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 ) .

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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 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 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.


SUD binds to Paip1 and Paip1M with the same binding intensity . n . s . : not significant .

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SUD binds to Paip1 and Paip1M with the same binding intensity. n. s.: not significant.


The Mac2 subdomain is crucial for interacting with Paip1M .

The Mac2 subdomain is crucial for interacting with Paip1M.


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

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


SUD 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 .

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SUD 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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GFP-trap-based co-immunoprecipitation (CoIP) assay demonstrating a complex formed by PABP, Paip1, and SUD. GFP: green fluorescent protein.


Left 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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Left 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).


SARS - CoV - 2 SUD ( SARS2 - SUD ) interacts with Paip1 in HEK - 293 cells as demonstrated by the split - YFP assay .

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SARS-CoV-2 SUD (SARS2-SUD) interacts with Paip1 in HEK-293 cells as demonstrated by the split-YFP assay.


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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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MERS-CoV SUD-MC (Mac3−DPUP) or XMC (Mac1−Mac3−DPUP) do not bind to Paip1. Scale bars represent 50 µM.


( 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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(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


( 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 .

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(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 .

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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 ) .

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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 .

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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

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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


STK38 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 .

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STK38 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 ) .

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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

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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 conditions


STK38 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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STK38 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).


( 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

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(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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(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.


( 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 .

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(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

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(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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(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.


( 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

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(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

O O O B-GENEPROD O O B-EXP_ASSAY O O O O O B-GENEPROD B-SUBCELLULAR O O O O O O O B-GENEPROD O B-CELL_LINE O O O O O O O O O O O O O O O O O O O O O O B-EXP_ASSAY O O B-GENEPROD O O O O O B-GENEPROD B-SUBCELLULAR B-EXP_ASSAY O B-SUBCELLULAR B-EXP_ASSAY O O O O O O O O O O O O O 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-GENEPROD O O O B-GENEPROD B-SUBCELLULAR O O O O O O O O O O O O O B-GENEPROD B-SUBCELLULAR

(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 O O O B-GENEPROD O B-CELL_LINE O O O O O O O O B-GENEPROD O O O O O O O O O O O O O O O O O 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-EXP_ASSAY O O B-GENEPROD O O O O O B-GENEPROD B-SUBCELLULAR B-EXP_ASSAY O B-SUBCELLULAR B-EXP_ASSAY O O O O O O O O O O O O O O B-GENEPROD O O O O B-GENEPROD B-SUBCELLULAR B-EXP_ASSAY O O O O O O O O O O O B-GENEPROD O O O O O O

(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


( 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

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(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

O O O B-GENEPROD O O B-EXP_ASSAY O O O O O B-GENEPROD B-SUBCELLULAR O O O O B-GENEPROD B-CELL_LINE O O O O O O O O O O O O O O O O O B-EXP_ASSAY O O B-GENEPROD O O O O B-GENEPROD B-SUBCELLULAR B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O 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-GENEPROD O O O B-GENEPROD B-SUBCELLULAR O O O O O O O O O O O O O B-GENEPROD B-SUBCELLULAR

(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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(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


B 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 .

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B 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 .

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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 .

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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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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.


A 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 .

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A 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 .

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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 .

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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 .

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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 .

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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.


A 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 .

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A 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 .

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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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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.


A 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 .

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A 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 ) .

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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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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.


A 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 .

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A 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 .

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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 .

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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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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.


A 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 .

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A 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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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.


A 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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A 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.


A 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 .

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A 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 .

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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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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.


A 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 .

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A 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.


A . 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 ) .

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A. 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 .

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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 .

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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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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.


A . 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 .

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A. 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 .

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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 .

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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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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.


A . 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 .

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A. 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 .

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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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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.


A . 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 .

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A. 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 .

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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 .

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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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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.


Control 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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Control 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.


A . 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 .

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A. 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 .

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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 .

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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 .

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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 .

O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O B-GENEPROD O B-GENEPROD I-GENEPROD I-GENEPROD O B-SUBCELLULAR O O O O O O B-GENEPROD O O O B-GENEPROD O O O O O B-SUBCELLULAR O O O B-GENEPROD O B-GENEPROD O O O O B-GENEPROD O B-GENEPROD I-GENEPROD I-GENEPROD O O O B-EXP_ASSAY O O O O O O B-GENEPROD O O B-SUBCELLULAR O O O O O O O O O O B-GENEPROD O O B-SUBCELLULAR O O O O O O O

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.


A . 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 .

O O B-CELL_LINE I-CELL_LINE I-CELL_LINE O O O O O O O O B-GENEPROD I-GENEPROD O O O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O B-GENEPROD O O O O B-SUBCELLULAR O O O O O O O O O O O O O

A. 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 .

O O B-CELL_LINE I-CELL_LINE I-CELL_LINE O O O O O O B-SMALL_MOLECULE O O O O O O B-GENEPROD O O O O B-SUBCELLULAR O O O O O O O O O

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 .

O O B-GENEPROD O O O B-SUBCELLULAR O O B-SUBCELLULAR O O B-CELL_LINE I-CELL_LINE I-CELL_LINE O O O O B-SMALL_MOLECULE O O O O O B-SUBCELLULAR O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O B-GENEPROD O O O O B-SUBCELLULAR O O B-GENEPROD O O O O O O O O O O O O O O O

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.


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 . 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 .

B-CELL_LINE I-CELL_LINE I-CELL_LINE O O O O O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O B-GENEPROD O B-GENEPROD O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O B-GENEPROD O O O O B-GENEPROD O O O O O O O B-GENEPROD O B-GENEPROD O O O O O O B-GENEPROD O O O O O O O O O O O O O O O

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.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 .

B-CELL_LINE I-CELL_LINE I-CELL_LINE O O O O O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O B-GENEPROD O B-GENEPROD O B-GENEPROD I-GENEPROD I-GENEPROD O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O B-GENEPROD O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O B-GENEPROD O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O O O O O O

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.


Huh - 7 cells were first starved with serum free medium and then incubated with oleic acids for 12 hours . Condition 1 ( control ) , the cells were not treated with BFA at any time point ( left column ) . Condition 2 , the cells were incubated with both 5 μg / ml of BFA and oleic acid incubation for 6 h and then in oleic acid alone for 10 h ( middle column ) . Condition 3 , the cells were treated with 5 μg / ml of BFA starting at 12 hours after oleic acid incubation ( right column ) . Bar = 10μm .

B-CELL_LINE I-CELL_LINE I-CELL_LINE O O O O O O O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O O O B-SMALL_MOLECULE O B-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O O B-SMALL_MOLECULE O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O O O O

Huh-7 cells were first starved with serum free medium and then incubated with oleic acids for 12 hours. Condition 1 (control), the cells were not treated with BFA at any time point (left column). Condition 2, the cells were incubated with both 5 μg/ml of BFA and oleic acid incubation for 6 h and then in oleic acid alone for 10 h (middle column). Condition 3, the cells were treated with 5 μg/ml of BFA starting at 12 hours after oleic acid incubation (right column). Bar = 10μm.


CRTH2 subcellular localization in GFP - fused CRTH2 expressing plasmid - transfected cells . Red arrows indicate CRTH2 localized in the ER and white arrows indicate CRTH2 localized in the plasma membrane . Green , GFP - CRTH2 ; blue , DAPI ; red , Calnexin ; Scale bar , 20 μm .

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CRTH2 subcellular localization in GFP-fused CRTH2 expressing plasmid-transfected cells. Red arrows indicate CRTH2 localized in the ER and white arrows indicate CRTH2 localized in the plasma membrane. Green, GFP-CRTH2; blue, DAPI; red, Calnexin; Scale bar, 20 μm.


Western blot analysis of CRTH2 expression in cell membrane and ER in NIH 3T3 and HEK293T cells . Myc - tagged CRTH2 expressing plasmid was transfected into NIH 3T3 and HEK293T cells . Whole cell lysate , plasma , and ER fractions were obtained 48 h after transfection . Calnexin and ATP1B were used as markers for ER and cell membrane , respectively .

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Western blot analysis of CRTH2 expression in cell membrane and ER in NIH 3T3 and HEK293T cells. Myc-tagged CRTH2 expressing plasmid was transfected into NIH 3T3 and HEK293T cells. Whole cell lysate, plasma, and ER fractions were obtained 48 h after transfection. Calnexin and ATP1B were used as markers for ER and cell membrane, respectively.


Representative images of CRTH2 subcellular localization in NIH 3T3 cells in response to TGF - β1 ( 10 ng / mL ) treatment . Red arrows indicate CRTH2 localized in the ER and white arrows indicate CRTH2 localized in the plasma membrane . Green , GFP - CRTH2 ; blue , DAPI ; red , Calnexin ; Scale bar , 20 μm .

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Representative images of CRTH2 subcellular localization in NIH 3T3 cells in response to TGF-β1 (10 ng/mL) treatment. Red arrows indicate CRTH2 localized in the ER and white arrows indicate CRTH2 localized in the plasma membrane. Green, GFP-CRTH2; blue, DAPI; red, Calnexin; Scale bar, 20 μm.


Co - immunoprecipitation analysis of association of CRTH2 with LARP6 . HEK293T cells were co - transfected with Myc - CRTH2 and Flag - LARP6 expressing plasmids . Whole cell lysates from HEK293T cells were immunoprecipitated and then immunoblotted with the indicated antibodies .

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Co-immunoprecipitation analysis of association of CRTH2 with LARP6. HEK293T cells were co-transfected with Myc-CRTH2 and Flag-LARP6 expressing plasmids. Whole cell lysates from HEK293T cells were immunoprecipitated and then immunoblotted with the indicated antibodies.


Co - IP analysis of association of CRTH2 with GPR78 . HEK293T cells were co - transfected with Myc - GPR78 expressing plasmids .

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Co-IP analysis of association of CRTH2 with GPR78. HEK293T cells were co-transfected with Myc-GPR78 expressing plasmids.


Effect of LARP6 knockdown on Col1a1 , Col1a2 and Col3a1 mRNA stability in WT and CRTH2 - / - fibroblasts . * P < 0 . 05 vs WT , ( two - way ANOVA ) ; n = 8 for all groups .

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Effect of LARP6 knockdown on Col1a1, Col1a2 and Col3a1 mRNA stability in WT and CRTH2-/- fibroblasts. *P < 0.05 vs WT, (two-way ANOVA); n = 8 for all groups.


Effect of LARP6 knockdown on Collagen I and III protein expression in WT and CRTH2 - / - fibroblasts .

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Effect of LARP6 knockdown on Collagen I and III protein expression in WT and CRTH2-/- fibroblasts.


, Representative images of Masson ′ s trichrome staining of lung sections from CRTH2flox / floxCol1a2 - CreERT ( F - CRTH2 KO ) and CRTH2flox / flox ( Control ) mice 14 days after bleomycin challenge . Blue indicates the collagen deposition regions . Scale bar , 100 μm . Quantification of collagen content in mouse lung sections after bleomycin challenge . * P < 0 . 05 vs control , # P < 0 . 05 vs saline group ( two - way ANOVA ) ; saline groups , n = 4 each ; bleomycin groups , n = 8 each .

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, Representative images of Masson′s trichrome staining of lung sections from CRTH2flox/floxCol1a2-CreERT (F-CRTH2 KO) and CRTH2flox/flox (Control) mice 14 days after bleomycin challenge. Blue indicates the collagen deposition regions. Scale bar, 100 μm. Quantification of collagen content in mouse lung sections after bleomycin challenge. *P < 0.05 vs control, #P < 0.05 vs saline group (two-way ANOVA); saline groups, n = 4 each; bleomycin groups, n = 8 each.


Representative images of Masson ′ s trichrome staining of liver sections from control and F - CRTH2 KO mice 4 weeks after CCl4 challenge ; Scale bar , 100 μm .

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Representative images of Masson′s trichrome staining of liver sections from control and F-CRTH2 KO mice 4 weeks after CCl4 challenge; Scale bar, 100 μm.


Representative images of Masson ′ s trichrome staining of heart sections from control and F - CRTH2 KO mice 21 days after ISO challenge ; Scale bar , 50 μm . Quantification of collagen content in mouse heart sections after ISO challenge . * P < 0 . 05 vs control , # P < 0 . 05 vs saline group ( two - way ANOVA ) ; PBS groups , n = 3 each , ISO groups , n = 7 - 8 .

O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O B-TISSUE O O O O O O B-GENEPROD O B-ORGANISM O O O B-SMALL_MOLECULE O O O O O O O O O O B-SMALL_MOLECULE O O B-ORGANISM B-TISSUE O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O B-SMALL_MOLECULE O O O O O O O B-SMALL_MOLECULE O O O O O O O O

Representative images of Masson′s trichrome staining of heart sections from control and F-CRTH2 KO mice 21 days after ISO challenge; Scale bar, 50 μm. Quantification of collagen content in mouse heart sections after ISO challenge. *P < 0.05 vs control, #P < 0.05 vs saline group (two-way ANOVA); PBS groups, n = 3 each, ISO groups, n = 7-8.


Representative images of Masson ' s trichrome staining of lung sections from F - LARP6 KO and F - LARP6 / F - CRTH2 double KO ( F - DKO ) mice 14 days after bleomycin challenge ; Scale bar , 100 μm . , Quantification of collagen content in mouse lung sections after bleomycin challenge . # P < 0 . 05 vs saline group ( two - way ANOVA ) ; saline groups , n = 4 each , bleomycin groups , n = 7 - 8 . Hydroxyproline content in lungs from F - LARP6 KO and F - DKO mice treated with bleomycin . # P < 0 . 05 vs saline group ( two - way ANOVA ) ; saline groups , n = 4 each , bleomycin groups , n = 8 each .

O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O B-TISSUE O O O O B-GENEPROD O O O O B-GENEPROD O O O B-GENEPROD O O O O O O O B-ORGANISM O O O B-SMALL_MOLECULE O O O O O O O O O O O B-SMALL_MOLECULE O O B-ORGANISM B-TISSUE O O B-SMALL_MOLECULE O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O B-SMALL_MOLECULE O O O O O O O B-SMALL_MOLECULE O O O O O O O O B-SMALL_MOLECULE O O B-TISSUE O O O B-GENEPROD O O O O O B-ORGANISM O O B-SMALL_MOLECULE O O O O O O O O B-SMALL_MOLECULE O O O O O O O O B-SMALL_MOLECULE O O O O O O O B-SMALL_MOLECULE O O O O O O O

Representative images of Masson's trichrome staining of lung sections from F-LARP6 KO and F-LARP6/F-CRTH2 double KO (F-DKO) mice 14 days after bleomycin challenge; Scale bar, 100 μm. , Quantification of collagen content in mouse lung sections after bleomycin challenge. #P < 0.05 vs saline group (two-way ANOVA); saline groups, n = 4 each, bleomycin groups, n = 7-8. Hydroxyproline content in lungs from F-LARP6 KO and F-DKO mice treated with bleomycin. #P < 0.05 vs saline group (two-way ANOVA); saline groups, n = 4 each, bleomycin groups, n = 8 each.


Representative images of Masson ' s trichrome staining of liver sections from F - LARP6 KO and F - DKO mice 4 weeks after CCl4 challenge ; Scale bar , 100 μm . Quantification of collagen content in mouse liver sections after CCl4 challenge . # P < 0 . 05 vs mineral oil group ( two - way ANOVA ) ; mineral oil groups , n = 4 each , CCl4 groups , n = 8 each . Hydroxyproline content in livers from F - LARP6 KO and F - DKO mice treated with CCl4 . # P < 0 . 05 vs mineral oil group ( two - way ANOVA ) ; saline groups , n = 4 each , CCl4 groups , n = 8 each .

O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O B-TISSUE O O O O B-GENEPROD O O O O O B-ORGANISM O O O B-SMALL_MOLECULE O O O O O O O O O O B-SMALL_MOLECULE O O B-ORGANISM B-TISSUE O O B-SMALL_MOLECULE O O O O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O B-SMALL_MOLECULE O O O O O O O B-SMALL_MOLECULE O O B-TISSUE O O O B-GENEPROD O O O O O B-ORGANISM O O B-SMALL_MOLECULE O O O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O O B-SMALL_MOLECULE O O O O O O O B-SMALL_MOLECULE O O O O O O O

Representative images of Masson's trichrome staining of liver sections from F-LARP6 KO and F-DKO mice 4 weeks after CCl4 challenge; Scale bar, 100 μm. Quantification of collagen content in mouse liver sections after CCl4 challenge. #P < 0.05 vs mineral oil group (two-way ANOVA); mineral oil groups, n = 4 each, CCl4 groups, n = 8 each. Hydroxyproline content in livers from F-LARP6 KO and F-DKO mice treated with CCl4. #P < 0.05 vs mineral oil group (two-way ANOVA); saline groups, n = 4 each, CCl4 groups, n = 8 each.