK38 ( wt ) were captured . Representative images are shown and scale bars are 40 µm . ( C ) Graphical representation of myc - STK38 ( wt ) nuclear staining / cytoplasmic staining ( n > 30 cells from 3 independent experiments , mean ± SEM ; * * * p < 0 . 001 , ns , not significant , Mann - Whitney test ) . Expression of wt XPO1 failed to induce cytoplasmic localization of STK38 upon nutrient starvation in presence of KPT - 185 where C528S mutant recapitulates results described in Figure 2B - C . Expression of phosphonegative XPO1 ( S1055A ) failed to induces STK38 cytoplasmic localization upon EBSS treatment while phosphomimetics XPO1 ( S1055D and S1055E ) were sufficient to promote STK38 cytoplasmic localization without autophagic stimuli . Finally , expression of XPO1 lacking in its 39 C - terminal residues mimicked phosphomimetics variants . ( D ) HAP1 cells carrying genomic XPO1 mutations of S1055 were subjected for IMDM ( nutrient rich medium ) or EBSS ( starvation ) incubation for 4 hours followed by western - blot analysis for p62 level measurement . This result indicates that p62 degradation in starvation conditions was inhibited in XPO1 _ S1055A HAP1 cells whereas p62 degradation was potentiated in both phosphomimetics variant ( S1055D & S1055E ) ( n = 4 independent experiments , mean ± SEM ; * p < 0 . 05 , ns , not significant , Mann - Whitney test ( E ) Hek - HT - iRFP - LC3 cells were transiently transfected with siRNA targeting endogenous STK38 ( or with non - targeting siRNA ( siNT ) ) . Two days after , cells were transiently transfected with indicated Flag - XPO1 mutant plasmids . 24 hours later , cells were incubated with DMEM or EBSS for 4 hours both supplemented with KPT - 185 ( final concentration = 1 µM ) in order to inhibit endogenous XPO1 activity and with chloroquine ( final concentration = 10 µM ) . Cells were then fixed and the number of iRFP - LC3 dots per cell was recorded only in cells positive for Flag - XPO1 mutants As expected , silencing of endogenous STK38 prevented the formation of LC3 dots upon starvation where introduction of phosphonegative XPO1 ( S1055A ) failed also to increase LC3 dots upon starvation . Interestingly , phosphomimetic XPO1 ( S1055D ) was sufficient to increase the number of LC3 dots even in complete and also STK38 - depleted conditions .

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Figure 5(A) Graphical representation of XPO1 protein. C528 to S amino acid mutation confers XPO1 resistance to both KPT-185 and KPT-330 chemical inhibitors [24]. S1055 amino acid correspond to the phosphorylation target of the STK38. Finally, the ΔCter region is also highlighted corresponding to a XPO1 construct lacking the 39 C-terminal residues (B-C) XPO1_S1055 phosphorylation is required and sufficient for STK38 nuclear exit upon nutrient starvation. (B) HeLa cells were transiently transfected with myc-STK38(wt) in addition to indicated Flag-XPO1 mutant plasmids. 24 hours later, cells were incubated with DMEM or EBSS for 4 hours both supplemented with KPT-185 (final concentration = 1 µM) in order to inhibit endogenous XPO1 activity. Cells were then fixed and stained for Flag and myc tags. Only cells positives for both Flag-XPO1 and myc-STK38(wt) were captured. Representative images are shown and scale bars are 40 µm. (C) Graphical representation of myc-STK38(wt) nuclear staining/cytoplasmic staining (n > 30 cells from 3 independent experiments, mean ± SEM; ***p<0.001, ns, not significant, Mann-Whitney test). Expression of wt XPO1 failed to induce cytoplasmic localization of STK38 upon nutrient starvation in presence of KPT-185 where C528S mutant recapitulates results described in Figure 2B-C. Expression of phosphonegative XPO1 (S1055A) failed to induces STK38 cytoplasmic localization upon EBSS treatment while phosphomimetics XPO1 (S1055D and S1055E) were sufficient to promote STK38 cytoplasmic localization without autophagic stimuli. Finally, expression of XPO1 lacking in its 39 C-terminal residues mimicked phosphomimetics variants.(D) HAP1 cells carrying genomic XPO1 mutations of S1055 were subjected for IMDM (nutrient rich medium) or EBSS (starvation) incubation for 4 hours followed by western-blot analysis for p62 level measurement. This result indicates that p62 degradation in starvation conditions was inhibited in XPO1_S1055A HAP1 cells whereas p62 degradation was potentiated in both phosphomimetics variant (S1055D & S1055E) (n = 4 independent experiments, mean ± SEM; *p<0.05, ns, not significant, Mann-Whitney test(E) Hek-HT-iRFP-LC3 cells were transiently transfected with siRNA targeting endogenous STK38 (or with non-targeting siRNA (siNT)). Two days after, cells were transiently transfected with indicated Flag-XPO1 mutant plasmids. 24 hours later, cells were incubated with DMEM or EBSS for 4 hours both supplemented with KPT-185 (final concentration = 1 µM) in order to inhibit endogenous XPO1 activity and with chloroquine (final concentration = 10 µM). Cells were then fixed and the number of iRFP-LC3 dots per cell was recorded only in cells positive for Flag-XPO1 mutants As expected, silencing of endogenous STK38 prevented the formation of LC3 dots upon starvation where introduction of phosphonegative XPO1 (S1055A) failed also to increase LC3 dots upon starvation. Interestingly, phosphomimetic XPO1 (S1055D) was sufficient to increase the number of LC3 dots even in complete and also STK38-depleted conditions.


Figure 6 ( A ) XPO1 and STK38 are required for Beclin1 cytoplasmic accumulation upon nutrient starvation - induced autophagy . HeLa cells were transiently transfected with the indicated siRNA ( control and KPT conditions = siNT ) . 72 hours later , cells were incubated with XPO1 inhibitors KPT - 185 or KPT - 330 as indicated ( final concentration = 1 µM ) or with DMSO for all other conditions for 2 hours prior incubation with DMEM or EBSS supplemented ( or not ) with inhibitors for 2 hours . Cells were fixed and stained for endogenous Beclin1 . Graphical representation of endogenous Beclin1 nuclear staining / cytoplasmic staining ( n > 30 cells from 3 independent experiments , mean ± SEM ; * p < 0 . 05 , * * * p < 0 . 001 , ns , not significant , Mann - Whitney test ) . XPO1 activity inhibition by both KPT - 185 and KPT - 330 as well as STK38 silencing failed to induce Beclin1 cytoplasmic accumulation upon nutrient starvation ( B ) XPO1 _ S1055 phosphorylation is required and sufficient for Beclin1 nuclear exit upon nutrient starvation . Genome edited XPO1 mutant HAP1 cells were incubated with IMDM ( complete ) or EBSS ( starvation ) for 2 hours . Cells were then fixed and stained for endogenous Beclin1 . Graphical representation of endogenous Beclin1 nuclear staining / cytoplasmic staining ( n > 30 cells from 3 independent experiments , mean ± SEM ; * * * p < 0 . 001 , ns , not significant , Mann - Whitney test ) . Here , phosphonegative XPO1 failed to induce Beclin1 cytoplasmic accumulation upon nutrient starvation whereas phosphomimetics variant ( S1055D & S1055E ) potentiated Beclin1 cytoplasmic accumulation ( C ) Genome edited XPO1 mutant HAP1 cells were transiently transfected with siRNA targeting endogenous STK38 ( siSTK38 # 206 or with non - targeting siRNA ( siNT ) ) . 72 hours later , cells were incubated with IMDM ( complete ) or EBSS ( starvation ) for 2 hours . Cells were then fixed and stained for endogenous Beclin1 . Graphical representation of endogenous Beclin1 nuclear staining / cytoplasmic staining ( n = 30 cells from 2 independent experiments ) . Here , phosphomimetic XPO1 ( S1055D ) potentiated Beclin1 cytoplasmic localization in both complete and starvation condition even in the absence of STK38 as compared to cells carrying wt

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

Figure 6(A) XPO1 and STK38 are required for Beclin1 cytoplasmic accumulation upon nutrient starvation-induced autophagy. HeLa cells were transiently transfected with the indicated siRNA (control and KPT conditions = siNT). 72 hours later, cells were incubated with XPO1 inhibitors KPT-185 or KPT-330 as indicated (final concentration = 1 µM) or with DMSO for all other conditions for 2 hours prior incubation with DMEM or EBSS supplemented (or not) with inhibitors for 2 hours. Cells were fixed and stained for endogenous Beclin1. Graphical representation of endogenous Beclin1 nuclear staining/cytoplasmic staining (n > 30 cells from 3 independent experiments, mean ± SEM; *p<0.05, ***p<0.001, ns, not significant, Mann-Whitney test). XPO1 activity inhibition by both KPT-185 and KPT-330 as well as STK38 silencing failed to induce Beclin1 cytoplasmic accumulation upon nutrient starvation(B) XPO1_S1055 phosphorylation is required and sufficient for Beclin1 nuclear exit upon nutrient starvation. Genome edited XPO1 mutant HAP1 cells were incubated with IMDM (complete) or EBSS (starvation) for 2 hours. Cells were then fixed and stained for endogenous Beclin1. Graphical representation of endogenous Beclin1 nuclear staining/cytoplasmic staining (n > 30 cells from 3 independent experiments, mean ± SEM; ***p<0.001, ns, not significant, Mann-Whitney test). Here, phosphonegative XPO1 failed to induce Beclin1 cytoplasmic accumulation upon nutrient starvation whereas phosphomimetics variant (S1055D & S1055E) potentiated Beclin1 cytoplasmic accumulation(C) Genome edited XPO1 mutant HAP1 cells were transiently transfected with siRNA targeting endogenous STK38 (siSTK38#206 or with non-targeting siRNA (siNT)). 72 hours later, cells were incubated with IMDM (complete) or EBSS (starvation) for 2 hours. Cells were then fixed and stained for endogenous Beclin1. Graphical representation of endogenous Beclin1 nuclear staining/cytoplasmic staining (n = 30 cells from 2 independent experiments). Here, phosphomimetic XPO1 (S1055D) potentiated Beclin1 cytoplasmic localization in both complete and starvation condition even in the absence of STK38 as compared to cells carrying wt XPO1


Figure 7 ( A ) XPO1 and STK38 are required for YAP1 nuclear export at high confluence . A549 cells were transiently transfected with the indicated siRNA ( control and KPT conditions = siNT ) at low or high confluency . 48 hours later , cells were incubated overnight in the presence of XPO1 inhibitors KPT - 185 and KPT - 330 ( final concentration = 1 µM ) or with DMSO for all other conditions . The next day , cells were fixed and stained for endogenous YAP1 . Quantitative representation of YAP1 nuclear fluorescence intensity ( n > 300 cells from 3 independent experiments , mean ± SEM ; * * * p < 0 . 001 , Mann - Whitney test ) . XPO1 activity inhibition by both KPT - 185 and KPT - 330 as well as STK38 silencing failed to induce YAP1 nuclear exit at high confluence ( B ) XPO1 _ S1055 phosphorylation is required and sufficient for YAP nuclear exit . Genome edited XPO1 HAP1 cells were cultured for two days at low versus high confluency . Cells were then fixed and stained for endogenous YAP1 . Quantitative representation of YAP1 nuclear fluorescence intensity ( n > 300 cells from 3 independent experiments , mean ± SEM ; * * * p < 0 . 001 , Mann - Whitney test ) . Here , phosphonegative XPO1 failed to induce YAP nuclear exit at high confluence whereas phosphomimetics variant ( S1055D & S1055E ) potentiated YAP1 nuclear exit ( C ) Genome edited XPO1 mutant HAP1 cells were transiently transfected with siRNA targeting endogenous STK38 ( siSTK38 # 206 or with non - targeting siRNA ( siNT ) ) at low or high confluence . 72 hours later , cells were fixed and stained for endogenous YAP1 . Quantitative representation of YAP1 nuclear fluorescence intensity ( n = 150 cells from 2 independent experiments ) . Here , phosphomimetic XPO1 ( S1055D ) potentiated YAP1 nuclear exit at both low and high confluences even in the absence of STK38 as compared to cells carrying wt

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Figure 7(A) XPO1 and STK38 are required for YAP1 nuclear export at high confluence. A549 cells were transiently transfected with the indicated siRNA (control and KPT conditions = siNT) at low or high confluency. 48 hours later, cells were incubated overnight in the presence of XPO1 inhibitors KPT-185 and KPT-330 (final concentration = 1 µM) or with DMSO for all other conditions. The next day, cells were fixed and stained for endogenous YAP1. Quantitative representation of YAP1 nuclear fluorescence intensity (n > 300 cells from 3 independent experiments, mean ± SEM; ***p<0.001, Mann-Whitney test). XPO1 activity inhibition by both KPT-185 and KPT-330 as well as STK38 silencing failed to induce YAP1 nuclear exit at high confluence(B) XPO1_S1055 phosphorylation is required and sufficient for YAP nuclear exit. Genome edited XPO1 HAP1 cells were cultured for two days at low versus high confluency. Cells were then fixed and stained for endogenous YAP1. Quantitative representation of YAP1 nuclear fluorescence intensity (n > 300 cells from 3 independent experiments, mean ± SEM; ***p<0.001, Mann-Whitney test). Here, phosphonegative XPO1 failed to induce YAP nuclear exit at high confluence whereas phosphomimetics variant (S1055D & S1055E) potentiated YAP1 nuclear exit(C) Genome edited XPO1 mutant HAP1 cells were transiently transfected with siRNA targeting endogenous STK38 (siSTK38#206 or with non-targeting siRNA (siNT)) at low or high confluence. 72 hours later, cells were fixed and stained for endogenous YAP1. Quantitative representation of YAP1 nuclear fluorescence intensity (n = 150 cells from 2 independent experiments). Here, phosphomimetic XPO1 (S1055D) potentiated YAP1 nuclear exit at both low and high confluences even in the absence of STK38 as compared to cells carrying wt XPO1


Figure 1B Endogenous USP45 was immunoprecipitated ( IP ) from wild ‐ type ( WT ) and USP45 knockout ( KO ) KBM7 cells lines . The immunoprecipitates were resolved on a polyacrylamide gel and stained with Instant Blue . The gel was divided into the indicated sections , and proteins identified by mass spectrometry are detailed in Supplementary Table S1 . The key proteins that we focus on in this study are indicated . C Endogenous USP45 was immunoprecipitated from the WT and KO KBM7 cell lines and subjected to immunoblotting with the indicated antibodies . D Endogenous USP45 was immunoprecipitated from WT and USP45 KO U2OS cells lines and analysed as in ( B ) . Mascot scores of the interactors are detailed in Supplementary Table S2 . E Endogenous USP45 was immunoprecipitated from the WT or KO U2OS cell lines and subjected to immunoblotting with the indicated antibodies . Similar results were obtained in at least two separate experiments .

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Figure 1B Endogenous USP45 was immunoprecipitated (IP) from wild‐type (WT) and USP45 knockout (KO) KBM7 cells lines. The immunoprecipitates were resolved on a polyacrylamide gel and stained with Instant Blue. The gel was divided into the indicated sections, and proteins identified by mass spectrometry are detailed in Supplementary Table S1. The key proteins that we focus on in this study are indicated.C Endogenous USP45 was immunoprecipitated from the WT and KO KBM7 cell lines and subjected to immunoblotting with the indicated antibodies.D Endogenous USP45 was immunoprecipitated from WT and USP45 KO U2OS cells lines and analysed as in (B). Mascot scores of the interactors are detailed in Supplementary Table S2.E Endogenous USP45 was immunoprecipitated from the WT or KO U2OS cell lines and subjected to immunoblotting with the indicated antibodies. Similar results were obtained in at least two separate experiments.


Figure 2A Extracts of HEK293 cells were analysed by gel filtration on a Superdex 200 10 / 300 GL preparative grade column ( GE Healthcare ) in buffer containing 0 . 15 M NaCl , and every second fraction denatured and analysed by immunoblotting with the indicated antibodies . The white dotted lines indicate that the samples were run on different polyacrylamide gels . B Endogenous USP45 or ERCC1 was immunoprecipitated from HEK293 cell lysates and subjected to immunoblotting with the indicated antibodies . C Yeast two ‐ hybrid ( Y2H ) assays were performed with a GAL4 DNA ‐ binding domain fusion and / or activation domain for each protein indicated in the table to detect interaction between these proteins . Cells grown on media lacking LEU , TRP and HIS ( to select for bait and prey plasmids ) or tested for lacZ reporter gene activity . D , E Endogenous SLX4 and ERCC1 were immunoprecipitated from extracts of WT or ERCC1 KO MEFs ( D ) , or WT or SLX4 KO MEFs ( E ) and subjected to immunoblotting with the indicated antibodies . F Extracts of WT or SLX4 KO MEFs were analysed by gel filtration as described in ( A ) . Similar results were obtained in at least two separate experiments .

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

Figure 2A Extracts of HEK293 cells were analysed by gel filtration on a Superdex 200 10/300 GL preparative grade column (GE Healthcare) in buffer containing 0.15 M NaCl, and every second fraction denatured and analysed by immunoblotting with the indicated antibodies. The white dotted lines indicate that the samples were run on different polyacrylamide gels.B Endogenous USP45 or ERCC1 was immunoprecipitated from HEK293 cell lysates and subjected to immunoblotting with the indicated antibodies.C Yeast two‐hybrid (Y2H) assays were performed with a GAL4 DNA‐binding domain fusion and/or activation domain for each protein indicated in the table to detect interaction between these proteins. Cells grown on media lacking LEU, TRP and HIS (to select for bait and prey plasmids) or tested for lacZ reporter gene activity.D, E Endogenous SLX4 and ERCC1 were immunoprecipitated from extracts of WT or ERCC1 KO MEFs (D), or WT or SLX4 KO MEFs (E) and subjected to immunoblotting with the indicated antibodies.F Extracts of WT or SLX4 KO MEFs were analysed by gel filtration as described in (A). Similar results were obtained in at least two separate experiments.


Figure 3A HEK293 cells were transiently transfected with the indicated GFP fusion constructs encoding full ‐ length or indicated fragments of USP45 . Thirty ‐ six hours post ‐ transfection cells were lysed and GFP immunoprecipitated . The immunoprecipitates were resolved on a polyacrylamide gel and stained with Instant Blue or subjected to immunoblotting with the indicated antibodies . GFP empty vector was used as a negative control . C , D HEK293 cells were transiently transfected with constructs expressing the indicated wild ‐ type or mutant GFP - USP45 [ 1 - 62 ] fragment ( C ) or GFP full ‐ length USP45 ( D ) or empty GFP ( C and D ) as a control and processed as described in ( A ) . LICOR - Odyssey quantitation of results shown in ( C ) is shown in Supplementary Fig S4 . E As in ( C ) except HEK293 cells were transfected with the indicated N ‐ terminal Znf ‐ UBP domain ‐ containing DUBs . Similar results were obtained in at least two separate experiments .

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Figure 3A HEK293 cells were transiently transfected with the indicated GFP fusion constructs encoding full‐length or indicated fragments of USP45. Thirty‐six hours post‐transfection cells were lysed and GFP immunoprecipitated. The immunoprecipitates were resolved on a polyacrylamide gel and stained with Instant Blue or subjected to immunoblotting with the indicated antibodies. GFP empty vector was used as a negative control.C, D HEK293 cells were transiently transfected with constructs expressing the indicated wild‐type or mutant GFP-USP45 [1-62] fragment (C) or GFP full‐length USP45 (D) or empty GFP (C and D) as a control and processed as described in (A). LICOR-Odyssey quantitation of results shown in (C) is shown in Supplementary Fig S4.E As in (C) except HEK293 cells were transfected with the indicated N‐terminal Znf‐UBP domain‐containing DUBs. Similar results were obtained in at least two separate experiments.


Figure 4A HEK293 cells were treated with the proteasome inhibitor lactacystin ( 5 μM ) or bortezomib ( 10 μM ) for the times indicated . Cells were lysed in the presence of 0 . 1 M N ‐ ethylmaleimide to inhibit deubiquitinylation and lysates were subjected to immunoblotting with indicated antibodies . B Wild ‐ type ( WT ) and USP45 knockout ( KO ) U2OS cells were lysed and subjected to immunoblotting as in ( A ) . C USP45 KO U2OS cells were transiently transfected with a construct encoding FLAG ‐ ERCC1 . Thirty ‐ six hours post ‐ transfection cells were treated with lactacystin ( 5 μM ) for 6 h and lysed with a buffer containing 0 . 1 M N ‐ ethylmaleimide . Flag ‐ ERCC1 was immunoprecipitated and treated in the absence or presence of 0 . 1 μg of recombinant USP45 wild ‐ type ( WT ) , non ‐ ERCC1 ‐ binding USP45 [ Asp25Ala , Glu26Ala ] ( AA ) or catalytically inactive USP45 [ Cys199Ala ] ( CA ) for 1 h . The reactions were terminated by addition of sample buffer and analysed by immunoblot and Coomassie staining . Similar results were obtained in at least two separate experiments .

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Figure 4A HEK293 cells were treated with the proteasome inhibitor lactacystin (5 μM) or bortezomib (10 μM) for the times indicated. Cells were lysed in the presence of 0.1 M N‐ethylmaleimide to inhibit deubiquitinylation and lysates were subjected to immunoblotting with indicated antibodies.B Wild‐type (WT) and USP45 knockout (KO) U2OS cells were lysed and subjected to immunoblotting as in (A).C USP45 KO U2OS cells were transiently transfected with a construct encoding FLAG‐ERCC1. Thirty‐six hours post‐transfection cells were treated with lactacystin (5 μM) for 6 h and lysed with a buffer containing 0.1 M N‐ethylmaleimide. Flag‐ERCC1 was immunoprecipitated and treated in the absence or presence of 0.1 μg of recombinant USP45 wild‐type (WT), non‐ERCC1‐binding USP45 [Asp25Ala, Glu26Ala] (AA) or catalytically inactive USP45 [Cys199Ala] (CA) for 1 h. The reactions were terminated by addition of sample buffer and analysed by immunoblot and Coomassie staining. Similar results were obtained in at least two separate experiments.


Figure 5A Clonogenic survival assays were carried out in wild ‐ type ( WT ) , USP45 knockout ( KO ) or USP45 KO cells re ‐ expressing wild ‐ type USP45 ( RESWT ) or catalytically inactive USP45 [ Cys199Ala ] ( RESCA ) . The cells were treated with the indicated doses of mitomycin C ( MMC ) 16 h . Medium was changed and number of colonies quantified after 10 days . Each data point is the average of 3 experiments undertaken with 6 replicates ± SD . B Control immunoblot analysis demonstrating that in the USP45 KO U2OS rescue cell lines , similar levels of wild ‐ type and catalytically inactive USP45 [ Cys199Ala ] were expressed . C , D As above , except rescue experiments are undertaken using the non ‐ ERCC1 ‐ binding USP45 [ Asp25Ala , Glu26Ala ] mutant . E , F As in ( A ) , except that DNA damage is induced using UV ‐ C irradiation ( E ) or 16 h hydroxyurea treatment ( F ) . Similar results were obtained in at two separate experiments .

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Figure 5A Clonogenic survival assays were carried out in wild‐type (WT), USP45 knockout (KO) or USP45 KO cells re‐expressing wild‐type USP45 (RESWT) or catalytically inactive USP45 [Cys199Ala] (RESCA). The cells were treated with the indicated doses of mitomycin C (MMC) 16 h. Medium was changed and number of colonies quantified after 10 days. Each data point is the average of 3 experiments undertaken with 6 replicates ± SD.B Control immunoblot analysis demonstrating that in the USP45 KO U2OS rescue cell lines, similar levels of wild‐type and catalytically inactive USP45 [Cys199Ala] were expressed.C, D As above, except rescue experiments are undertaken using the non‐ERCC1‐binding USP45 [Asp25Ala, Glu26Ala] mutant.E, F As in (A), except that DNA damage is induced using UV‐C irradiation (E) or 16 h hydroxyurea treatment (F). Similar results were obtained in at two separate experiments.


Figure 6A Wild ‐ type ( WT ) , USP45 knockout ( KO ) or USP45 KO cells re ‐ expressing wild ‐ type USP45 ( RESWT ) or catalytically inactive USP45 [ Cys199Ala ] ( RESCA ) were used to analyse ERCC1 foci formation . Staining and analysis of endogenous ERCC1 foci formation was undertaken before ( left panel ) , or after DNA damage induction by mitomycin C ( 40 ng / ml , 16 h , middle panel ) or UV ( 20 J / m2 followed by 3 ‐ h recovery , right panel ) . Three independent experiments were performed in which 500 cells per experiment were analysed . Scale bar , 10 μm . B Proportion of cells displaying more than 10 endogenous ERCC1 foci were quantified . Three independent experiments were performed in which 500 cells per experiment were analysed . Results are the mean of 3 experiments ± SD . C The wild ‐ type and USP45 knockout U2OS cells were treated with mitomycin C ( 40 ng / ml , 16 h ) and total number of ERCC1 staining foci per cell was quantified in at least 50 independent cells at the indicated times . Similar results were obtained in two separate experiments . The data are presented as the average number of ERCC1 staining foci per cell . D As in ( C ) , except that γ ‐ H2AX staining foci were analysed . The data are presented as the average number of γ ‐ H2AX staining foci per cell .

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Figure 6A Wild‐type (WT), USP45 knockout (KO) or USP45 KO cells re‐expressing wild‐type USP45 (RESWT) or catalytically inactive USP45 [Cys199Ala] (RESCA) were used to analyse ERCC1 foci formation. Staining and analysis of endogenous ERCC1 foci formation was undertaken before (left panel), or after DNA damage induction by mitomycin C (40 ng/ml, 16 h, middle panel) or UV (20 J/m2 followed by 3‐h recovery, right panel). Three independent experiments were performed in which 500 cells per experiment were analysed. Scale bar, 10 μm.B Proportion of cells displaying more than 10 endogenous ERCC1 foci were quantified. Three independent experiments were performed in which 500 cells per experiment were analysed. Results are the mean of 3 experiments ± SD.C The wild‐type and USP45 knockout U2OS cells were treated with mitomycin C (40 ng/ml, 16 h) and total number of ERCC1 staining foci per cell was quantified in at least 50 independent cells at the indicated times. Similar results were obtained in two separate experiments. The data are presented as the average number of ERCC1 staining foci per cell.D As in (C), except that γ‐H2AX staining foci were analysed. The data are presented as the average number of γ‐H2AX staining foci per cell.


Figure 7A Representative immunofluorescence microscopy images of endogenous wild ‐ type ( WT ) and USP45 knockout ( KO ) U2OS cells treated with no drug , BrdU ( 10 μM , 24 h ) , psoralen or angelicin ( 25 μM , 3 h ) . Cells were fixed 10 min after laser micro ‐ irradiation and stained for localisation of endogenous USP45 , phosphorylated γ ‐ H2AX and DAPI . The right ‐ hand panel shows magnification of one micro ‐ irradiated cell . Similar results were obtained in at least three independent experiments . Scale bar , 10 μm .

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Figure 7A Representative immunofluorescence microscopy images of endogenous wild‐type (WT) and USP45 knockout (KO) U2OS cells treated with no drug, BrdU (10 μM, 24 h), psoralen or angelicin (25 μM, 3 h). Cells were fixed 10 min after laser micro‐irradiation and stained for localisation of endogenous USP45, phosphorylated γ‐H2AX and DAPI. The right‐hand panel shows magnification of one micro‐irradiated cell. Similar results were obtained in at least three independent experiments. Scale bar, 10 μm.


Figure 8A U2OS cells were transiently transfected with the GFP - USP45 or GFP alone . Thirty ‐ six hours post ‐ transfection cells were irradiated with UV ‐ C ( 150 J / m2 ) through a micropore filter ( lower panels ) or left untreated ( upper panels ) . Five minutes post ‐ irradiation , cells were fixed and co ‐ immunostained with antibodies recognising cyclobutane pyrimidine dimer ( CPD ) DNA lesions and GFP . The white arrows indicate co ‐ localisation between GFP - USP45 and CPD . Scale bar , 10 μm . B U2OS wild ‐ type ( WT ) and USP45 knockout ( KO ) cells were irradiated with UV ‐ C ( 20 J / m2 ) . At the indicated post ‐ irradiation times , genomic DNA was extracted and subjected to Southern dot blot analysis using antibodies recognising CPD and dsDNA total antibody . Data are shown from a triplicate experiment in which each blot is derived from independent cells . C Same as ( B ) but using a High Sensitivity CPD / Cyclobutane Pyrimidine Dimer Elisa kit ( NM ‐ MA ‐ K001 ) from Cosmo Bio . For the DNA damage detection , the manufacturer protocol was followed . Absorbance at 492 was measured that represent amount of CPDs in each sample at indicated times . Calf thymus DNA , UV ‐ Cirradiated ( 10 J / m2 ) and not irradiated was used as positive and negative samples , respectively .

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Figure 8A U2OS cells were transiently transfected with the GFP-USP45 or GFP alone. Thirty‐six hours post‐transfection cells were irradiated with UV‐C (150 J/m2) through a micropore filter (lower panels) or left untreated (upper panels). Five minutes post‐irradiation, cells were fixed and co‐immunostained with antibodies recognising cyclobutane pyrimidine dimer (CPD) DNA lesions and GFP. The white arrows indicate co‐localisation between GFP-USP45 and CPD. Scale bar, 10 μm.B U2OS wild‐type (WT) and USP45 knockout (KO) cells were irradiated with UV‐C (20 J/m2). At the indicated post‐irradiation times, genomic DNA was extracted and subjected to Southern dot blot analysis using antibodies recognising CPD and dsDNA total antibody. Data are shown from a triplicate experiment in which each blot is derived from independent cells.C Same as (B) but using a High Sensitivity CPD/Cyclobutane Pyrimidine Dimer Elisa kit (NM‐MA‐K001) from Cosmo Bio. For the DNA damage detection, the manufacturer protocol was followed. Absorbance at 492 was measured that represent amount of CPDs in each sample at indicated times. Calf thymus DNA, UV‐Cirradiated (10 J/m2) and not irradiated was used as positive and negative samples, respectively.


Figure 9A Live ‐ cell fluorescence microscopy analysis of U2OS cells stably expressing either GFPNLS empty , wild ‐ type ( WT ) USP45 or catalytically inactive USP45 [ Cys199Ala ] . Cells were treated with BrdU ( 10 μM , 24 h ) , psoralen or angelicin ( 25 μM , 3 h ) and images captured at the indicated times after laser micro ‐ irradiation . The location of the micro ‐ irradiation stripe in each cell is indicated with a yellow line . Similar results were obtained in at least two independent experiments . Scale bar , 10 μm .

Figure 9A Live‐cell fluorescence microscopy analysis of U2OS cells stably expressing either GFPNLS empty, wild‐type (WT) USP45 or catalytically inactive USP45 [Cys199Ala]. Cells were treated with BrdU (10 μM, 24 h), psoralen or angelicin (25 μM, 3 h) and images captured at the indicated times after laser micro‐irradiation. The location of the micro‐irradiation stripe in each cell is indicated with a yellow line. Similar results were obtained in at least two independent experiments. Scale bar, 10 μm.


Figure 1A . Lysates from 293T cells transfected with the indicated DNA constructs were incubated with 1 mM Mg + + plus 0 . 1 mM GTPγS , GDP or 2 mM EDTA and immunoprecipitated with anti - Myc antibody . Rab18 - HA co - immunoprecipitated with Myc - TRAPPC9 when it was nucleotide free ( + EDTA ) . B . A mutant of Rab18 unable to bind nucleotide , N122I , was co - immunoprecipitated with Myc - TRAPPC9 by anti - Myc antibody . C . HA - tagged Rab1 and Rab18 , but not Rab2 , could co - immunoprecipitate endogenous TRAPPII complex but not TRAPPIII complex . D . GST - Rab18 could pull down endogenous TRAPPII complex in an in vitro binding assay . GST - Rab18 was pre - loaded with GTPγS or GDP , or stripped off nucleotide by EDTA . Lysates from 293T cells were subjected to GST - Rab18 pull - down . The presence of various TRAPP subunits was detected by immunoblotting . TRAPPII , but not TRAPPIII complex was co - isolated with GST - Rab18 , most strongly with Rab18 in nucleotide - free state .

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Figure 1A. Lysates from 293T cells transfected with the indicated DNA constructs were incubated with 1 mM Mg++ plus 0.1 mM GTPγS, GDP or 2 mM EDTA and immunoprecipitated with anti-Myc antibody. Rab18-HA co-immunoprecipitated with Myc-TRAPPC9 when it was nucleotide free (+EDTA).B. A mutant of Rab18 unable to bind nucleotide, N122I, was co-immunoprecipitated with Myc-TRAPPC9 by anti-Myc antibody.C. HA-tagged Rab1 and Rab18, but not Rab2, could co-immunoprecipitate endogenous TRAPPII complex but not TRAPPIII complex.D. GST-Rab18 could pull down endogenous TRAPPII complex in an in vitro binding assay. GST-Rab18 was pre-loaded with GTPγS or GDP, or stripped off nucleotide by EDTA. Lysates from 293T cells were subjected to GST-Rab18 pull-down. The presence of various TRAPP subunits was detected by immunoblotting. TRAPPII, but not TRAPPIII complex was co-isolated with GST-Rab18, most strongly with Rab18 in nucleotide-free state.


Figure 2A . TRAPPII and TRAPPIII complexes were immunoprecipitated by antibodies specifically recognizing TRAPPC9 or TRAPPC12 , and the presence of various co - precipitating TRAPP subunits was detected by immunoblotting . B - D . TRAPP stimulated guanine nucleotide exchange on purified Rab18 , Rab1 and Rab2 GST fusion proteins measured by release of 3H - GDP that was pre - loaded onto the Rab proteins . E - G . Nucleotide exchange reactions measured by time - dependent binding of [ 35S ] GTPγS . Immunoprecipitates using the indicated antibodies were included in the assay as the GEF to be tested . In the 3H - GDP release experiments , the reactions were incubated for 30 minutes at 37 ° C before the Rab proteins were subjected to nitrocellulose filter binding . In the [ 35S ] GTPγS binding experiments , the reactions were incubated for the indicated amount of time before filter binding . H - I . Anti - Myc antibody immunoprecipitates from lysates of HEK293T cells transfected Myc - TRAPPC9 were used as the GEF for GST - Rab1 or GST - Rab18 in 30 min incubations at 37 ° C with [ 35S ] GTPγS .

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Figure 2A. TRAPPII and TRAPPIII complexes were immunoprecipitated by antibodies specifically recognizing TRAPPC9 or TRAPPC12, and the presence of various co-precipitating TRAPP subunits was detected by immunoblotting.B-D. TRAPP stimulated guanine nucleotide exchange on purified Rab18, Rab1 and Rab2 GST fusion proteins measured by release of 3H-GDP that was pre-loaded onto the Rab proteins.E-G. Nucleotide exchange reactions measured by time-dependent binding of [35S]GTPγS. Immunoprecipitates using the indicated antibodies were included in the assay as the GEF to be tested. In the 3H-GDP release experiments, the reactions were incubated for 30 minutes at 37°C before the Rab proteins were subjected to nitrocellulose filter binding. In the [35S]GTPγS binding experiments, the reactions were incubated for the indicated amount of time before filter binding.H-I. Anti-Myc antibody immunoprecipitates from lysates of HEK293T cells transfected Myc-TRAPPC9 were used as the GEF for GST-Rab1 or GST-Rab18 in 30 min incubations at 37°C with [35S]GTPγS.


Figure 3A . Depleting TRAPPC9 , but not TRAPPC8 , increased lipid droplet size in HEK293T cells . The efficiency of siRNA depletion of TRAPPC8 and TRAPPC9 in Hela cells is shown by immunoblotting in the top left panel . A . Depleting TRAPPC9 , but not TRAPPC8 , increased lipid droplet size in HEK293T cells . Representative fluorescence images show the status of lipid droplets in these siRNA treated cells after oleic acid incubation . Bar = 10 μm . B . After loading with oleic acid , human skin fibroblasts containing a R475 * mutation in TRAPPC9 accumulated lipid droplets of greater sizes than skin fibroblasts similarly isolated from a TRAPPC9 wildtype human individual . Bar = 10 μm .

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Figure 3A. Depleting TRAPPC9, but not TRAPPC8, increased lipid droplet size in HEK293T cells. The efficiency of siRNA depletion of TRAPPC8 and TRAPPC9 in Hela cells is shown by immunoblotting in the top left panel.A. Depleting TRAPPC9, but not TRAPPC8, increased lipid droplet size in HEK293T cells. Representative fluorescence images show the status of lipid droplets in these siRNA treated cells after oleic acid incubation. Bar =10 μm.B. After loading with oleic acid, human skin fibroblasts containing a R475* mutation in TRAPPC9 accumulated lipid droplets of greater sizes than skin fibroblasts similarly isolated from a TRAPPC9 wildtype human individual. Bar = 10 μm.


Figure 4A . HEK293T cells with the indicated genotypes were loaded with 400 μM oleic acid for 24 h before staining with bodipy 493 / 503 . Bar = 10 μm . B . Wildtype or TRAPPII deleted cells were loaded with oleic acid and stained with bodipy 493 / 503 ( green ) and endogenous Rab18 by immunofluorescence ( red ) . Bar = 10 μm . C . DsRed - Rab18 was transfected into wildtype ( top panels and insets ) or TRAPPII deleted HEK293T cells ( bottom panels and insets ) that were loaded with oleic acid and stained with bodipy 493 / 503 ( green ) . Bar = 10 μm . D . Homogenates of wildtype or TRAPPII deleted cells were subjected to sucrose gradient fractionation . Fractions 1 and 2 were enriched with LDs . The presence of Rab18 , TRAPPC9 , TRAPPC2 , LD marker TIP47 , and ER marker Calnexin were detected by SDS - PAGE followed by immunoblotting .

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Figure 4A. HEK293T cells with the indicated genotypes were loaded with 400 μM oleic acid for 24 h before staining with bodipy 493/503. Bar = 10 μm.B. Wildtype or TRAPPII deleted cells were loaded with oleic acid and stained with bodipy 493/503 (green) and endogenous Rab18 by immunofluorescence (red). Bar = 10 μm.C. DsRed-Rab18 was transfected into wildtype (top panels and insets) or TRAPPII deleted HEK293T cells (bottom panels and insets) that were loaded with oleic acid and stained with bodipy 493/503 (green). Bar = 10 μm.D. Homogenates of wildtype or TRAPPII deleted cells were subjected to sucrose gradient fractionation. Fractions 1 and 2 were enriched with LDs. The presence of Rab18, TRAPPC9, TRAPPC2, LD marker TIP47, and ER marker Calnexin were detected by SDS-PAGE followed by immunoblotting.


Figure 5Control siRNA depletion ( siCtrl ) was conducted with siRNA oligoes specific to a firefly luciferase sequence ; siCOPB = depletion of γ - COP ; siRab18 = depletion of Rab18 . C9C10KO = HEK293T cells with TRAPPC9 and TRAPPC10 genes deleted . N = 3 , Error bars = S . D .

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Figure 5Control siRNA depletion (siCtrl) was conducted with siRNA oligoes specific to a firefly luciferase sequence; siCOPB = depletion of γ-COP; siRab18 = depletion of Rab18. C9C10KO = HEK293T cells with TRAPPC9 and TRAPPC10 genes deleted. N= 3, Error bars = S.D.


Figure 6A . Myc - TRAPPC9 directly interacted with FLAG - γ - COP . The presence of FLAG - γ - COP in immunoprecipitations of Myc - TRAPPC9 and Myc - TRAPPC10 from cell lysates of the indicated genetic backgrounds was investigated by immunoblotting . B , C . Co - immunoprecipitation between FLAG - γ - COP and HA - Rab18 in HEK293T cells with indicated genetic backgrounds . The presence of FLAG - γ - COP in immunoprecipitates of HA - Rab18 ( and reciprocal IP in C ) was investigated by immunoblotting . D . The interaction between endogenous COPI and Rab18 was TRAPPII dependent . Endogenous Rab18 was immunoprecipitated by anti - Rab18 antibody from lysates of indicated genetic background . The presence of γ - COP and various TRAPP subunits were detected by their specific antibodies as indicated . E . Co - immunoprecipitation between TRAPPII and HA - Rab18 in control ( siFFL ) and γ - COP ( si - γ - COP ) siRNA depleted cells . TRAPPII was precipitated with transfected Myc - TRAPPC10 and the presence of HA - Rab18 was determined by immunoblotting . F . Co - immunoprecipitation between FLAG - γ - COP and TRAPPII in control ( siFFL ) and Rab18 ( si - Rab18 ) siRNA depleted cells . TRAPPII was precipitated with Myc - TRAPPC10 and the presence of FLAG - γ - COP was determined by immunoblotting . The molecular weight of endogenous Rab18 overlaps with IgG light chain , and therefore , the amount of endogenous Rab18 precipitated with TRAPPII cannot be determined in this experiment .

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Figure 6A. Myc-TRAPPC9 directly interacted with FLAG-γ-COP. The presence of FLAG-γ-COP in immunoprecipitations of Myc-TRAPPC9 and Myc-TRAPPC10 from cell lysates of the indicated genetic backgrounds was investigated by immunoblotting.B,C. Co-immunoprecipitation between FLAG-γ-COP and HA-Rab18 in HEK293T cells with indicated genetic backgrounds. The presence of FLAG-γ-COP in immunoprecipitates of HA-Rab18 (and reciprocal IP in C) was investigated by immunoblotting.D. The interaction between endogenous COPI and Rab18 was TRAPPII dependent. Endogenous Rab18 was immunoprecipitated by anti-Rab18 antibody from lysates of indicated genetic background. The presence of γ-COP and various TRAPP subunits were detected by their specific antibodies as indicated.E. Co-immunoprecipitation between TRAPPII and HA-Rab18 in control (siFFL) and γ-COP (si-γ-COP) siRNA depleted cells. TRAPPII was precipitated with transfected Myc-TRAPPC10 and the presence of HA-Rab18 was determined by immunoblotting.F. Co-immunoprecipitation between FLAG-γ-COP and TRAPPII in control (siFFL) and Rab18 (si-Rab18) siRNA depleted cells. TRAPPII was precipitated with Myc-TRAPPC10 and the presence of FLAG-γ-COP was determined by immunoblotting. The molecular weight of endogenous Rab18 overlaps with IgG light chain, and therefore, the amount of endogenous Rab18 precipitated with TRAPPII cannot be determined in this experiment.


Figure 7A . Huh - 7 cells were depleted with control siRNA specific to firefly luciferase sequence ( top panels ) or with siRNA specific to γ - COP ( bottom panels ) . Endogenous Rab18 ( red ) and LD ( green ) were stained and visualized . Bar = 10 μm . B . Huh - 7 cells were treated with or without BFA for 6 hours before staining for Rab18 ( red ) and LD ( green ) . Bar = 10 μm . C . TRAPPC9 were detected on Golgi but not LD surface . Huh - 7 cells were permeabilized with digitonin before fixation and staining . LDs were labeled with bodipy 493 / 503 and pseudocolored in white . TRAPPC9 ( red ) and LD surface marker ADRP ( green ) were detected by their respective antibodies . Bar = 10 μm .

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Figure 7A. Huh-7 cells were depleted with control siRNA specific to firefly luciferase sequence (top panels) or with siRNA specific to γ-COP (bottom panels). Endogenous Rab18 (red) and LD (green) were stained and visualized. Bar = 10 μm.B. Huh-7 cells were treated with or without BFA for 6 hours before staining for Rab18 (red) and LD (green). Bar = 10 μm.C. TRAPPC9 were detected on Golgi but not LD surface. Huh-7 cells were permeabilized with digitonin before fixation and staining. LDs were labeled with bodipy 493/503 and pseudocolored in white. TRAPPC9 (red) and LD surface marker ADRP (green) were detected by their respective antibodies. Bar = 10 μm.


Figure 8Huh - 7 cells were first serum - starved and then incubated with oleic acid for the indicated time before staining with Bodipy 493 / 503 , TRAPPC9 , Rab18 and γ - COP . A . In the merged images , bodipy 493 / 503 was pseudocolored in green , TRAPPC9 in red , and ADRP in blue . In the colocalization between TRAPPC9 and ADRP on the right side , the ADRP signal was re - colored to green for easy visualization . Bar = 10μm . Huh - 7 cells were first serum - starved and then incubated with oleic acid for the indicated time before staining with Bodipy 493 / 503 , TRAPPC9 , Rab18 and γ - COP . B . Bodipy 493 / 503 was pseudocolored in green , Rab18 in red , and γ - COP in blue . In the colocalization between Rab18 and γ - COP on the right side , the γ - COP signal was re - colored to green for easy visualization . Bar = 10μm .

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Figure 8Huh-7 cells were first serum-starved and then incubated with oleic acid for the indicated time before staining with Bodipy 493/503, TRAPPC9, Rab18 and γ-COP.A. In the merged images, bodipy 493/503 was pseudocolored in green, TRAPPC9 in red, and ADRP in blue. In the colocalization between TRAPPC9 and ADRP on the right side, the ADRP signal was re-colored to green for easy visualization. Bar = 10μm.Huh-7 cells were first serum-starved and then incubated with oleic acid for the indicated time before staining with Bodipy 493/503, TRAPPC9, Rab18 and γ-COP.B. Bodipy 493/503 was pseudocolored in green, Rab18 in red, and γ-COP in blue. In the colocalization between Rab18 and γ-COP on the right side, the γ-COP signal was re-colored to green for easy visualization. Bar = 10μm.