Figure 9Huh - 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 .

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Figure 9Huh-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.


Figure 1CRTH2 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 . Representative images of CRTH2 subcellular localization in NIH 3T3 cells in response to TGF - β1 ( 10 ng / mL ) treatment . Red arrows indicate CRTH2 localized in the ER and white arrows indicate CRTH2 localized in the plasma membrane . Green , GFP - CRTH2 ; blue , DAPI ; red , Calnexin ; Scale bar , 20 μm .

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Figure 1CRTH2 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.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.


Figure 2Co - immunoprecipitation analysis of association of CRTH2 with LARP6 . HEK293T cells were co - transfected with Myc - CRTH2 and Flag - LARP6 expressing plasmids . Whole cell lysates from HEK293T cells were immunoprecipitated and then immunoblotted with the indicated antibodies . Co - IP analysis of association of CRTH2 with GPR78 . HEK293T cells were co - transfected with Myc - GPR78 expressing plasmids .

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Figure 2Co-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.


Figure 3Effect of LARP6 knockdown on Col1a1 , Col1a2 and Col3a1 mRNA stability in WT and CRTH2 - / - fibroblasts . * P < 0 . 05 vs WT , ( two - way ANOVA ) ; n = 8 for all groups . Effect of LARP6 knockdown on Collagen I and III protein expression in WT and CRTH2 - / - fibroblasts .

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Figure 3Effect 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.


Figure 4 , 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 . 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 .

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Figure 4, 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.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.


Figure 5Representative 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 . 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 .

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Figure 5Representative 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.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.


Figure 6Microscale thermophoresis ( MST ) analysis of the binding affinity of Bumetanide with MBP - LARP6Quantification 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 .

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Figure 6Microscale thermophoresis (MST) analysis of the binding affinity of Bumetanide with MBP-LARP6Quantification 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.


Figure 7Bumetanide 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 .

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Figure 7Bumetanide 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.


Figure 1B - 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 ) , 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 ) .

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Figure 1B-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),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).


Figure 2A - 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 . 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 ) .

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Figure 2A-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.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).


Figure 3A - 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 ) . 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 .

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Figure 3A-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).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.


Figure 4A - 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 ) 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 ) 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

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Figure 4A-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)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)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 NBs


Figure 5A - 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 . 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 . 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 B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 5A-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.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.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.


Figure 6A ) 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 ' ) . 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 ) . 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 B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 6A) 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').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).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).


Figure 7B ) 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 > ) . 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 ) 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 B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 7B) 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>).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)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).


Figure 1 ( 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 B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 1(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).


Figure 2 ( 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 ) . ( 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 ) .

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

Figure 2(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).(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).


Figure 3 ( 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 ) . ( 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 ) . ( 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 B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 3(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).(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).(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).


Figure 4Transcript 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 B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 4Transcript 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).


Figure 5Stomatal 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 ) . ( 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 B-PANEL_START O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 5Stomatal 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).(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).


Figure 1A / 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 B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 1A/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.


Figure 2Effects 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 . 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 B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 2Effects 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.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.


Figure 3A - D . Correlative light and electron microscopy ( CLEM ) on A . untransfected ( WT ) HeLa cells harbouring endogenous CD63 labeled with GFP as compared to those ectopically expressing B . HA - RILP , C . HA - SKIP or D . both HA - RILP and HA - SKIP . Left panels : wide field fluorescence images of fixed cells showing endogenous GFP - CD63 ( green ) and SiR - lysosome - stained endosomes and / or lysosomes ( red ) . Cell and nuclear boundaries are demarcated with solid and dashed lines respectively , zoom insets highlight regions selected for EM imaging . Middle panels : overview electron micrographs of perinuclear ( PN ) and peripheral ( PP ) cell regions selected for further analysis . Various endolysosomal subtypes are designated by arrowheads : MVBs ( white ) , lysosomes ( black ) , endolysosomes ( yellow ) and abnormal / enlarged endolysosomes ( red ) . Right panels : graphical representations of endosomal distribution under the indicated conditions based on ultrastructural characterization .

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Figure 3A-D. Correlative light and electron microscopy (CLEM) on A. untransfected (WT) HeLa cells harbouring endogenous CD63 labeled with GFP as compared to those ectopically expressing B. HA-RILP, C. HA-SKIP or D. both HA-RILP and HA-SKIP. Left panels: wide field fluorescence images of fixed cells showing endogenous GFP-CD63 (green) and SiR-lysosome-stained endosomes and/or lysosomes (red). Cell and nuclear boundaries are demarcated with solid and dashed lines respectively, zoom insets highlight regions selected for EM imaging. Middle panels: overview electron micrographs of perinuclear (PN) and peripheral (PP) cell regions selected for further analysis. Various endolysosomal subtypes are designated by arrowheads: MVBs (white), lysosomes (black), endolysosomes (yellow) and abnormal/enlarged endolysosomes (red). Right panels: graphical representations of endosomal distribution under the indicated conditions based on ultrastructural characterization.


Figure 4A . Electron micrograph of sections immunolabelled against RFP - SKIP ( 15nm gold ) and GFP - Rab7 ( 10nm gold ) . Arrowheads and zoom inset ( 1 . 75x ) highlight presence of RFP - SKIP and GFP - Rab7 on the same endosomal membrane , scale bar : 200nm . B . Co - immunoprecipitations ( Co - IP ) of HA - RILP and RFP - SKIP with GFP - Rab7 ( R7 ) versus its mutants Q67L ( QL ) and T22N ( TN ) from HEK293T cells using GFP - trap beads . Representative immunoblots against GFP , HA and RFP are shown , EV : empty vector , IP : immunoprecipitation , TL : total lysate ( see also Fig EV1E ) . C . Quantification of interaction between SKIP and Rab7 mutants expressed as fraction Co - IP relative to wild type Rab7 , n = 3 independent experiments . D . Pull - down ( PD ) of RFP - SKIP or RFP - RILP from HEK293T cell lysates using recombinant GST - Rab7 versus GST - Arl8b and free GST . Representative immunoblots against RFP and GST are shown ( see also EV1F ) . E . In vitro glutathione precipitation assays . E . SKIP truncation analysis by PD against GST - Rab7 . Top panels : representative immunoblots against Myc and GST ( see also Fig EV1G ) . Bottom panels : schematic representation of SKIP domain organization . Regions of SKIP capable of interacting with Arl8b versus Rab7 are demarcated with solid black lines . An alignment of human ( h ) and murine ( m ) SKIP sequences to known effectors of Rab7 surrounding the conserved KML / I effector motif at residues 610 - 612 of SKIP is provided . H . Graphical summary of SKIP as a dual effector of Arl8b and Rab7 . F / G . Co - IP of RFP - SKIP versus its KMI motif mutants AAI and AAA with constitutively active GFP - Rab7 Q67L using RFP - trap beads ( see also Fig EV1H ) . F . Representative immunoblots against GFP and RFP . G . Quantification of interaction between SKIP mutants with Rab7 expressed as fraction Co - IP relative to wild type SKIP , n = 3 independent experiments . I - K . Time - lapse of SKIP - mediated transport of late endosomes . I . Schematic representation of tamoxifen - induced activation of SKIP onto endosomal membranes . J / K . Live HeLa cells co - expressing GFP - ER - SKIP ( green ) and mCherry - Rab7 ( magenta ) together with HA - RILP ( unstained ) expressed at low levels ( cells transfected at 1 : 5 RILP : SKIP ratio ) were imaged in the J . absence or K . presence of tamoxifen , allowing on - demand association of SKIP with endosomal membranes . Confocal frames from time - lapses taken at the indicated time - points following treatment are shown . Cell and nuclear boundaries are demarcated with solid and dashed lines respectively , zoom insets ( 3x ) highlight select peripheral ( PP ) and perinuclear ( PN ) cell regions , scale bars :

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

Figure 4A. Electron micrograph of sections immunolabelled against RFP-SKIP (15nm gold) and GFP-Rab7 (10nm gold). Arrowheads and zoom inset (1.75x) highlight presence of RFP-SKIP and GFP-Rab7 on the same endosomal membrane, scale bar: 200nm. B. Co-immunoprecipitations (Co-IP) of HA-RILP and RFP-SKIP with GFP-Rab7 (R7) versus its mutants Q67L (QL) and T22N (TN) from HEK293T cells using GFP-trap beads. Representative immunoblots against GFP, HA and RFP are shown, EV: empty vector, IP: immunoprecipitation, TL: total lysate (see also Fig EV1E). C. Quantification of interaction between SKIP and Rab7 mutants expressed as fraction Co-IP relative to wild type Rab7, n=3 independent experiments.  D. Pull-down (PD) of RFP-SKIP or RFP-RILP from HEK293T cell lysates using recombinant GST-Rab7 versus GST-Arl8b and free GST. Representative immunoblots against RFP and GST are shown (see also EV1F).  E. In vitro glutathione precipitation assays. E. SKIP truncation analysis by PD against GST-Rab7. Top panels: representative immunoblots against Myc and GST (see also Fig EV1G). Bottom panels: schematic representation of SKIP domain organization. Regions of SKIP capable of interacting with Arl8b versus Rab7 are demarcated with solid black lines. An alignment of human (h) and murine (m) SKIP sequences to known effectors of Rab7 surrounding the conserved KML/I effector motif at residues 610-612 of SKIP is provided. H. Graphical summary of SKIP as a dual effector of Arl8b and Rab7.  F/G. Co-IP of RFP-SKIP versus its KMI motif mutants AAI and AAA with constitutively active GFP-Rab7 Q67L using RFP-trap beads (see also Fig EV1H). F. Representative immunoblots against GFP and RFP. G. Quantification of interaction between SKIP mutants with Rab7 expressed as fraction Co-IP relative to wild type SKIP, n=3 independent experiments. I-K. Time-lapse of SKIP-mediated transport of late endosomes. I. Schematic representation of tamoxifen-induced activation of SKIP onto endosomal membranes. J/K. Live HeLa cells co-expressing GFP-ER-SKIP (green) and mCherry-Rab7 (magenta) together with HA-RILP (unstained) expressed at low levels (cells transfected at 1:5 RILP:SKIP ratio) were imaged in the J. absence or K. presence of tamoxifen, allowing on-demand association of SKIP with endosomal membranes. Confocal frames from time-lapses taken at the indicated time-points following treatment are shown. Cell and nuclear boundaries are demarcated with solid and dashed lines respectively, zoom insets (3x) highlight select peripheral (PP) and perinuclear (PN) cell regions, scale bars: 10µm


Figure 5A - C . Consequences of effector depletion on the endogenous Arl8b / Rab7 hybrid compartment . A . Representative confocal images of fixed HeLa cells harbouring GFP - tagged endogenous Arl8b ( G - eArl8b , green ) , transfected with the indicated siRNAs and immunolabeled against endogenous Rab7 ( eRab7 , magenta ) . B . Colocalization ( Mander ' s overlap ) between endogenous Arl8b and Rab7 in response to effector depletion , n siC = 10 , n siRILP = 9 , n siSKIP = 9 images ( 4 ≥ cells per image ) analysed from 2 independent experiments . C . Immunoblot analysis for depletion efficiency of SKIP and RILP , with actin as loading control . D / E . Effect of Rab7 GTPase activity status on its association with the peripheral SKIP compartment . D . Left panels : Representative confocal images of fixed HeLa cells expressing GFP - Rab7 or its mutants Q67L or T22N ( green ) together with HA - RILP ( red ) and Myc - SKIP ( blue ) , immunolabeled against the indicated epitope tags . Right panels : Schematic overview per condition . E . Colocalization ( Mander ' s overlap ) between the indicated protein pairs , n Rab7 = 5 , n QL = 5 , n TN = 7 images ( 2 ≥ cells per image ) analysed from 2 independent experiments . F . Graphical summary of Rab7 removal from the SKIP compartment .

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

Figure 5A-C. Consequences of effector depletion on the endogenous Arl8b/Rab7 hybrid compartment. A. Representative confocal images of fixed HeLa cells harbouring GFP-tagged endogenous Arl8b (G-eArl8b, green), transfected with the indicated siRNAs and immunolabeled against endogenous Rab7 (eRab7, magenta). B. Colocalization (Mander's overlap) between endogenous Arl8b and Rab7 in response to effector depletion, n siC=10, n siRILP=9, n siSKIP=9 images (4≥ cells per image) analysed from 2 independent experiments. C. Immunoblot analysis for depletion efficiency of SKIP and RILP, with actin as loading control.D/E. Effect of Rab7 GTPase activity status on its association with the peripheral SKIP compartment. D. Left panels: Representative confocal images of fixed HeLa cells expressing GFP-Rab7 or its mutants Q67L or T22N (green) together with HA-RILP (red) and Myc-SKIP (blue), immunolabeled against the indicated epitope tags. Right panels: Schematic overview per condition. E. Colocalization (Mander's overlap) between the indicated protein pairs, n Rab7=5, n QL=5, n TN=7 images (2≥ cells per image) analysed from 2 independent experiments. F. Graphical summary of Rab7 removal from the SKIP compartment.


Figure 6B . Representative confocal images of fixed HeLa cells depleted of TBC1D2 , 1D5 , or 1D15 using siRNA oligo pools and immunolabeled against CD63 ( white ) , scale bar : 10µm . C . Plot of CD63 pixel distribution as a function of TBC1D2 / 1D5 / 1D15 depletion 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 ≥ 8 cells per condition analysed from 2 independent experiments . D / E . Rescue of TBC1D15 depletion phenotype . D . Representative confocal images of fixed HeLa cells transfected with either control siRNA ( siC ) or oligo # 3 targeting TBC1D15 ( siTBC1D15 ) and ectopically expressing either GFP - EV , GFP - TBC1D5 or siRNA - resistant GFP - TBC1D15res ( green ) , immunolabeled for CD63 ( magenta ) , scale bar : 10µm . E . Quantification of rescue expressed as % cells ( average ) in the population exhibiting one of 3 phenotypes : clustered , dispersed or intermediate ; total numbers of cells analysed per condition appear above each bar , n = 3 independent experiments . Effects of TBC1D15 depletion on the morphology and dynamics of late organelles . F . Representative electron micrographs of fixed HeLa cells transfected with either control siRNA ( siC ) or a pool of oligos targeting TBC1D15 ( siTBC1D15 ) are shown , scale bars as indicated . G . Analysis of late compartment dynamics as a function of TBC1D15 depletion . Tracks followed by SiR - lysosome - positive vesicles during a time - lapse lasting 255 sec ( 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 ( 3x ) highlight select peripheral ( PP ) and perinuclear ( PN ) regions , scale bar : 10µm EV10 ) . H . Quantification of high motility fraction ( % tracks with displacement rates above 0 . 9µm / sec ) , n siC = 27 , n si1D15 = 37 images ( 2 ≥ cells per image ) analysed from 2 independent experiments . Effectiveness of TBC1D15 ( si1D15 ) depletion is confirmed by immunoblot against endogenous TBC1D15 ( e1D15 ) following its immunoprecipitation ( IP ) , TL : total lysate . I . FRAP of GFP - Rab7 in live HeLa cells transfected with either control siRNA ( siC , black line ) or a pool of oligos targeting TBC1D15 ( si1D15 , red line ) . Plotted is average GFP - Rab7 signal recovery during 200 s following bleaching , expressed as % of pre - bleach signal , n = 3 bleach regions per sample .

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

Figure 6B. Representative confocal images of fixed HeLa cells depleted of TBC1D2, 1D5, or 1D15 using siRNA oligo pools and immunolabeled against CD63 (white), scale bar: 10µm. C. Plot of CD63 pixel distribution as a function of TBC1D2/1D5/1D15 depletion 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≥8 cells per condition analysed from 2 independent experiments.D/E. Rescue of TBC1D15 depletion phenotype. D. Representative confocal images of fixed HeLa cells transfected with either control siRNA (siC) or oligo #3 targeting TBC1D15 (siTBC1D15) and ectopically expressing either GFP-EV, GFP-TBC1D5 or siRNA-resistant GFP-TBC1D15res (green), immunolabeled for CD63 (magenta), scale bar: 10µm. E. Quantification of rescue expressed as % cells (average) in the population exhibiting one of 3 phenotypes: clustered, dispersed or intermediate; total numbers of cells analysed per condition appear above each bar, n=3 independent experiments.Effects of TBC1D15 depletion on the morphology and dynamics of late organelles. F. Representative electron micrographs of fixed HeLa cells transfected with either control siRNA (siC) or a pool of oligos targeting TBC1D15 (siTBC1D15) are shown, scale bars as indicated.G. Analysis of late compartment dynamics as a function of TBC1D15 depletion. Tracks followed by SiR-lysosome-positive vesicles during a time-lapse lasting 255 sec (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 (3x) highlight select peripheral (PP) and perinuclear (PN) regions, scale bar: 10µm EV10). H. Quantification of high motility fraction (% tracks with displacement rates above 0.9µm/sec), n siC=27, n si1D15=37 images (2≥ cells per image) analysed from 2 independent experiments. Effectiveness of TBC1D15 (si1D15) depletion is confirmed by immunoblot against endogenous TBC1D15 (e1D15) following its immunoprecipitation (IP), TL: total lysate.I. FRAP of GFP-Rab7 in live HeLa cells transfected with either control siRNA (siC, black line) or a pool of oligos targeting TBC1D15 (si1D15, red line). Plotted is average GFP-Rab7 signal recovery during 200 s following bleaching, expressed as % of pre-bleach signal, n=3 bleach regions per sample.


Figure 7Effects of depleting known GAPs for Rab7 on late compartment segregation mediated by RILP and SKIP . A . Representative confocal images of fixed HeLa cells transfected with either control siRNA ( siC ) , two different siRNA oligos targeting TBC1D15 ( # 1 and # 3 ) or oligo pool targeting TBC1D17 and ectopically expressing GFP - Arl8b ( green ) in combination with HA - RILP ( cyan ) and Myc - SKIP ( 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 of SKIP with endogenous Rab7 ( Mander ' s overlap ) in response to depletion of TBC - containing proteins , n ≥ 4 images ( 3 ≥ cells per image ) analysed per condition from 2 or more independent experimentsC . Plot of Rab7 - positive pixel distribution 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 ≥ 8 cells analysed per condition from 2 independent experiments . D . Validation of depletion efficiencies expressed as fraction of the indicated mRNA remaining relative to siC assayed by qPCR , n = 3 independent experiments . E . Effect of TBC1D15 depletion on Rab7 - dependent MHC - II receptor trafficking to the SKIP - positive compartment , n siC = 21 , n si1D15 = 21 cells analysed from 3 independent

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Figure 7Effects of depleting known GAPs for Rab7 on late compartment segregation mediated by RILP and SKIP. A. Representative confocal images of fixed HeLa cells transfected with either control siRNA (siC), two different siRNA oligos targeting TBC1D15 (#1 and #3) or oligo pool targeting TBC1D17 and ectopically expressing GFP-Arl8b (green) in combination with HA-RILP (cyan) and Myc-SKIP (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 of SKIP with endogenous Rab7 (Mander's overlap) in response to depletion of TBC-containing proteins, n≥4 images (3≥ cells per image) analysed per condition from 2 or more independent experimentsC. Plot of Rab7-positive pixel distribution 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≥8 cells analysed per condition from 2 independent experiments.D. Validation of depletion efficiencies expressed as fraction of the indicated mRNA remaining relative to siC assayed by qPCR, n=3 independent experiments.E. Effect of TBC1D15 depletion on Rab7-dependent MHC-II receptor trafficking to the SKIP-positive compartment, n siC=21, n si1D15=21 cells analysed from 3 independent experiments


Figure 8Representative confocal images of either control ( siC ) Hela cells harbouring endogenous TBC1D15 tagged with GFP ( Ge1D15 , green ) or those depleted of FIS1 using a pool of siRNA oligos , fixed and immunolabeled for the mitochondrial marker TOM20 ( blue ) in combination with either endogenous A . Rab7 Zoom insets ( 2 . 5x ) highlight select cell regions , arrowheads point to vesicles positive for TBC1D15 and negative for TOM20 ( yellow ) . Representative confocal images of either control ( siC ) Hela cells harbouring endogenous TBC1D15 tagged with GFP ( Ge1D15 , green ) or those depleted of FIS1 using a pool of siRNA oligos , fixed and immunolabeled for the mitochondrial marker TOM20 ( blue ) in combination with either endogenous B . CD63 ( red ) . Zoom insets ( 2 . 5x ) highlight select cell regions , arrowheads point to vesicles positive for TBC1D15 and negative for TOM20 ( yellow ) . Effect of FIS1 on late compartment distribution . C . Representative confocal images of fixed HeLa cells depleted of FIS1 using a siRNA oligo pool and immunolabeled against CD63 ( white ) . D . Plot of CD63 pixel distribution as a function of FIS1 depletion 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 siC = 14 , n siFis1 = 17 cells analysed from 2 independent experiments . E . Colocalization of SKIP with endogenous Rab7 ( Mander ' s overlap ) in response to FIS1 depletion , n siC = 13 , n siFis1 = 9 images ( 3 ≥ cells per image ) analysed from 2 independent experimentsF . Validation of FIS1 depletion efficiency assayed by qPCR and expressed as fraction FIS1 mRNA remaining relative to siC , n = 3 independent experiments . G - J . Recruitment of endogenous TBC1D15 to the SKIP compartment . G . Left panels : representative confocal images of fixed HeLa cells harbouring endogenous TBC1D15 tagged with GFP ( G - e1D15 , green ) and ectopically expressing either empty vector , HA - SKIP or PLEKHM1 - HA ( red ) , immunolabeled against HA . Zoom insets ( 2 . 7x ) highlight select peripheral ( PP ) and perinuclear ( PN ) cell regions . Right panels : pixel plots of endogenous GFP - TBC1D15 ( green line ) and HA signals ( red line ) corresponding to the dashed white lines in G . H - J . Colocalization ( Mander ' s overlap ) between the indicated pairs of proteins , n ≥ 5 images , 3 ≥ cells per image , analysed per condition from 2 independent

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

Figure 8Representative confocal images of either control (siC) Hela cells harbouring endogenous TBC1D15 tagged with GFP (Ge1D15, green) or those depleted of FIS1 using a pool of siRNA oligos, fixed and immunolabeled for the mitochondrial marker TOM20 (blue) in combination with either endogenous A. Rab7 Zoom insets (2.5x) highlight select cell regions, arrowheads point to vesicles positive for TBC1D15 and negative for TOM20 (yellow).Representative confocal images of either control (siC) Hela cells harbouring endogenous TBC1D15 tagged with GFP (Ge1D15, green) or those depleted of FIS1 using a pool of siRNA oligos, fixed and immunolabeled for the mitochondrial marker TOM20 (blue) in combination with either endogenous B. CD63 (red). Zoom insets (2.5x) highlight select cell regions, arrowheads point to vesicles positive for TBC1D15 and negative for TOM20 (yellow).Effect of FIS1 on late compartment distribution. C. Representative confocal images of fixed HeLa cells depleted of FIS1 using a siRNA oligo pool and immunolabeled against CD63 (white).D. Plot of CD63 pixel distribution as a function of FIS1 depletion 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 siC=14, n siFis1=17 cells analysed from 2 independent experiments.E. Colocalization of SKIP with endogenous Rab7 (Mander's overlap) in response to FIS1 depletion, n siC=13, n siFis1=9 images (3≥ cells per image) analysed from 2 independent experimentsF. Validation of FIS1 depletion efficiency assayed by qPCR and expressed as fraction FIS1 mRNA remaining relative to siC, n=3 independent experiments.G-J. Recruitment of endogenous TBC1D15 to the SKIP compartment. G. Left panels: representative confocal images of fixed HeLa cells harbouring endogenous TBC1D15 tagged with GFP (G-e1D15, green) and ectopically expressing either empty vector, HA-SKIP or PLEKHM1-HA (red), immunolabeled against HA. Zoom insets (2.7x) highlight select peripheral (PP) and perinuclear (PN) cell regions. Right panels: pixel plots of endogenous GFP-TBC1D15 (green line) and HA signals (red line) corresponding to the dashed white lines in G. H-J. Colocalization (Mander's overlap) between the indicated pairs of proteins, n≥5 images, 3≥ cells per image, analysed per condition from 2 independent experiments


Figure 9Effects of SKIP KMI motif on HOPS complex recruitment . A . Analysis of SKIP / HOPS interactions by co - IP of RFP / mCherry - HOPS subunits with Myc - SKIP versus the KMI motif mutant AAA from HEK293T cells using RFP - trap beads . Representative immunoblots against RFP and Myc are shown , IP : immunoprecipitation , TL : total lysate . B . Quantification of SKIP / HOPS interactions , n = 3 independent experiments . Top graph : fold change in binding of HOPS subunits to AAA mutant relative to wild type ( WT ) SKIP ( 1 . 0 ) , significance : one - way ANOVA test ( relative to VPS41 ) . Bottom graph : Co - IP per HOPS subunit relative to VPS39 , significance : one - way ANOVA test ( relative to VPS39 ) , * p < 0 . 05 , * * * p < 0 . 001 , ns : not significant . C . Colocalization ( Mander ' s overlap ) between SKIP and RFP - VPS39 ( images or mCherry - VPS41 ( images , n ≥ 10 images ( 2 ≥ cells per image ) analysed per condition from 2 independent experiments . D . Representative confocal images of fixed HeLa cells ectopically expressing Myc - SKIP , KMI motif mutant ( AAA ) or RUN domain truncation mutant ( ∆ RUN ) ( red ) together with RFP - VPS39 ( green ) , immunolabeled for endogenous VPS11 ( eVPS11 , blue ) . Effect of HOPS over - expression on recruitment of TBC1D15 to the SKIP compartment . E . Representative confocal images of fixed HeLa cells harbouring endogenous GFP - TBC1D15 ( Ge1D15 , green ) and ectopically expressing HA - SKIP ( red ) together with mCh - VPS18 or RFP - VPS39 ( white ) , immunolabeled for endogenous VPS18 or VPS11 ( blue ) , as indicatedF - I . Colocalization of SKIP and HOPS complex with TBC1D15 as a function of HOPS subunit over - expression . F / H . Mander ' s overlap between F . SKIP or H . eVPS18 and endogenous TBC1D15 , n ≥ 5 images ( 2 ≥ cells per image ) analysed per condition from 2 independent experiments , significance : one - way ANOVA test ( relative to EV ) , * * p < 0 . 01 , * * * p < 0 . 001 , ns : not significant . G / I . Mander ' s overlap between G . SKIP or I . eVPS11 and endogenous TBC1D15 as a function VPS18 overabundance , with or without FIS1 depletion ( siFIS1 ) , n ≥ 3 images ( 2 ≥ cells per image ) analysed per condition from 2 or more independent experiments .

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Figure 9Effects of SKIP KMI motif on HOPS complex recruitment. A. Analysis of SKIP/HOPS interactions by co-IP of RFP/mCherry-HOPS subunits with Myc-SKIP versus the KMI motif mutant AAA from HEK293T cells using RFP-trap beads. Representative immunoblots against RFP and Myc are shown, IP: immunoprecipitation, TL: total lysate. B. Quantification of SKIP/HOPS interactions, n=3 independent experiments. Top graph: fold change in binding of HOPS subunits to AAA mutant relative to wild type (WT) SKIP (1.0), significance: one-way ANOVA test (relative to VPS41). Bottom graph: Co-IP per HOPS subunit relative to VPS39, significance: one-way ANOVA test (relative to VPS39), * p<0.05, *** p<0.001, ns: not significant.C. Colocalization (Mander's overlap) between SKIP and RFP-VPS39 (images or mCherry-VPS41 (images , n≥10 images (2≥ cells per image) analysed per condition from 2 independent experiments.D. Representative confocal images of fixed HeLa cells ectopically expressing Myc-SKIP, KMI motif mutant (AAA) or RUN domain truncation mutant (∆RUN) (red) together with RFP-VPS39 (green), immunolabeled for endogenous VPS11 (eVPS11, blue).Effect of HOPS over-expression on recruitment of TBC1D15 to the SKIP compartment. E. Representative confocal images of fixed HeLa cells harbouring endogenous GFP-TBC1D15 (Ge1D15, green) and ectopically expressing HA-SKIP (red) together with mCh-VPS18 or RFP-VPS39 (white), immunolabeled for endogenous VPS18 or VPS11 (blue), as indicatedF-I. Colocalization of SKIP and HOPS complex with TBC1D15 as a function of HOPS subunit over-expression. F/H. Mander's overlap between F. SKIP or H. eVPS18 and endogenous TBC1D15, n≥5 images (2≥ cells per image) analysed per condition from 2 independent experiments, significance: one-way ANOVA test (relative to EV), ** p<0.01, *** p<0.001, ns: not significant. G/I. Mander's overlap between G. SKIP or I. eVPS11 and endogenous TBC1D15 as a function VPS18 overabundance, with or without FIS1 depletion (siFIS1), n≥3 images (2≥ cells per image) analysed per condition from 2 or more independent experiments.


Figure 10A / B . In situ SKIP / TBC1D15 complex formation assayed using proximity - based biotin ligation ( BioID ) . A . Neutravidin precipitates ( PD ) from biotin - treated HEK293T cells ectopically expressing GFP - TBC1D15 ( GFP - 1D15 ) together with HA - BioID - SKIP or HA - BioID - EV . Representative immunoblots against GFP , HA and VPS18 , TL : total lysate . B . Quantification of biotinylation of GFP - TBC1D15 ( top graph ) and endogenous VPS18 ( eVPS18 , bottom graph ) by BioID - SKIP , expressed relative to BioID - EV ( 1 . 0 ) , n = 4 independent experiments . C . Analysis of TBC1D15 / HOPS interactions by co - IP of RFP / mCherry - HOPS subunits with GFP - TBC1D15 from HEK293T cells using GFP - trap beads . Left panel : schematic of HOPS complex composition , with subunits tested marked in yellow . Right panel : representative immunoblots against GFP and RFP ; IP : immunoprecipitation , TL : total lysate . Effects of VPS18 depletion on the recruitment of TBC1D15 to the SKIP complex . D . Representative confocal images of fixed HeLa cells harbouring endogenous GFP - TBC1D15 ( G - e1D15 , green ) , transfected with either control siRNA ( siC ) or oligo pool targeting VPS18 ( siVPS18 ) and ectopically expressing HA - SKIP ( red ) , immunolabeled against endogenous VPS18 ( eVPS18 , blue ) and HA , scale bar : 10µm . 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 . E . Colocalization ( Mander ' s overlap ) between SKIP and endogenous TBC1D15 as a function of VPS18 depletion , n siC = 9 , n siVPS18 = 11 images ( 2 ≥ cells per image ) analysed from 2 independent experiments . F . Neutravidin precipitates ( PD ) from biotin - treated HeLa cells ectopically expressing GFP - TBC1D15 ( GFP - 1D15 ) together with BioID - SKIP or BioID - EV . Representative immunoblots against GFP , HA and VPS18 are shown , TL : total lysate . G . Quantification of GFP - TBC1D15 biotinylation by BioID - SKIP over BioID - EV in response to VPS18 depletion expressed as % of control ( siC ) , n = 4 independent experiments performed in HeLa or HEK293T cells . H . Quantification of VPS18 protein abundance from experiments in G , n = 4 . . Effect of TBC1D15 activity on Rab7 displacement from the SKIP compartment . Colocalization ( Mander ' s overlap ) between Rab7 and TBC1D15 ( WT or R417A mutant ) in the presence of either SKIP or PLEKHM1 combined with the indicated VPS proteins , n ≥ 5 images ( 2 ≥ cells per image ) analysed from 2 independent

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

Figure 10A/B. In situ SKIP/TBC1D15 complex formation assayed using proximity-based biotin ligation (BioID). A. Neutravidin precipitates (PD) from biotin-treated HEK293T cells ectopically expressing GFP-TBC1D15 (GFP-1D15) together with HA-BioID-SKIP or HA-BioID-EV. Representative immunoblots against GFP, HA and VPS18 , TL: total lysate. B. Quantification of biotinylation of GFP-TBC1D15 (top graph) and endogenous VPS18 (eVPS18, bottom graph) by BioID-SKIP, expressed relative to BioID-EV (1.0), n=4 independent experiments.C. Analysis of TBC1D15/HOPS interactions by co-IP of RFP/mCherry-HOPS subunits with GFP-TBC1D15 from HEK293T cells using GFP-trap beads. Left panel: schematic of HOPS complex composition, with subunits tested marked in yellow. Right panel: representative immunoblots against GFP and RFP; IP: immunoprecipitation, TL: total lysate.Effects of VPS18 depletion on the recruitment of TBC1D15 to the SKIP complex. D. Representative confocal images of fixed HeLa cells harbouring endogenous GFP-TBC1D15 (G-e1D15, green), transfected with either control siRNA (siC) or oligo pool targeting VPS18 (siVPS18) and ectopically expressing HA-SKIP (red), immunolabeled against endogenous VPS18 (eVPS18, blue) and HA, scale bar: 10µm. 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. E. Colocalization (Mander's overlap) between SKIP and endogenous TBC1D15 as a function of VPS18 depletion, n siC=9, n siVPS18=11 images (2≥ cells per image) analysed from 2 independent experiments.F. Neutravidin precipitates (PD) from biotin-treated HeLa cells ectopically expressing GFP-TBC1D15 (GFP-1D15) together with BioID-SKIP or BioID-EV. Representative immunoblots against GFP, HA and VPS18 are shown, TL: total lysate. G. Quantification of GFP-TBC1D15 biotinylation by BioID-SKIP over BioID-EV in response to VPS18 depletion expressed as % of control (siC), n=4 independent experiments performed in HeLa or HEK293T cells. H. Quantification of VPS18 protein abundance from experiments in G, n=4.. Effect of TBC1D15 activity on Rab7 displacement from the SKIP compartment. Colocalization (Mander's overlap) between Rab7 and TBC1D15 (WT or R417A mutant) in the presence of either SKIP or PLEKHM1 combined with the indicated VPS proteins, n≥ 5 images (2≥ cells per image) analysed from 2 independent experiments