( 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O

(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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) 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

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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)) 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 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 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 .

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

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 .

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

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 .

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 .

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

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 .

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 .

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

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 .

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

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

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 .

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

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 .

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

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 .

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

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 .

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

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 ) .

O O O 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 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 .

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

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 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

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 .

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 .

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

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 .

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

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 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

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 .

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

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 .

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

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 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O B-MEASURED_VAR O B-MEASURED_VAR O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O

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 .

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

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 .

O O O O O O O O O O O B-MEASURED_VAR O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O

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 .

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

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 ) .

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

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 .

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 .

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

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 .

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

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 .

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

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 .

O 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-MEASURED_VAR O O O O O O O O O O

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 .

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

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 .

O O O O O O O O O O O O O O O O O O O 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-MEASURED_VAR O

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 .

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

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 .

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

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 .

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 .

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 .

O O O O O O O O O O O O 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. 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 .

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

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 .

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

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 .

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

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 .

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

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 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O 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,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 .

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 .

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

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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 .

O O O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 .

O O O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 .

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

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 .

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 .

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

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 .

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

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 .

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

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 .

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

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 .

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 .

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 .

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

, 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 .

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

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


Representative images of Masson ' s trichrome staining of heart sections from F - LARP6 KO and F - DKO 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 saline group ( two - way ANOVA ) ; saline groups , n = 3 each , ISO groups , n = 7 each . , Hydroxyproline content in hearts from F - LARP6 KO and F - DKO mice treated with ISO . # P < 0 . 05 vs saline group ( two - way ANOVA ) ; saline groups , n = 4 each , ISO groups , n = 8 each .

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

Representative images of Masson's trichrome staining of heart sections from F-LARP6 KO and F-DKO 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 saline group (two-way ANOVA); saline groups, n = 3 each, ISO groups, n = 7 each. , Hydroxyproline content in hearts from F-LARP6 KO and F-DKO mice treated with ISO. #P < 0.05 vs saline group (two-way ANOVA); saline groups, n = 4 each, ISO groups, n = 8 each.


Microscale thermophoresis ( MST ) analysis of the binding affinity of Bumetanide with MBP -

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

Microscale thermophoresis (MST) analysis of the binding affinity of Bumetanide with MBP-LARP6


Quantification of collagen content in lung sections from Bumetanide - treated control ( CRTH2flox / flox ) and F - CRTH2 KO mice . * P < 0 . 05 vs control , # P < 0 . 05 vs Vehicle ( two - way ANOVA ) ; n = 7 for all groups . Effect of Bumetanide treatment on pulmonary hydroxyproline levels in control and F - CRTH2 KO mice . * P < 0 . 05 vs control ; # P < 0 . 05 vs Vehicle ( two - way ANOVA ) ; n = 7 for all groups .

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

Quantification of collagen content in lung sections from Bumetanide-treated control (CRTH2flox/flox) and F-CRTH2 KO mice. *P < 0.05 vs control, #P < 0.05 vs Vehicle (two-way ANOVA); n = 7 for all groups. Effect of Bumetanide treatment on pulmonary hydroxyproline levels in control and F-CRTH2 KO mice. *P < 0.05 vs control; #P < 0.05 vs Vehicle (two-way ANOVA); n = 7 for all groups.


Bumetanide interferes with the interaction of CRTH2 with LARP6 in MRC - 5 human lung fibroblasts MRC - 5 . MRC - 5 cells were co - transfected with myc - LARP6 and flag - CRTH2 expressing plasmids for 24 h , and then treated with Bumetanide ( 0 . 1 , 2 μM ) for an additional 24 h . Whole cell lysates from MRC - 5 cells were immunoprecipitated and immunoblotted with the indicated antibodies .

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

Bumetanide interferes with the interaction of CRTH2 with LARP6 in MRC-5 human lung fibroblasts MRC-5. MRC-5 cells were co-transfected with myc-LARP6 and flag-CRTH2 expressing plasmids for 24 h, and then treated with Bumetanide (0.1, 2 μM) for an additional 24 h. Whole cell lysates from MRC-5 cells were immunoprecipitated and immunoblotted with the indicated antibodies.


Effect of Bumetanide on Collagen ( Col ) I and III protein expression in MRC - 5 cells .

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

Effect of Bumetanide on Collagen (Col) I and III protein expression in MRC-5 cells.


B - D ' ' ) Representative images showing that Hh accumulates on the surface of LDs in glial cells of the CB ( yellow arrows ) Glial cells are marked by repo - GAL4 > GFP and CB is circled in ( B ) .

O O O O O O O O O O O O O O O O O O O O O O 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-D'') Representative images showing that Hh accumulates on the surface of LDs in glial cells of the CB (yellow arrows) Glial cells are marked by repo-GAL4>GFP and CB is circled in (B).


E - G ' ' ) In the posterior compartment of the developing wing disc ( WD ) pouch region where Hh is expressed , LDs and Hh are not tightly associated ( white arrows ) ,

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

E-G'') In the posterior compartment of the developing wing disc (WD) pouch region where Hh is expressed, LDs and Hh are not tightly associated (white arrows),


H - J ) Hh - LD association is quantified by plotting the pixel intensities of both Hh ( cyan ) and LDs ( red ) along a line across LDs . Y axis represents gray intensity values , and X axis represents relative LD position .

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

H-J) Hh-LD association is quantified by plotting the pixel intensities of both Hh (cyan) and LDs (red) along a line across LDs. Y axis represents gray intensity values, and X axis represents relative LD position.


K - L ' ' ) Hh - LD associations are observed in the cortex glia ( yellow arrows , NP2222 - GAL4 > mGFP ) .

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

K-L'') Hh-LD associations are observed in the cortex glia (yellow arrows, NP2222-GAL4>mGFP).


A - D ) Representative images showing that upon knockdown of Hh in cortex glial cells ( NP2222 - GAL4 > mGFP with UAS - dcr2 ) , cortex glial membrane and overall Repo + glial cell number are significantly reduced , quantified in ( C ) ( n = 7 , 8 brain lobes ) and ( D ) ( n = 7 , 8 brain lobes ) , respectively . E - G ) Hh knockdown in glia ( repo - GAL4 > GFP ) results in niche disruption and clustering of NBs ( circled with yellow dashed line ) , as well as an increase in the percentage of NBs in M phase ( pH3 + ) , quantified in ( E ) ( n = 12 , 10 brain lobes ) .

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

A-D) Representative images showing that upon knockdown of Hh in cortex glial cells (NP2222-GAL4>mGFP with UAS-dcr2), cortex glial membrane and overall Repo+ glial cell number are significantly reduced, quantified in (C) (n=7, 8 brain lobes) and (D) (n=7, 8 brain lobes), respectively. E-G) Hh knockdown in glia (repo-GAL4>GFP) results in niche disruption and clustering of NBs (circled with yellow dashed line), as well as an increase in the percentage of NBs in M phase (pH3+), quantified in (E) (n=12, 10 brain lobes).


H - K ) Representative images showing that Hh overexpression using pan - glial ( repo - GAL4 ) and cortex - glial ( NP2222 - GAL4 ) drivers both result in a decrease in NB EdU index , quantified in ( J ) ( n = 16 , 20 brain lobes ) and ( K ) ( n = 11 , 14 brain lobes ) , respectively .

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

H-K) Representative images showing that Hh overexpression using pan-glial (repo-GAL4) and cortex-glial (NP2222-GAL4) drivers both result in a decrease in NB EdU index, quantified in (J) (n=16, 20 brain lobes) and (K) (n=11, 14 brain lobes), respectively.


L - M ' ' ) Representative images showing ci - lacZ is expressed in NBs ( yellow arrows ) . ( M - M ' ' ) are zoomed in images of ( L ) .

L-M'') Representative images showing ci-lacZ is expressed in NBs (yellow arrows). (M-M'') are zoomed in images of (L).


N - P ) Overexpression of ciACT in NBs ( dnab - GAL4 ) reduces EdU index , quantified in ( P ) ( n = 12 , 10 brain lobes ) .

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

N-P) Overexpression of ciACT in NBs (dnab-GAL4) reduces EdU index, quantified in (P) (n=12, 10 brain lobes).


A - B ' ' ) Representative images showing that Lsd2YFP is localised to the surface of LDs ( yellow arrows ) .

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

A-B'') Representative images showing that Lsd2YFP is localised to the surface of LDs (yellow arrows).


C - D ' ' ) Lsd2YFP co - localises with Hh antibody staining in the cortex glia ( yellow arrows ) but not surface glial cells ( white arrow ) .

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

C-D'') Lsd2YFP co-localises with Hh antibody staining in the cortex glia (yellow arrows) but not surface glial cells (white arrow).


E - G ) Lsd - 2 knockdown in cortex glial cells ( NP2222 - GAL4 ) where hh is overexpressed effectively reduces LD number in CB ( outlined in yellow dashed lines ) . H - K ) Representative images showing that NB EdU index is rescued upon Lsd - 2 knockdown in cortex glial cells ( NP2222 - GAL4 ) where hh is overexpressed , quantified in ( K ) ( n = 11 , 14 ; 16 , 14 ; 6 , 14 brain lobes ) . The NP2222 - GAL4 > w1118 vs hhOE columns depict the same data as Fig 2K . EdU + NBs are circled with yellow , dashed lines .

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

E-G) Lsd-2 knockdown in cortex glial cells (NP2222-GAL4) where hh is overexpressed effectively reduces LD number in CB (outlined in yellow dashed lines). H-K) Representative images showing that NB EdU index is rescued upon Lsd-2 knockdown in cortex glial cells (NP2222-GAL4) where hh is overexpressed, quantified in (K) (n=11, 14; 16, 14; 6, 14 brain lobes). The NP2222-GAL4>w1118 vs hhOE columns depict the same data as Fig 2K. EdU+ NBs are circled with yellow, dashed lines.


A - D ' ) Representative images showing that pan - glial overexpression of htlACT , but not InRwt or EgfrACT causes an expansion of cortex glia that enwraps NBs . Glial cells are marked with repo - GAL4 > GFP , and NBs are marked with Mira . ( A ' , B ' , C ' and D ' ) are zoomed in images of ( A , B , C and D ) , respectively .

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

A-D') Representative images showing that pan-glial overexpression of htlACT, but not InRwt or EgfrACT causes an expansion of cortex glia that enwraps NBs. Glial cells are marked with repo-GAL4 >GFP, and NBs are marked with Mira. (A', B', C' and D') are zoomed in images of (A, B, C and D), respectively.


Representative images showing that cortex glial overexpression of htlACT but not InRwt or EgfrACT causes an increase in cortex glial membrane size ( H ) ( n = 10 , 10 brain lobes ) NP2222 - GAL4 > mGFP is used to mark cortex glial membrane in ( E - G )

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

Representative images showing that cortex glial overexpression of htlACT but not InRwt or EgfrACT causes an increase in cortex glial membrane size (H) (n=10, 10 brain lobes) NP2222-GAL4>mGFP is used to mark cortex glial membrane in (E-G)


Representative images showing that cortex glial overexpression of EgfrACT to mark cortex glial membrane wrapper - GAL4 > is used in ( I , J ) .

Representative images showing that cortex glial overexpression of EgfrACT to mark cortex glial membrane wrapper-GAL4> is used in (I, J).


cortex glial overexpression of htlACT but not InRwt or EgfrACT causes an increase in glial cell ( Repo + ) numbers quantified in ( K ) ( n = 10 , 10 , 10 ; 7 , 12 brain lobes )

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

cortex glial overexpression of htlACT but not InRwt or EgfrACT causes an increase in glial cell (Repo+) numbers quantified in (K) (n=10, 10, 10; 7, 12 brain lobes)


L ) Glial ( repo - GAL4 > ) overexpression of InRwt but not htlACT or EgfrACT significantly reduces the number of CB NBs ( n = 10 , 10 , 8 , 11 brain lobes ) .

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

L) Glial (repo-GAL4>) overexpression of InRwt but not htlACT or EgfrACT significantly reduces the number of CB NBs (n= 10, 10, 8, 11 brain lobes).


M ) Glial ( repo - GAL4 > ) overexpression of htlACT but not InRwt or EgfrACT significantly reduces the pH3 index of CB

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

M) Glial (repo-GAL4>) overexpression of htlACT but not InRwt or EgfrACT significantly reduces the pH3 index of CB NBs


A - E ) Representative images showing that both pan - glial ( repo - GAL4 > ) and cortex glial ( NP2222 - GAL4 > ) htlACT overexpression significantly reduce NB EdU index , quantified in ( E ) ( n = 15 , 14 ; 10 , 16 brain lobes ) . ( A , B , C , D ) are single sections with Mira and EdU staining , and ( A ' , B ' C ' , D ' ) are Z - projection of the EdU staining .

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

A-E) Representative images showing that both pan-glial (repo-GAL4>) and cortex glial (NP2222-GAL4>) htlACT overexpression significantly reduce NB EdU index, quantified in (E) (n=15 ,14; 10, 16 brain lobes). (A, B, C, D) are single sections with Mira and EdU staining, and (A', B' C', D') are Z-projection of the EdU staining.


pan - glial ( repo - GAL4 ) htlACT overexpression lengthens NB cell cycle , quantified in ( F ) The cell cycle length is measured as the length between consecutive divisions .

pan-glial (repo-GAL4) htlACT overexpression lengthens NB cell cycle, quantified in (F) The cell cycle length is measured as the length between consecutive divisions.


Representative still images from ex vivo CNS live imaging at 72ALH showing that pan - glial ( repo - GAL4 ) htlACT overexpression lengthens NB cell cycle , ( n = 25 , 6 NBs imaged from 3 brains per genotype ) . NBs ( Dpn : : GFP , red ; Histone RFP , grey ) are circled with blue dashed lines .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-MEASURED_VAR O O O O O O O O O O O

Representative still images from ex vivo CNS live imaging at 72ALH showing that pan-glial (repo-GAL4) htlACT overexpression lengthens NB cell cycle, (n= 25, 6 NBs imaged from 3 brains per genotype). NBs (Dpn::GFP, red; Histone RFP, grey) are circled with blue dashed lines.


J - L ) Representative images showing that the percentage of NBs in G1 - S transition and G2 / M phase are both significantly increased with significantly less cells remaining in G1 phase , quantified in ( L ) ( n = 9 , 10 brain lobes ) . NBs in G1 phase ( Mira + , GFP + ) are circled by green dashed lines ; NBs in G1 - S transition ( Mira + , GFP - RFP - ) are circled by grey dashed lines ; NBs in S phase ( Mira + , RFP + ) are circled by red dashed lines ; and NBs in G2 / M phase ( Mira + , GFP + RFP + ) are circled by yellow dashed lines .

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

J-L) Representative images showing that the percentage of NBs in G1-S transition and G2/M phase are both significantly increased with significantly less cells remaining in G1 phase, quantified in (L) (n= 9, 10 brain lobes). NBs in G1 phase (Mira+, GFP+) are circled by green dashed lines; NBs in G1-S transition (Mira+, GFP-RFP-) are circled by grey dashed lines; NBs in S phase (Mira+, RFP+) are circled by red dashed lines; and NBs in G2/M phase (Mira+, GFP+RFP+) are circled by yellow dashed lines.


M - O ) Representative images showing that the number of EdU + neurons generated per NB is significantly reduced upon pan - glial overexpression of FGF ( repo - LexA > LexAop - htlACT ; yellow arrows ) , quantified in ( O ) n = 94 , 127 NB lineages imaged from 5 and 7 brain lobes , respectively ) . NB lineages are marked with dnab - gal4 > GFP .

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

M-O) Representative images showing that the number of EdU+ neurons generated per NB is significantly reduced upon pan-glial overexpression of FGF (repo-LexA > LexAop-htlACT; yellow arrows), quantified in (O) n= 94, 127 NB lineages imaged from 5 and 7 brain lobes, respectively). NB lineages are marked with dnab-gal4 > GFP.


A ) Pan - glial ( repo - GAL4 ) htlACT overexpression causes upregulation of hh , fasn1 and lsd2 transcripts ( n = 3 biological replicates pooled from 20 brains for each genotype ; for each biological replicate , we ran 3 technical replicates for each PCR reaction ) . The lipogenesis genes ( acc , lipin ) , and lipolysis gene bmm transcripts are not significantly altered . We utilised rpl32 as a reference gene in these experiments , as it is not altered by htlACT overexpression . The data are represented by log2 fold change relative to the control ( repo - GAL4 > w1118 ) .

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

A) Pan-glial (repo-GAL4) htlACT overexpression causes upregulation of hh, fasn1 and lsd2 transcripts (n = 3 biological replicates pooled from 20 brains for each genotype; for each biological replicate, we ran 3 technical replicates for each PCR reaction). The lipogenesis genes (acc, lipin), and lipolysis gene bmm transcripts are not significantly altered. We utilised rpl32 as a reference gene in these experiments, as it is not altered by htlACT overexpression. The data are represented by log2 fold change relative to the control (repo-GAL4> w1118).


C - G ) Representative images showing that Hh staining normally localised to a ring like structure ( yellow arrows ) , becomes delocalises to the glial cytoplasm upon htlACT overexpression , quantified in ( E ) ( n = 4 , 6 brain lobes ) . Glial cells are marked with repo - GAL4 > GFP . ( E - F ' ) are zoomed in images of ( C - D ' ) .

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

C-G) Representative images showing that Hh staining normally localised to a ring like structure (yellow arrows), becomes delocalises to the glial cytoplasm upon htlACT overexpression, quantified in (E) (n=4, 6 brain lobes). Glial cells are marked with repo-GAL4>GFP. (E-F') are zoomed in images of (C-D').


H ) Cortex glial ( NP2222 - GAL4 > ) overexpression of two independent hh RNAis significantly rescue EdU incorporation defects caused by htlACT overexpression ( n = 10 , 16 ; 7 , 12 ; 14 , 11 brain lobes ) .

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

H) Cortex glial (NP2222-GAL4>) overexpression of two independent hh RNAis significantly rescue EdU incorporation defects caused by htlACT overexpression (n=10, 16; 7, 12; 14, 11 brain lobes).


I ) Knockdown of NB Hh signalling pathway ( dnab - GAL4 > UAS - ciRNAi ) rescues NB EdU incorporation defects induced by glial htlACT overexpression ( repo - LexA > LexAop - htlACT ) . Induction of ciRNAi in NBs alone increases NB EdU incorporation ( n = 8 , 13 ; 10 , 10 ; 30 , 26 ; 12 , 7 brain lobes ) .

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

I) Knockdown of NB Hh signalling pathway (dnab-GAL4> UAS-ciRNAi) rescues NB EdU incorporation defects induced by glial htlACT overexpression (repo-LexA> LexAop-htlACT). Induction of ciRNAi in NBs alone increases NB EdU incorporation (n=8, 13; 10, 10; 30, 26; 12, 7 brain lobes).


J ) The NB EdU incorporation defects due to cortex glial ( NP2222 - GAL4 > ) overexpression of htlACT , is significantly rescued by overexpression of RNAis against fasn1 and lsd2 , compared to corresponding control RNAis ( n = 10 , 16 ; 25 , 17 ; 14 , 8 brain lobes ) .

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

J) The NB EdU incorporation defects due to cortex glial (NP2222-GAL4>) overexpression of htlACT, is significantly rescued by overexpression of RNAis against fasn1 and lsd2, compared to corresponding control RNAis (n=10, 16; 25, 17; 14, 8 brain lobes).


B ) Inhibition of palmitoylation ( via two independent rasp RNAis ) rescues NB EdU incorporation defects induced by cortex glial ( NP2222 - GAL4 > ) htlACT overexpression , while knockdown of Rasp in cortex glial cells alone does not alter NB EdU index ( n = 10 , 15 ; 10 , 16 ; 18 , 21 ; 26 , 29 brain lobes ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Inhibition of palmitoylation (via two independent rasp RNAis) rescues NB EdU incorporation defects induced by cortex glial (NP2222-GAL4>) htlACT overexpression, while knockdown of Rasp in cortex glial cells alone does not alter NB EdU index (n=10, 15; 10, 16; 18, 21; 26, 29 brain lobes).


C - C ' ) Representative images showing that Hh . N . EGFP ( which cannot undergo cholesterol modification ) are found as puncta on the surface of NBs ( yellow arrows ) when overexpressed in neighbouring cortex glial cells ( NP2222 - GAL4 > ) .

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

C-C') Representative images showing that Hh.N.EGFP (which cannot undergo cholesterol modification) are found as puncta on the surface of NBs (yellow arrows) when overexpressed in neighbouring cortex glial cells (NP2222-GAL4>).


Representative images showing that knockdown of Fasn1 in glial cells ( repo - GAL4 > ) , where Hh . N . EGFP is overexpressed , significantly reduces the number of Hh . N . EGFP puncta on the surface of NBs ( yellow arrows ) n = 38 , 38 , 57 NBs from 4 , 4 , 8 brain lobes , respectively ) .

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

Representative images showing that knockdown of Fasn1 in glial cells (repo-GAL4>), where Hh.N.EGFP is overexpressed, significantly reduces the number of Hh.N.EGFP puncta on the surface of NBs (yellow arrows) n=38, 38, 57 NBs from 4, 4, 8 brain lobes, respectively).


knockdown of Fasn1 in glial cells ( repo - GAL4 > ) , where Hh . N . EGFP is overexpressed , significantly reduces the number of Hh . N . EGFP puncta on the surface of NBs phenocopying the effect of Rasp knockdown , quantified in ( G )

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

knockdown of Fasn1 in glial cells (repo-GAL4>), where Hh.N.EGFP is overexpressed, significantly reduces the number of Hh.N.EGFP puncta on the surface of NBs phenocopying the effect of Rasp knockdown, quantified in (G)


H ) Cortex glial ( NP2222 - GAL4 > ) overexpression of Hh . N . EGFP significantly reduces NB EdU incorporation ( n = 13 , 12 brain lobes ) .

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

H) Cortex glial (NP2222-GAL4>) overexpression of Hh.N.EGFP significantly reduces NB EdU incorporation (n= 13,12 brain lobes).


I ) Knockdown of Fasn1 in glial cells rescues NB EdU incorporation defects , caused by glial Hh . N . EGFP overexpression , quantified in ( I ) ( n = 15 , 13 brain lobes ) .

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

I) Knockdown of Fasn1 in glial cells rescues NB EdU incorporation defects, caused by glial Hh.N.EGFP overexpression, quantified in (I) (n=15, 13 brain lobes).


( A ) ANAC032expression in WT plants sprayed with Pst , 6 and 24 hpi , compared to control ( sprayed with 10 mM MgCl2 ( Mock ) ) . Error bars represent means ± SD ( n = 3 ; ' n ' represents independently performed experiments , each including the rosette leaves of at least three plants grown in individual pots ) . ANAC032expression in WT treated with ( B ) SA or MeJA or ( C ) COR for 3 and 6 h compared to non - treated controls . In B and C , means ± SD are given ( n = 3 ; ' n ' represents independently performed experiments , each including at least 20 seedlings ) . FCh , fold change . Asterisks indicate a significant difference from their respective controls ( p < 0 . 01 ; Student ´ s t - test ) .

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

(A) ANAC032expression in WT plants sprayed with Pst, 6 and 24 hpi, compared to control (sprayed with 10 mM MgCl2 (Mock)). Error bars represent means ± SD (n = 3; 'n' represents independently performed experiments, each including the rosette leaves of at least three plants grown in individual pots). ANAC032expression in WT treated with (B) SA or MeJA or (C) COR for 3 and 6 h compared to non-treated controls. In B and C, means ± SD are given (n = 3; 'n' represents independently performed experiments, each including at least 20 seedlings). FCh, fold change. Asterisks indicate a significant difference from their respective controls (p < 0.01; Student´s t-test).


( A ) Plants after pressure infiltration with Pst DC3000 , at 4 dpi . The experiment was repeated three times ( each time 9 - 12 plants ) with similar results .

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

(A) Plants after pressure infiltration with Pst DC3000, at 4 dpi. The experiment was repeated three times (each time 9 - 12 plants) with similar results.


( B ) Pst DC3000 growth in ANAC032 transgenics and WT plants 3 dpi after pressure infiltration . Two independent experiments were performed with three replications per experiment , each replicate consisting of three plants grown in individual pots ( six measurements in total ) . The graph shows data points of the two individual experiments ( I and II ) along with their mean ( Avg ) .

(B) Pst DC3000 growth in ANAC032 transgenics and WT plants 3 dpi after pressure infiltration. Two independent experiments were performed with three replications per experiment, each replicate consisting of three plants grown in individual pots (six measurements in total). The graph shows data points of the two individual experiments (I and II) along with their mean (Avg).


( C ) Disease status of plants sprayed with Pst DC3000 , 5 dpi . The experiment was repeated four times with similar results . ( D ) Disease severity index ( 1 , small chlorotic lesions ; 5 , large lesions ) scored 5 dpi after spraying with Pst DC3000 . Data are from three independent experiments with at least six plants per genotype in each . Means are shown ± SD . Asterisks indicate significant ( * p < 0 . 05 and * * p < 0 . 005 ) differences between transgenic and WT plants in chi - square analysis .

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

(C) Disease status of plants sprayed with Pst DC3000, 5 dpi. The experiment was repeated four times with similar results. (D) Disease severity index (1, small chlorotic lesions; 5, large lesions) scored 5 dpi after spraying with Pst DC3000. Data are from three independent experiments with at least six plants per genotype in each. Means are shown ± SD. Asterisks indicate significant (*p < 0.05 and **p < 0.005) differences between transgenic and WT plants in chi-square analysis.


( E ) Heat map showing the fold change ( log2 basis ) in the expression ratio of defense - / stress - related differentially expressed genes in anac032 - 1 and 35S : ANAC032 compared to WT after spraying with Pst DC3000 ( 6 hpi ) normalised to their respective controls . Blue , downregulated ; red , upregulated . Data represent means of three independent experiments , each including the rosettes leaves of at least three plants grown in individual pots . Asterisks indicate significant differences from WT plants ( Student ' s t test , p ≤ 0 . 05 ) .

(E) Heat map showing the fold change (log2 basis) in the expression ratio of defense-/stress-related differentially expressed genes in anac032-1 and 35S:ANAC032 compared to WT after spraying with Pst DC3000 (6 hpi) normalised to their respective controls. Blue, downregulated; red, upregulated. Data represent means of three independent experiments, each including the rosettes leaves of at least three plants grown in individual pots. Asterisks indicate significant differences from WT plants (Student's t test, p ≤ 0.05).


( A ) MYC2 , PDF1 . 2A and NIMIN1 expression in ANAC032 transgenics compared to WT after spraying with Pst DC3000 ( 24 hpi ) normalised to their respective controls . FCh , fold change . Means ± SD ( n = 3 ; ' n ' represents independently performed experiments , each including the rosette leaves of at least three plants grown in individual pots ) .

(A) MYC2, PDF1.2A and NIMIN1 expression in ANAC032 transgenics compared to WT after spraying with Pst DC3000 (24 hpi) normalised to their respective controls. FCh, fold change. Means ± SD (n = 3; 'n' represents independently performed experiments, each including the rosette leaves of at least three plants grown in individual pots).


( B ) EMSA showing binding of ANAC032 to MYC2 , PDF1 . 2A and NIMIN1 promoter regions ( in 5 ´ - DY682 - labelled double - stranded oligonucleotides ) harbouring ANAC032 binding sites . 1 , labelled probe only ; 2 , labelled probe plus GST protein 3 , labelled probe plus ANAC032 - GST protein ; 4 , labelled probe , ANAC032 - GST protein and 100 x competitor ( unlabelled oligonucleotide containing ANAC032 binding site ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) EMSA showing binding of ANAC032 to MYC2, PDF1.2A and NIMIN1 promoter regions (in 5´-DY682-labelled double-stranded oligonucleotides) harbouring ANAC032 binding sites. 1, labelled probe only; 2, labelled probe plus GST protein 3, labelled probe plus ANAC032-GST protein; 4, labelled probe, ANAC032-GST protein and 100 x competitor (unlabelled oligonucleotide containing ANAC032 binding site).


( C ) Confocal microscopy image showing nuclear localization of ANAC032 - GFP fusion protein expressed from the ANAC032 promoter in ANAC032prom : ANAC032 - GFP / anac032 - 1 seedlings treated with Pst at 6 hpi . Left , bright field ; right , chlorophyll auto - fluorescence ( red ) and GFP fluorescence ( green ) under bright field .

O O O O O O O O O O O O O O O O O O O O O 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-MEASURED_VAR O O O O O O O O O O O O O O O O

(C) Confocal microscopy image showing nuclear localization of ANAC032-GFP fusion protein expressed from the ANAC032 promoter in ANAC032prom:ANAC032-GFP/anac032-1 seedlings treated with Pst at 6 hpi. Left, bright field; right, chlorophyll auto-fluorescence (red) and GFP fluorescence (green) under bright field.


( D ) Expression of MYC2 , PDF1 . 2A , NIMIN1 and ANAC032 in two - week - old ANAC032prom : ANAC032 - GFP / anac032 - 1 seedlings compared to WT at 6 hpi with Pst , normalised to their respective controls . FCh , fold change . Means ± SD ( n = 3 ; ' n ' represents independently performed experiments , each including the rosette leaves of at least three plants grown in individual pots ) . Asterisks indicate a significant difference from WT , normalised to their respective controls ( p < 0 . 05 ; Student ´ s t - test ) .

(D) Expression of MYC2, PDF1.2A, NIMIN1 and ANAC032 in two-week-old ANAC032prom:ANAC032-GFP/anac032-1 seedlings compared to WT at 6 hpi with Pst, normalised to their respective controls. FCh, fold change. Means ± SD (n = 3; 'n' represents independently performed experiments, each including the rosette leaves of at least three plants grown in individual pots). Asterisks indicate a significant difference from WT, normalised to their respective controls (p < 0.05; Student´s t-test).


( E ) ChIP - qPCR shows enrichment of MYC2 , PDF1 . 2Aand NIMIN1 promoter regions containing ANAC032 binding site compared to a promoter region lacking the ANAC032 binding site ( AT5G09810 ; Neg Cont ) . Means ± SD ( n = 3 ; ' n ' represents independently performed experiments , each including the rosette leaves of at least three plants grown in individual pots ) . Asterisks indicate a significant difference from negative control ( p < 0 . 01 ; Student ´ s t - test ) .

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

(E) ChIP-qPCR shows enrichment of MYC2, PDF1.2Aand NIMIN1 promoter regions containing ANAC032 binding site compared to a promoter region lacking the ANAC032 binding site (AT5G09810; Neg Cont). Means ± SD (n = 3; 'n' represents independently performed experiments, each including the rosette leaves of at least three plants grown in individual pots). Asterisks indicate a significant difference from negative control (p < 0.01; Student´s t-test).


Transcript levels of ( A ) NIMIN1 and ( B ) PR1 in WT , anac032 - 1 and 35S : ANAC032 plants after 3 and 6 h of treatment with SA compared to their respective controls .

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

Transcript levels of (A) NIMIN1 and (B) PR1 in WT, anac032-1 and 35S:ANAC032 plants after 3 and 6 h of treatment with SA compared to their respective controls.


( C ) , Expression of PDF1 . 2A in WT and anac032 - 1 plants after 3 and 6 h of treatment with SA compared to their respective controls . Transcript levels were measured using qRT - PCR and numbers on the y - axis indicate fold change ( FCh ; log2 basis ) . Means are shown ± SD ( n = 3 ; ' n ' represents independently performed experiments ) . Asterisks indicate a significant difference from wild type ( p < 0 . 05 ; Student ' s t test ) .

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

(C), Expression of PDF1.2A in WT and anac032-1 plants after 3 and 6 h of treatment with SA compared to their respective controls. Transcript levels were measured using qRT-PCR and numbers on the y-axis indicate fold change (FCh; log2 basis). Means are shown ± SD (n = 3; 'n' represents independently performed experiments). Asterisks indicate a significant difference from wild type (p < 0.05; Student's t test).


Stomatal aperture 1 h ( A ) or 4 h ( B ) after spraying with Pst DC3000 or mock treatment .

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

Stomatal aperture 1 h (A) or 4 h (B) after spraying with Pst DC3000 or mock treatment.


( C ) Microscope images of stomata at 1 and 4 hpi with Pst DC3000 ( Mock at 4 h ) .

(C) Microscope images of stomata at 1 and 4 hpi with Pst DC3000 (Mock at 4 h).


( D ) , Stomatal aperture 1 h after treatment with COR , ABA , or COR plus ABA , compared to mock . In ( A ) , ( B ) , and ( D ) , data are means ± SD of 12 to16 measurements ; in each measurement the rosette leaves from at least 6 - 8 plants were used ) . Asterisks indicate a significant difference from mock treatment ( p < 0 . 0001 ; Student ´ s t - test ) .

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

(D), Stomatal aperture 1 h after treatment with COR, ABA, or COR plus ABA, compared to mock. In (A), (B), and (D), data are means ± SD of 12 to16 measurements; in each measurement the rosette leaves from at least 6 - 8 plants were used). Asterisks indicate a significant difference from mock treatment (p < 0.0001; Student´s t-test).


A / B . Hybrid Rab7 / Arl8b compartment . A . Representative confocal images of fixed HeLa cells expressing Arl8b - GFP ( green ) , immunolabeled against endogenous Rab7 ( eRab7 , magenta ) . Zoom insets ( 3x ) highlight select regions of colocalization ( white ) , white arrowheads point to vesicles positive for both GTPases . B . Colocalization ( Mander ' s overlap ) of endogenous Rab7 with Arl8b - GFP versus free GFP ( EV ) , n EV = 4 , n Arl8b = 9 images ( 3 ≥ cells per image ) analysed from 2 independent experiments . Significance : 2 - tailed student ' s t - test , * p < 0 . 05 .

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

A/B. Hybrid Rab7/Arl8b compartment. A. Representative confocal images of fixed HeLa cells expressing Arl8b-GFP (green), immunolabeled against endogenous Rab7 (eRab7, magenta). Zoom insets (3x) highlight select regions of colocalization (white), white arrowheads point to vesicles positive for both GTPases. B. Colocalization (Mander's overlap) of endogenous Rab7 with Arl8b-GFP versus free GFP (EV), n EV=4, n Arl8b=9 images (3≥ cells per image) analysed from 2 independent experiments. Significance: 2-tailed student's t-test, * p<0.05.


C - F . Analysis of Arl8b compartment organization and dynamics as a function of Rab7 activity status . C . Left and middle panels : representative confocal images of live HeLa cells expressing mCherry - Rab7 or its mutants Q67L and T22N ( white ) , together with Arl8b - GFP ( green ) , taken at the start of time - lapse ( t0 ) . Right panels : tracks followed by Arl8b - positive null during the time - lapse lasting 50 s recorded at 0 . 5 s per frame , with highest displacement rates for each track depicted on a rainbow colour scale ( blue : immobile ; red : maximum mobility per time interval ) . Zoom insets ( 2 . 8x ) highlight select peripheral ( PP ) and perinuclear ( PN ) cell regions D . Plot of Arl8b - positive pixel distribution expressed as fractional distance along a straight line from center of nucleus ( 0 ) to the plasma membrane ( 1 . 0 ) , numbers of ( pixels ) plotted given above each scatter , n = 7 cells analysed per condition from 2 independent experiments . E / F . Quantification of mean Arl8b vesicle displacement and maximum speed , respectively , n Rab7 = 21 , n QL = 25 , n TN = 24 images ( 3 ≥ cells per image ) analysed from 2 independent experiments .

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

C-F. Analysis of Arl8b compartment organization and dynamics as a function of Rab7 activity status. C. Left and middle panels: representative confocal images of live HeLa cells expressing mCherry-Rab7 or its mutants Q67L and T22N (white), together with Arl8b-GFP (green), taken at the start of time-lapse (t0). Right panels: tracks followed by Arl8b-positive null during the time-lapse lasting 50 s recorded at 0.5 s per frame, with highest displacement rates for each track depicted on a rainbow colour scale (blue: immobile; red: maximum mobility per time interval). Zoom insets (2.8x) highlight select peripheral (PP) and perinuclear (PN) cell regions D. Plot of Arl8b-positive pixel distribution expressed as fractional distance along a straight line from center of nucleus (0) to the plasma membrane (1.0), numbers of (pixels) plotted given above each scatter, n=7 cells analysed per condition from 2 independent experiments. E/F. Quantification of mean Arl8b vesicle displacement and maximum speed, respectively, n Rab7=21, n QL=25, n TN=24 images (3≥ cells per image) analysed from 2 independent experiments.


Effects of select transport route activation on the spatial organization of late compartments . A . Representative confocal images of fixed HeLa cells ectopically expressing Arl8b - GFP or Arl8b - Myc ( green ) , in combination with HA - RILP ( cyan ) and / or Myc - SKIP , PLEKHM1 - FLAG , or GFP - FYCO1 ( red ) , immunolabeled against endogenous Rab7 ( eRab7 , magenta ) and the indicated epitope tags . Cell and nuclear boundaries are demarcated with solid and dashed lines respectively , zoom insets ( 3 . 5x ) highlight select peripheral ( PP ) and perinuclear ( PN ) cell regions , scale bar : 10µm . B . Colocalization ( Mander ' s overlap ) between the indicated pairs of proteins , n ≥ 3 images ( 3 ≥ cells per image ) analysed per condition from 2 independent experiments .

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

Effects of select transport route activation on the spatial organization of late compartments. A. Representative confocal images of fixed HeLa cells ectopically expressing Arl8b-GFP or Arl8b-Myc (green), in combination with HA-RILP (cyan) and/or Myc-SKIP, PLEKHM1-FLAG, or GFP-FYCO1 (red), immunolabeled against endogenous Rab7 (eRab7, magenta) and the indicated epitope tags. Cell and nuclear boundaries are demarcated with solid and dashed lines respectively, zoom insets (3.5x) highlight select peripheral (PP) and perinuclear (PN) cell regions, scale bar: 10µm. B. Colocalization (Mander's overlap) between the indicated pairs of proteins, n≥3 images (3≥ cells per image) analysed per condition from 2 independent experiments.


C . Plot of eRab7 - or Arl8b - GFP / Myc - Arl8b - positive pixel distribution in response to the indicated effector perturbations , expressed as fractional distance along a straight line from center of nucleus ( 0 ) to the plasma membrane ( 1 . 0 ) , number of ( pixels ) plotted given above each scatter , n ≥ 6 cells per condition analysed from 2 independent experiments .

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

C. Plot of eRab7- or Arl8b-GFP / Myc-Arl8b-positive pixel distribution in response to the indicated effector perturbations, expressed as fractional distance along a straight line from center of nucleus (0) to the plasma membrane (1.0), number of (pixels) plotted given above each scatter, n≥6 cells per condition analysed from 2 independent experiments.


D . Colocalization ( Mander ' s overlap ) between Arl8b and Rab7 as a function of the indicated effector perturbations , n ≥ 5 images ( 3 ≥ cells per image ) analysed per condition from 2 independent experiments .

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

D. Colocalization (Mander's overlap) between Arl8b and Rab7 as a function of the indicated effector perturbations, n≥5 images (3≥ cells per image) analysed per condition from 2 independent experiments.