qPCR analysis of UPR target genes in the absence of ER stress . Wild type and ERp18 knockout HEK293T cells were untreated prior to isolation of mRNA for qPCR analysis . mRNA levels for Grp78 , Grp94 , ATF6α , ATF4 , XBP1s ( spliced form of XBP1 ) and ERp72 were normalized to GAPDH and then ERp18 KO levels compared to wild type . Error bars represent ± standard deviation for three independent experiments . qPCR analysis of UPR target genes following ER stress . Wild type and ERp18 knockout HEK293T cells were treated with 1 µM TG for approximately 16 h prior to isolation of mRNA for qPCR analysis . mRNA levels for Grp78 , Grp94 , ATF6α , ATF4 , XBP1s ( spliced form of XBP1 ) and ERp72 were normalized to GAPDH and then ERp18 KO levels compared to wild type controls . Error bars represent ± standard deviation for three independent experiments .

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qPCR analysis of UPR target genes in the absence of ER stress. Wild type and ERp18 knockout HEK293T cells were untreated prior to isolation of mRNA for qPCR analysis. mRNA levels for Grp78, Grp94, ATF6α, ATF4, XBP1s (spliced form of XBP1) and ERp72 were normalized to GAPDH and then ERp18 KO levels compared to wild type. Error bars represent ± standard deviation for three independent experiments.   qPCR analysis of UPR target genes following ER stress. Wild type and ERp18 knockout HEK293T cells were treated with 1 µM TG for approximately 16 h prior to isolation of mRNA for qPCR analysis. mRNA levels for Grp78, Grp94, ATF6α, ATF4, XBP1s (spliced form of XBP1) and ERp72 were normalized to GAPDH and then ERp18 KO levels compared to wild type controls. Error bars represent ± standard deviation for three independent experiments.


qPCR analysis of UPR target genes in the absence of ER stress . Wild type and ERp18 knockout HEK293T cells were untreated prior to isolation of mRNA for qPCR analysis . mRNA levels for Grp78 , Grp94 , ATF6α , ATF4 , XBP1s ( spliced form of XBP1 ) and ERp72 were normalized to GAPDH and then ERp18 KO levels compared to wild type . Error bars represent ± standard deviation for three independent experiments .

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qPCR analysis of UPR target genes in the absence of ER stress. Wild type and ERp18 knockout HEK293T cells were untreated prior to isolation of mRNA for qPCR analysis. mRNA levels for Grp78, Grp94, ATF6α, ATF4, XBP1s (spliced form of XBP1) and ERp72 were normalized to GAPDH and then ERp18 KO levels compared to wild type. Error bars represent ± standard deviation for three independent experiments.


Levels of BiP protein in wild type or ERp18 KO cells before and after ER stress induction with thapsigargin ( TG ) . Panel 1 compares wild type and ERp18 KO cells as indicated . Panel 2 compares ERp18 KO cells to ERp18 KO cells that have been transfected with ERp18 ( RV ) . Normalised BiP levels from ( D ) were quantified . Error bars represent ± standard deviation for three independent experiments .

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Levels of BiP protein in wild type or ERp18 KO cells before and after ER stress induction with thapsigargin (TG). Panel 1 compares wild type and ERp18 KO cells as indicated. Panel 2 compares ERp18 KO cells to ERp18 KO cells that have been transfected with ERp18 (RV).   Normalised BiP levels from (D ) were quantified. Error bars represent ± standard deviation for three independent experiments.


Levels of BiP protein in wild type or ERp18 KO cells before and after ER stress induction with thapsigargin ( TG ) . Panel 1 compares wild type and ERp18 KO cells as indicated . Panel 2 compares ERp18 KO cells to ERp18 KO cells that have been transfected with ERp18 ( RV ) .

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Levels of BiP protein in wild type or ERp18 KO cells before and after ER stress induction with thapsigargin (TG). Panel 1 compares wild type and ERp18 KO cells as indicated. Panel 2 compares ERp18 KO cells to ERp18 KO cells that have been transfected with ERp18 (RV).


Levels of ERp72 protein in wild type or ERp18 KO cells before and after ER stress induction with thapsigargin ( TG ) . Panel 1 compares wild type and ERp18 KO cells as indicated . Panel 2 compares ERp18 KO cells to ERp18 KO cells that have been transfected with ERp18 ( RV ) . Normalised ERp72 levels from ( F ) were quantified . Error bars represent ± standard deviation for three independent experiments .

Levels of ERp72 protein in wild type or ERp18 KO cells before and after ER stress induction with thapsigargin (TG). Panel 1 compares wild type and ERp18 KO cells as indicated. Panel 2 compares ERp18 KO cells to ERp18 KO cells that have been transfected with ERp18 (RV).   Normalised ERp72 levels from (F) were quantified. Error bars represent ± standard deviation for three independent experiments.


Levels of ERp72 protein in wild type or ERp18 KO cells before and after ER stress induction with thapsigargin ( TG ) . Panel 1 compares wild type and ERp18 KO cells as indicated . Panel 2 compares ERp18 KO cells to ERp18 KO cells that have been transfected with ERp18 ( RV ) .

Levels of ERp72 protein in wild type or ERp18 KO cells before and after ER stress induction with thapsigargin (TG). Panel 1 compares wild type and ERp18 KO cells as indicated. Panel 2 compares ERp18 KO cells to ERp18 KO cells that have been transfected with ERp18 (RV).


A , B Wild type and ERp18 KO cells stably expressing ATF6α were treated with 5 µM TG for the indicated times . Cells were then subject to differential centrifugation to obtain membrane ( A ) and nuclear fractions ( B ) which were separated by SDS - PAGE under reducing conditions and analysed by western blotting for ATF6α . The positions of full - length ATF6α ( ATF6 - FL ) , cleaved membrane - associated ATF6α ( ATF6 - P ) and nuclear translocated S2P cleaved ATF6α ( ATF6 - N ) are as indicated . HDAC2 was used as a nuclear marker .

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A, B Wild type and ERp18 KO cells stably expressing ATF6α were treated with 5 µM TG for the indicated times. Cells were then subject to differential centrifugation to obtain membrane (A) and nuclear fractions (B) which were separated by SDS-PAGE under reducing conditions and analysed by western blotting for ATF6α. The positions of full-length ATF6α (ATF6-FL), cleaved membrane-associated ATF6α (ATF6-P) and nuclear translocated S2P cleaved ATF6α (ATF6-N) are as indicated. HDAC2 was used as a nuclear marker.


( C ) ATF6α was immunoisolated from wild type or ERp18 KO cells expressing ATF6α . Samples were separated by SDS - PAGE and ATF6α was detected by western blotting . HC indicates immunoglobulin heavy chains .

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(C) ATF6α was immunoisolated from wild type or ERp18 KO cells expressing ATF6α. Samples were separated by SDS-PAGE and ATF6α was detected by western blotting. HC indicates immunoglobulin heavy chains.


( D , E ) Blots of membrane and nuclear fractions respectively from ERp18 KO cells overexpressing ATF6α , either untreated ( - ) or treated ( + ) with 30 µM PF429242 ( S1P inhibitor ) prior to and following induction of ER stress with 10 mM DTT . The positions of ATF6 - P , ATF6 - M and ATF6 - N are as indicated . The unprocessed ER - localised ATF6α ( designated ER ) and the O - linked glycan modified Golgi - translocated ATF6α ( designated G ) are indicated . HDAC2 was included as a nuclear marker . The blots in ( D , E ) confirm that ATF6 - P is produced independently of S1P and also the absence of ATF6α processing to ATF6 - N in the presence of the S1P inhibitor .

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(D, E) Blots of membrane and nuclear fractions respectively from ERp18 KO cells overexpressing ATF6α, either untreated (-) or treated (+) with 30 µM PF429242 (S1P inhibitor) prior to and following induction of ER stress with 10 mM DTT. The positions of ATF6-P, ATF6-M and ATF6-N are as indicated. The unprocessed ER-localised ATF6α (designated ER) and the O-linked glycan modified Golgi-translocated ATF6α (designated G) are indicated. HDAC2 was included as a nuclear marker. The blots in (D, E) confirm that ATF6-P is produced independently of S1P and also the absence of ATF6α processing to ATF6-N in the presence of the S1P inhibitor.


HEK293T cells stably overexpressing wild type ATF6α or a D564G mutant were treated with 10 mM DTT as indicated to induce ER stress in the presence of the S1P inhibitor . Cell lysates were prepared , separated by SDS - PAGE and then probed with anti - ATF6α . Blot reveals the presence of O - linked glycan modified Golgi - translocated ATF6 ( designated G ) , which is absent for the D564G mutant , confirming a lack of ER to Golgi trafficking .

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HEK293T cells stably overexpressing wild type ATF6α or a D564G mutant were treated with 10 mM DTT as indicated to induce ER stress in the presence of the S1P inhibitor. Cell lysates were prepared, separated by SDS-PAGE and then probed with anti-ATF6α. Blot reveals the presence of O-linked glycan modified Golgi-translocated ATF6 (designated G), which is absent for the D564G mutant, confirming a lack of ER to Golgi trafficking.


Wild type and ERp18 KO cell lines stably expressing ATF6α D564G were either left untreated ( - ) or treated ( + ) with 5 µg / ml brefeldin A for1 h prior to cell lysis . The anti - ATF6α western blot of membrane fractions reveals proteolytic processing by S1P to produce ATF6 - M , only in the brefeldin A treated cells . Nuclear fractions confirm the presence of S2P cleavage product , ATF6 - N following brefeldin A treatment . Anti - HDAC2 was used as a loading control for the nuclear fraction .

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Wild type and ERp18 KO cell lines stably expressing ATF6α D564G were either left untreated (-) or treated (+) with 5 µg/ml brefeldin A for1 h prior to cell lysis. The anti-ATF6α western blot of membrane fractions reveals proteolytic processing by S1P to produce ATF6-M, only in the brefeldin A treated cells. Nuclear fractions confirm the presence of S2P cleavage product, ATF6-N following brefeldin A treatment. Anti-HDAC2 was used as a loading control for the nuclear fraction.


A - C Wild type cells stably expressing ATF6α ( A ) or ERp18 KO cells stably expressing ATF6α ( B ) , and ERp18 KO cells stably expressing ATF6α transiently transfected with ERp18 ( RV cells ) ( C ) , were pre - treated with 30 µM PF429242 ( S1P inhibitor ) for 1 h prior to induction of ER stress with 10 mM DTT as indicated .

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A-C Wild type cells stably expressing ATF6α (A) or ERp18 KO cells stably expressing ATF6α (B), and ERp18 KO cells stably expressing ATF6α transiently transfected with ERp18 (RV cells) (C), were pre-treated with 30 µM PF429242 (S1P inhibitor) for 1 h prior to induction of ER stress with 10 mM DTT as indicated.


D - F Wild type cells transiently expressing ATF6 C467A ( D ) , ATF6 C618A ( E ) , or ATF6 DM ( F ) were treated as above . Whole cell lysates were separated by SDS - PAGE under reducing conditions and analysed by western blotting for ATF6α . The positions of unprocessed ER - localised ATF6α ( ER ) and the O - linked glycan modified Golgi - translocated ATF6α ( Gol ) are indicated as is ATF6 - P .

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D-F Wild type cells transiently expressing ATF6 C467A (D), ATF6 C618A (E), or ATF6 DM (F) were treated as above. Whole cell lysates were separated by SDS-PAGE under reducing conditions and analysed by western blotting for ATF6α. The positions of unprocessed ER-localised ATF6α (ER) and the O-linked glycan modified Golgi-translocated ATF6α (Gol) are indicated as is ATF6-P.


G The percentage of Golgi - ATF6α was quantified , the error bars represent ± standard deviation for at least three independent experiments .

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G The percentage of Golgi-ATF6α was quantified, the error bars represent ± standard deviation for at least three independent experiments.


Relative expression of Rab39a in siRNA - treated cells after 48 hours of knockdown . Rab39a expression in I - Ab or Rab39a siRNA - treated cells was normalized to expression in cells treated with control β2m siRNA . Error bars show SD between three independent experiments . * p < 0 . 05 using two - way Student ' s t - test .

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Relative expression of Rab39a in siRNA-treated cells after 48 hours of knockdown. Rab39a expression in I-Ab or Rab39a siRNA-treated cells was normalized to expression in cells treated with control β2m siRNA. Error bars show SD between three independent experiments. * p < 0.05 using two-way Student's t-test.


XPT of siRNA treated DC3 . 2R cells after 48 hours of knockdown . Treated cells were fed with the indicated amounts of Ova - Fe The cells were then exposed to RF33 . 70 - Luc Reporter CD8 T cells overnight . RLU indicates relative luminescence units produced by reporter T cell stimulation . Error bars show SD of > 3 replicate wells . * p < 0 . 05 vs control I - Ab using two - way ANOVA . Representative plot of 3 independent experiments .

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XPT of siRNA treated DC3.2R cells after 48 hours of knockdown. Treated cells were fed with the indicated amounts of Ova-Fe The cells were then exposed to RF33.70-Luc Reporter CD8 T cells overnight. RLU indicates relative luminescence units produced by reporter T cell stimulation. Error bars show SD of >3 replicate wells. * p < 0.05 vs control I-Ab using two-way ANOVA. Representative plot of 3 independent experiments.


XPT of siRNA treated DC3 . 2R cells after 48 hours of knockdown . Treated cells were fed with the indicated amounts of Ova - latex The cells were then exposed to RF33 . 70 - Luc Reporter CD8 T cells overnight . RLU indicates relative luminescence units produced by reporter T cell stimulation . Error bars show SD of > 3 replicate wells . * p < 0 . 05 vs control I - Ab using two - way ANOVA . Representative plot of 3 independent experiments .

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XPT of siRNA treated DC3.2R cells after 48 hours of knockdown. Treated cells were fed with the indicated amounts of Ova-latex The cells were then exposed to RF33.70-Luc Reporter CD8 T cells overnight. RLU indicates relative luminescence units produced by reporter T cell stimulation. Error bars show SD of >3 replicate wells. * p < 0.05 vs control I-Ab using two-way ANOVA. Representative plot of 3 independent experiments.


XPT of siRNA treated DC3 . 2R cells after 48 hours of knockdown . Treated cells were fed with the indicated amounts of unconjugated soluble Ova The cells were then exposed to RF33 . 70 - Luc Reporter CD8 T cells overnight . RLU indicates relative luminescence units produced by reporter T cell stimulation . Error bars show SD of > 3 replicate wells . * p < 0 . 05 vs control I - Ab using two - way ANOVA . Representative plot of 3 independent experiments .

XPT of siRNA treated DC3.2R cells after 48 hours of knockdown. Treated cells were fed with the indicated amounts of unconjugated soluble Ova The cells were then exposed to RF33.70-Luc Reporter CD8 T cells overnight. RLU indicates relative luminescence units produced by reporter T cell stimulation. Error bars show SD of >3 replicate wells. * p < 0.05 vs control I-Ab using two-way ANOVA. Representative plot of 3 independent experiments.


DC3 . 2 - Rab39a cells ( Rab39a KO with dox - inducible Rab39a ) were incubated overnight with or without 1 µg / ml dox to induce Rab39a expression . 1x105 Cells were then fed with Ova - Fe and exposed to reporter T cells as in ( B ) . Error bars show SD of duplicate wells . * p < 0 . 05 using two - way ANOVA . Representative plot of 3 independent experiments .

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DC3.2-Rab39a cells (Rab39a KO with dox-inducible Rab39a) were incubated overnight with or without 1 µg/ml dox to induce Rab39a expression. 1x105 Cells were then fed with Ova-Fe and exposed to reporter T cells as in (B). Error bars show SD of duplicate wells. * p < 0.05 using two-way ANOVA. Representative plot of 3 independent experiments.


MHC - II presentation of siRNA treated DC3 . 2R cells after 48 hours of knockdown . Treated cells were fed with the indicated amounts of Ova - Fe . The cells were then exposed to MF2 . 2D9 - Luc Reporter CD4 T cells overnight . Error bars show SD of > 3 replicate wells . * p < 0 . 05 vs negative control β2m using two - way ANOVA . None of the bead concentrations are p < 0 . 05 for Rab39a siRNA vs β2m . Representative plot of 3 independent

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MHC-II presentation of siRNA treated DC3.2R cells after 48 hours of knockdown. Treated cells were fed with the indicated amounts of Ova-Fe. The cells were then exposed to MF2.2D9-Luc Reporter CD4 T cells overnight. Error bars show SD of >3 replicate wells. * p < 0.05 vs negative control β2m using two-way ANOVA. None of the bead concentrations are p < 0.05 for Rab39a siRNA vs β2m. Representative plot of 3 independent experiments


DC3 . 2 - Rab39a cells ( Rab39a KO with dox - inducible Rab39a ) were incubated overnight with or without 1 µg / ml dox to induce Rab39a expression . 1x105 cells were then fed with Ova - Fe and exposed to reporter T cells as in ( A ) . Error bars show SD of duplicate wells . None of the bead concentrations are p < 0 . 05 for Rab39a siRNA vs β2m using two - way ANOVA . Representative plot of 3 independent

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 B-CONTROLLED_VAR 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

DC3.2-Rab39a cells (Rab39a KO with dox-inducible Rab39a) were incubated overnight with or without 1 µg/ml dox to induce Rab39a expression. 1x105 cells were then fed with Ova-Fe and exposed to reporter T cells as in (A). Error bars show SD of duplicate wells. None of the bead concentrations are p < 0.05 for Rab39a siRNA vs β2m using two-way ANOVA. Representative plot of 3 independent experiments


MHC - I Presentation of SIINFEKL - H2 - Kb complexes on siRNA treated DC3 . 2 NS - Ova ( C ) or DC3 . 2 UbS8L ( D ) cells . 48 hours after siRNA transfection , the indicated amounts of dox was added to the cells to induce Ova expression . After 2 hours at 37oC , RF33 . 70 - Luc CD8 T cells were added along with Brefeldin A ( Golgiplug ) to a final concentration of 1 : 1000 . The cells were incubated overnight for reporter T cell luciferase expression . Error bars show SD of > 3 replicate wells . * p < 0 . 05 vs negative control I - Ab using two - way ANOVA . None of the dox concentrations are p < 0 . 05 for Rab39a siRNA vs I - Ab . Representative plot of 3 independent

O O O 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 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 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

MHC-I Presentation of SIINFEKL-H2-Kb complexes on siRNA treated DC3.2 NS-Ova (C) or DC3.2 UbS8L (D) cells. 48 hours after siRNA transfection, the indicated amounts of dox was added to the cells to induce Ova expression. After 2 hours at 37oC, RF33.70-Luc CD8 T cells were added along with Brefeldin A (Golgiplug) to a final concentration of 1:1000. The cells were incubated overnight for reporter T cell luciferase expression. Error bars show SD of >3 replicate wells. * p < 0.05 vs negative control I-Ab using two-way ANOVA. None of the dox concentrations are p < 0.05 for Rab39a siRNA vs I-Ab. Representative plot of 3 independent experiments


Flow cytometric analysis of surface H2 - Kb and I - Ab levels of siRNA treated DC3 . 2R cells . Shown are results after 48 hours of knockdown . Representative plot of 3 independent

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Flow cytometric analysis of surface H2-Kb and I-Ab levels of siRNA treated DC3.2R cells. Shown are results after 48 hours of knockdown. Representative plot of 3 independent experiments


Phagocytosis assay after 48 hours of siRNA knockdown . DC3 . 2R cells treated with the indicated siRNAs were incubated on ice for 30 minutes with 4 beads per cell of Alexa Fluor 647 conjugated beads . The mixture was then placed in media at 37oC for 1 hour . Cells were harvested and incubated on ice with 1 mg / ml of Trypan Blue to quench uneaten beads before analysis through flow cytometry . As a control , some cells were incubated with 10 µM Cytochalasin D during the feeding period . Shown are percentages of cells that have taken up beads . Representative plot of two independent experiments .

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Phagocytosis assay after 48 hours of siRNA knockdown. DC3.2R cells treated with the indicated siRNAs were incubated on ice for 30 minutes with 4 beads per cell of Alexa Fluor 647 conjugated beads. The mixture was then placed in media at 37oC for 1 hour. Cells were harvested and incubated on ice with 1 mg/ml of Trypan Blue to quench uneaten beads before analysis through flow cytometry. As a control, some cells were incubated with 10 µM Cytochalasin D during the feeding period. Shown are percentages of cells that have taken up beads. Representative plot of two independent experiments.


Rab39a was induced in DC3 . 2 - Rab39a cells ( Rab39a KO with dox - inducible Rab39a ) via overnight incubation with 1µg / ml dox . KO and rescued cells were then harvested and assayed as in ( A ) for phagocytosis . Representative plot of two independent experiments .

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Rab39a was induced in DC3.2-Rab39a cells (Rab39a KO with dox-inducible Rab39a) via overnight incubation with 1µg / ml dox. KO and rescued cells were then harvested and assayed as in (A) for phagocytosis. Representative plot of two independent experiments.


DC3 . 2 - Rab39a cells were induced to express Rab39a overnight . Cells on coverslips were washed , fixed and stained according to the listed protocol and antibodies . Green = Rab39a ( HA tag ) , Magenta = Lamp2 . Arrows highlight punctate structures with colocalization . Scale bar = 10 µm . Representative image of two independent experiments .

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DC3.2-Rab39a cells were induced to express Rab39a overnight. Cells on coverslips were washed, fixed and stained according to the listed protocol and antibodies. Green = Rab39a (HA tag), Magenta = Lamp2. Arrows highlight punctate structures with colocalization. Scale bar = 10 µm. Representative image of two independent experiments.


DC3 . 2 Rab39a KO cells were transduced with dox inducible mcherry - tagged Rab39a . Cells were plated in 35 mm glass bottom dishes and induced with 1 µg / ml dox . The next day , 1 µm latex beads conjugated with Alexa 488 - NHS ( 1 bead / cell ) were added . After 2 hours at 37oC , cells were imaged under a Leica TCS SP5 confocal microscope . Green = Alexa 488 labeled beads . Magenta = mcherry - Rab39a . Arrows highlight internalized beads surrounded by Rab39a . Scale bar = 10 µm . Representative image of two independent experiments .

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DC3.2 Rab39a KO cells were transduced with dox inducible mcherry-tagged Rab39a. Cells were plated in 35 mm glass bottom dishes and induced with 1 µg/ml dox. The next day, 1 µm latex beads conjugated with Alexa 488-NHS (1 bead/cell) were added. After 2 hours at 37oC, cells were imaged under a Leica TCS SP5 confocal microscope. Green = Alexa 488 labeled beads. Magenta = mcherry-Rab39a. Arrows highlight internalized beads surrounded by Rab39a. Scale bar = 10 µm. Representative image of two independent experiments.


5x105 of DC3 . 2 - Rab39a cells were induced to express Rab39a overnight with 1 µg / ml dox in a 6 well plate . Cells were fed at 1 bead / cell of biotinylated - 6µm magnetic beads for the indicated lengths of time . Magnetic - bead - phagosomes were isolated using the listed protocol . Phagosomes were washed and stained for Rab39a ( HA - tag ) . Representative data for 3 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

5x105 of DC3.2-Rab39a cells were induced to express Rab39a overnight with 1 µg/ml dox in a 6 well plate. Cells were fed at 1 bead/cell of biotinylated-6µm magnetic beads for the indicated lengths of time. Magnetic-bead-phagosomes were isolated using the listed protocol. Phagosomes were washed and stained for Rab39a (HA-tag). Representative data for 3 independent experiments.


1 . 25 x 105 of DC3 . 2 - Rab39a cells were incubated in a 12 well plate overnight ± dox ( 1µg / ml ) to induce Rab39a . Cells were fed at 4 beads / cell of biotinylated - ova - 6µm - magnetic - beads for 3 hours with or without 1 : 1000 BFA ( golgiplug , BD ) . Phagosomes were isolated using the listed protocol . Isolated phagosomes were washed , permeabilized and stained . Shown are histograms of indicated stains as well as % of Rab39a positive phagosomes . Representative plot of two independent experiments .

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 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

1.25 x 105 of DC3.2-Rab39a cells were incubated in a 12 well plate overnight ± dox (1µg/ml) to induce Rab39a. Cells were fed at 4 beads/cell of biotinylated-ova-6µm-magnetic-beads for 3 hours with or without 1:1000 BFA (golgiplug, BD). Phagosomes were isolated using the listed protocol. Isolated phagosomes were washed, permeabilized and stained. Shown are histograms of indicated stains as well as % of Rab39a positive phagosomes. Representative plot of two independent experiments.


XPT of siRNA treated DC3 . 2R cells after 48 hours of knockdown . Treated cells were fed with the indicated amounts of C8L peptid conjugated to iron oxide beads via a disulfide bond . The cells were then exposed to RF33 . 70 - Luc Reporter CD8 T cells overnight . Error bars show SD of > 3 replicate wells . * p < 0 . 05 for siRNA vs control I - Ab using two - way ANOVA . Representative plot of 3 independent

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

XPT of siRNA treated DC3.2R cells after 48 hours of knockdown. Treated cells were fed with the indicated amounts of C8L peptid conjugated to iron oxide beads via a disulfide bond. The cells were then exposed to RF33.70-Luc Reporter CD8 T cells overnight. Error bars show SD of >3 replicate wells. * p < 0.05 for siRNA vs control I-Ab using two-way ANOVA. Representative plot of 3 independent experiments


XPT of siRNA treated DC3 . 2R cells after 48 hours of knockdown . Treated cells were fed with the indicated amounts o Ova ( B ) conjugated to iron oxide beads via a disulfide bond . The cells were then exposed to RF33 . 70 - Luc Reporter CD8 T cells overnight . Error bars show SD of > 3 replicate wells . * p < 0 . 05 for siRNA vs control I - Ab using two - way ANOVA . Representative plot of 3 independent

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

XPT of siRNA treated DC3.2R cells after 48 hours of knockdown. Treated cells were fed with the indicated amounts o Ova (B) conjugated to iron oxide beads via a disulfide bond. The cells were then exposed to RF33.70-Luc Reporter CD8 T cells overnight. Error bars show SD of >3 replicate wells. * p < 0.05 for siRNA vs control I-Ab using two-way ANOVA. Representative plot of 3 independent experiments


DC3 . 2 - Rab39a cells ( Rab39a KO with dox - inducible Rab39a ) were incubated overnight with or without 1 µg / ml dox to induce Rab39a expression . 1x105 cells were then fed with C8L peptide beads and exposed to reporter T cells as in ( A ) . Error bars show SD of duplicate wells . * p < 0 . 05 between no dox and + dox using two - way ANOVA . Representative plot of 3 independent

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

DC3.2-Rab39a cells (Rab39a KO with dox-inducible Rab39a) were incubated overnight with or without 1 µg/ml dox to induce Rab39a expression. 1x105 cells were then fed with C8L peptide beads and exposed to reporter T cells as in (A). Error bars show SD of duplicate wells. * p < 0.05 between no dox and + dox using two-way ANOVA. Representative plot of 3 independent experiments


Same as in ( A ) but cells were fed with 8 µg of A9M peptide bead and exposed to 12 . 64 - Luc Reporter T cells . Experiments were combined by normalizing RLU values to the average of the negative control I - Ab . Error bars show SD of 3 independent experiments . * p < 0 . 05 for Rab39a or β2m siRNA vs negative control I - Ab using

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

Same as in (A) but cells were fed with 8 µg of A9M peptide bead and exposed to 12.64-Luc Reporter T cells. Experiments were combined by normalizing RLU values to the average of the negative control I-Ab. Error bars show SD of 3 independent experiments. * p < 0.05 for Rab39a or β2m siRNA vs negative control I-Ab using ANOVA


DC3 . 2 - Rab39a - H2 - Ld cells ( Rab39a KO with dox - inducible Rab39a ) were incubated with or without 1 µg / ml dox overnight to induce Rab39a expression . 1x106 cells were fed at 4 beads / cell of biotinylated - magnetic - beads for 4 hours . Magnetic bead phagosomes were isolated using the listed protocol . Phagosomes were washed , fixed , permeabilized and stained for Open ( 64 - 3 - 7 ) and Closed ( 30 - 5 - 7s ) forms of H2 - Ld . Representative histograms shown in ( B ) . The background fluorescence of unstained beads was subtracted from the resulting phagosome gMFI and the fold change of open or closed H2 - Ld ( dox / no dox ) was calculated . Three independent experiments were combined for analysis and error bars show the SD between experiments . Open H2 - Ld , but not Closed H2 - Ld was statistically significant ( p < 0 . 05 ) between KO and rescued cells using two - tailed ratio - paired Student ' s t - test .

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 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 O O O O O 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 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

DC3.2-Rab39a-H2-Ld cells (Rab39a KO with dox-inducible Rab39a) were incubated with or without 1 µg/ml dox overnight to induce Rab39a expression. 1x106 cells were fed at 4 beads/cell of biotinylated-magnetic-beads for 4 hours. Magnetic bead phagosomes were isolated using the listed protocol. Phagosomes were washed, fixed, permeabilized and stained for Open (64-3-7) and Closed (30-5-7s) forms of H2-Ld. Representative histograms shown in (B). The background fluorescence of unstained beads was subtracted from the resulting phagosome gMFI and the fold change of open or closed H2-Ld (dox/no dox) was calculated. Three independent experiments were combined for analysis and error bars show the SD between experiments. Open H2-Ld, but not Closed H2-Ld was statistically significant (p<0.05) between KO and rescued cells using two-tailed ratio-paired Student's t-test.


DC3 . 2 - Rab39a - H2 - Ld cells ( Rab39a KO with dox - inducible Rab39a ) were incubated with or without 1 µg / ml dox overnight to induce Rab39a expression . 1x106 cells were fed at 4 beads / cell of biotinylated - magnetic - beads for 4 hours . Magnetic bead phagosomes were isolated using the listed protocol . Phagosomes were washed , fixed , permeabilized and stained for Open ( 64 - 3 - 7 ) and Closed ( 30 - 5 - 7s ) forms of H2 - Ld . H2 - Ld cell surface levels of KO and induced cells as in ( A ) .

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

DC3.2-Rab39a-H2-Ld cells (Rab39a KO with dox-inducible Rab39a) were incubated with or without 1 µg/ml dox overnight to induce Rab39a expression. 1x106 cells were fed at 4 beads/cell of biotinylated-magnetic-beads for 4 hours. Magnetic bead phagosomes were isolated using the listed protocol. Phagosomes were washed, fixed, permeabilized and stained for Open (64-3-7) and Closed (30-5-7s) forms of H2-Ld. H2-Ld cell surface levels of KO and induced cells as in (A).


Phagosomes from KO and induced cells were isolated as in ( A ) . Permeabilized , unfixed phagosomes were incubated with 10 µM of the indicated peptides for 3 hours at 25oC in Perm buffer . Phagosomes were then washed and stained for H2 - Ld . Shown are histograms of indicated stains .

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

Phagosomes from KO and induced cells were isolated as in (A). Permeabilized, unfixed phagosomes were incubated with 10 µM of the indicated peptides for 3 hours at 25oC in Perm buffer. Phagosomes were then washed and stained for H2-Ld. Shown are histograms of indicated stains.


Phagosomes from KO and induced cells were isolated as in ( A ) . Permeabilized , unfixed phagosomes were incubated with 10 µM of the indicated peptides for 3 hours at 25oC in Perm buffer . Phagosomes were then washed and stained for H2 - Ld . Shown are histograms of indicated stains . Flow cytometry plots of ( D ) .

Phagosomes from KO and induced cells were isolated as in (A). Permeabilized, unfixed phagosomes were incubated with 10 µM of the indicated peptides for 3 hours at 25oC in Perm buffer. Phagosomes were then washed and stained for H2-Ld. Shown are histograms of indicated stains. Flow cytometry plots of (D).


Analysis of phagosomes shown in ( D ) and ( E ) . The background fluorescence of unstained beads was subtracted from the resulting phagosome gMFI and the fold change of pulsed / unpulsed phagosomes was calculated .

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

Analysis of phagosomes shown in (D) and (E). The background fluorescence of unstained beads was subtracted from the resulting phagosome gMFI and the fold change of pulsed / unpulsed phagosomes was calculated.


DC3 . 2 Rab39a - H2 - Ld cells were incubated with or without 1 µg / ml dox overnight to induce Rab39a expression . 1x106 cells were fed at 4 beads / cell of biotinylated - Ova - magnetic - beads for 4 hours in the presence or absence of BFA . Isolated phagosomes were stained for H2 - Ld . Representative of two independent experiments .

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

DC3.2 Rab39a-H2-Ld cells were incubated with or without 1 µg/ml dox overnight to induce Rab39a expression. 1x106 cells were fed at 4 beads/cell of biotinylated-Ova-magnetic-beads for 4 hours in the presence or absence of BFA. Isolated phagosomes were stained for H2-Ld. Representative of two independent experiments.


DC3 . 2 - Rab39a - H2 - Ld cells ( Rab39a KO with dox - inducible Rab39a ) were incubated with 1 µg / ml dox overnight to induce Rab39a expression . 1x106 cells were fed at 4 beads / cell of biotinylated - Ova - magnetic - beads for 4 hours with the indicated inhibitors . Magnetic - bead phagosomes were isolated using the listed protocol . Phagosomes were washed , fixed , permeabilized and stained for Open and Closed ( 30 - 5 - 7s ) forms of H2 - Ld . Representative histograms of two independent experiments shown .

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

DC3.2-Rab39a-H2-Ld cells (Rab39a KO with dox-inducible Rab39a) were incubated with 1 µg/ml dox overnight to induce Rab39a expression. 1x106 cells were fed at 4 beads/cell of biotinylated-Ova-magnetic-beads for 4 hours with the indicated inhibitors. Magnetic-bead phagosomes were isolated using the listed protocol. Phagosomes were washed, fixed, permeabilized and stained for Open and Closed (30-5-7s) forms of H2-Ld. Representative histograms of two independent experiments shown.


Cells as in ( A ) were incubated with or without 1 µg / ml dox overnight to induce Rab39a expression . 1x106 cells were fed at 4 beads / cell of biotinylated - Ova - magnetic - beads for 4 hours . Phagosomes were isolated and stained for Ova and Open H2 - Ld . Representative of 3 independent experiments .

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

Cells as in (A) were incubated with or without 1 µg/ml dox overnight to induce Rab39a expression. 1x106 cells were fed at 4 beads/cell of biotinylated-Ova-magnetic-beads for 4 hours. Phagosomes were isolated and stained for Ova and Open H2-Ld. Representative of 3 independent experiments.


Quantification of Ova remaining in bead - phagosomes ( C ) and Nox2 staining ( D ) . Representative Histogram shown . For analysis , the background fluorescence of unstained beads was subtracted from the resulting phagosome gMFI and the fold change of target protein ( dox / no dox ) was calculated . Three independent experiments were combined . P < 0 . 05 using two - tailed ratio - paired Student ' s t - test . Error bars show SD between 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 B-CONTROLLED_VAR 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

Quantification of Ova remaining in bead-phagosomes (C) and Nox2 staining (D). Representative Histogram shown. For analysis, the background fluorescence of unstained beads was subtracted from the resulting phagosome gMFI and the fold change of target protein (dox/no dox) was calculated. Three independent experiments were combined. P<0.05 using two-tailed ratio-paired Student's t-test. Error bars show SD between experiments.


Cells as in ( A ) were incubated with or without 1 µg / ml dox overnight to induce Rab39a expression . 1x106 cells were incubated on ice with 4 beads / cell of biotinylated - Ova - beads for bead attachment . Cells were then transferred into 37oC media containing 5 µM CellROX Deep Red . After 4 hours of incubation , unfixed phagosomes were extracted and analyzed via flow cytometry for CellROX fluorescence . For analysis , the background fluorescence of unstained beads was subtracted from the resulting phagosome gMFI and the fold change ( dox / no dox ) was calculated . Three independent experiments were combined . P < 0 . 05 using two - tailed ratio - paired Student ' s t - test . Error bars show SD between experiments .

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

Cells as in (A) were incubated with or without 1 µg/ml dox overnight to induce Rab39a expression. 1x106 cells were incubated on ice with 4 beads/cell of biotinylated-Ova-beads for bead attachment. Cells were then transferred into 37oC media containing 5 µM CellROX Deep Red. After 4 hours of incubation, unfixed phagosomes were extracted and analyzed via flow cytometry for CellROX fluorescence. For analysis, the background fluorescence of unstained beads was subtracted from the resulting phagosome gMFI and the fold change (dox/no dox) was calculated. Three independent experiments were combined. P<0.05 using two-tailed ratio-paired Student's t-test. Error bars show SD between experiments.


Same as in C with Sec22b staining . Three independent experiments were combined . P < 0 . 05 using two - tailed ratio - paired Student ' s t - test . Error bars show SD between 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

Same as in C with Sec22b staining. Three independent experiments were combined. P<0.05 using two-tailed ratio-paired Student's t-test. Error bars show SD between experiments.


Cells as in ( A ) were fed at 1 bead per cell with pHrodo conjugated beads that increase fluorescence at low pH . After 4 hours of incubation , whole cells were harvested and run through flow cytometry to measure fluorescence of eaten beads . For analysis , the background fluorescence of uneaten beads was subtracted from the resulting gMFI and the fold change of dox / no dox was calculated . Three independent experiments were combined . p < 0 . 05 using two - tailed ratio - paired Student ' s t - test . Error bars show SD between experiments . Also shown are histograms of whole cells compared to background fluorescence of pH beads .

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

Cells as in (A) were fed at 1 bead per cell with pHrodo conjugated beads that increase fluorescence at low pH. After 4 hours of incubation, whole cells were harvested and run through flow cytometry to measure fluorescence of eaten beads. For analysis, the background fluorescence of uneaten beads was subtracted from the resulting gMFI and the fold change of dox/no dox was calculated. Three independent experiments were combined. p<0.05 using two-tailed ratio-paired Student's t-test. Error bars show SD between experiments. Also shown are histograms of whole cells compared to background fluorescence of pH beads.


DC3 . 2R cells were treated with the indicated siRNA . 48 hours after transfection , cells were fed with Ova - Fe ( 2 µg Ova ) with or without 100 µM leupeptin . RF33 . 70 - Luc reporter T cells were added . After overnight incubation , T cell luciferase was measured . Representative plot of two independent experiments . Error bars show SD between 3 replicate wells . p values were calculated using 2 - way ANOVA .

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-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

DC3.2R cells were treated with the indicated siRNA. 48 hours after transfection, cells were fed with Ova-Fe (2 µg Ova) with or without 100 µM leupeptin. RF33.70-Luc reporter T cells were added. After overnight incubation, T cell luciferase was measured. Representative plot of two independent experiments. Error bars show SD between 3 replicate wells. p values were calculated using 2-way ANOVA.


5x104 DC3 . 2 - Rab39a - H2 - Ld cells were fed with Ova - Fe ( 1µg Ova ) with or without 1 µg dox and / or 100 µM leupeptin . RF33 . 70 - Luc reporter T cells were added . After overnight incubation , T cell luciferase was measured . Representative plot of two independent experiments . Error bars show SD between 3 replicate wells . p values were calculated using 2 - way ANOVA .

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-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 O O O O O O O O O O O O

5x104 DC3.2-Rab39a-H2-Ld cells were fed with Ova-Fe (1µg Ova) with or without 1 µg dox and/or 100 µM leupeptin. RF33.70-Luc reporter T cells were added. After overnight incubation, T cell luciferase was measured. Representative plot of two independent experiments. Error bars show SD between 3 replicate wells. p values were calculated using 2-way ANOVA.


Rab39a expression in splenocyte subsets was measured using an FDG assay . Shown are relative FDG fluorescence of Rab39a KO cells / WT background . Summary of two 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

Rab39a expression in splenocyte subsets was measured using an FDG assay. Shown are relative FDG fluorescence of Rab39a KO cells/WT background. Summary of two independent experiments.


Splenic DCs were expanded in Rab39a KO and WT mice by injection of B16 - Flt3L tumor . 10 days post tumor implantation , DCIR2 + ( Cd8α - ) or XCR1 + ( Cd8α + ) dendritic cells were isolated from spleens via magnetic positive selection . 5 x 104 cells were plated in a 96 well plate and incubated with the indicated amounts of Ova - Fe and 1 : 1 of Reporter CD8 or CD4 T - cells overnight before measuring luciferase activity . Error bars indicate SD of 2 replicate wells . Shown are representative graphs of 3 independent experiments . * p < 0 . 05 using two - way ANOVA .

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

Splenic DCs were expanded in Rab39a KO and WT mice by injection of B16-Flt3L tumor. 10 days post tumor implantation, DCIR2+ (Cd8α-) or XCR1+ (Cd8α+) dendritic cells were isolated from spleens via magnetic positive selection. 5 x 104 cells were plated in a 96 well plate and incubated with the indicated amounts of Ova-Fe and 1:1 of Reporter CD8 or CD4 T-cells overnight before measuring luciferase activity. Error bars indicate SD of 2 replicate wells. Shown are representative graphs of 3 independent experiments. * p < 0.05 using two-way ANOVA.


CD11b + CD11c + dendritic cells were enriched from WT or Rab39a KO spleens via magnetic negative selection . Cells were fed with 3 µm biotin - ova - magnetic beads for 4 hours . Phagosomes were extracted , permeabilized and stained for the indicated proteins . For ROS measurements , phagocytosis was performed with 5 µM CellROX and the phagosomes were not permeabilized . For analysis , the background fluorescence of unstained beads was subtracted from the resulting phagosome gMFI and the fold change of target ( dox / no dox ) was calculated . Shown are three independent experiments combined . Error bars show SD between experiments . p values were calculated using two - tailed ratio - paired 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 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 O O O O O O O O O O O O O O O O O O O O O O O O O O

CD11b+ CD11c+ dendritic cells were enriched from WT or Rab39a KO spleens via magnetic negative selection. Cells were fed with 3 µm biotin-ova-magnetic beads for 4 hours. Phagosomes were extracted, permeabilized and stained for the indicated proteins. For ROS measurements, phagocytosis was performed with 5 µM CellROX and the phagosomes were not permeabilized. For analysis, the background fluorescence of unstained beads was subtracted from the resulting phagosome gMFI and the fold change of target (dox/no dox) was calculated. Shown are three independent experiments combined. Error bars show SD between experiments. p values were calculated using two-tailed ratio-paired Student's t-test.


CD11b + CD11c + dendritic cells were enriched from WT or Rab39a KO spleens via magnetic negative selection . Cells were fed with 3 µm magnetic beads conjugated to pHrodo . After 4 hours , cells were harvested for flow cytometry to measure fluorescence of eaten beads due to phagosomal pH . For analysis , the background fluorescence of unstained beads was subtracted from the resulting gMFI and the fold change ( dox / no dox ) was calculated . Shown are three independent experiments with error bars indicating the SD . p values were calculated using two - tailed ratio - paired 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 O O O O O O O O O O O O O

CD11b+ CD11c+ dendritic cells were enriched from WT or Rab39a KO spleens via magnetic negative selection. Cells were fed with 3 µm magnetic beads conjugated to pHrodo. After 4 hours, cells were harvested for flow cytometry to measure fluorescence of eaten beads due to phagosomal pH. For analysis, the background fluorescence of unstained beads was subtracted from the resulting gMFI and the fold change (dox/no dox) was calculated. Shown are three independent experiments with error bars indicating the SD. p values were calculated using two-tailed ratio-paired Student's t-test.


CD11b + CD11c + dendritic cells were enriched from WT or Rab39a KO spleens via magnetic negative selection . Cells were fed with 3 µm magnetic beads conjugated to Alexa fluor dye . After 4 hours , cells were harvested and resuspended in 1 mg / ml Trypan blue to quench fluorescence of uneaten beads . Cells were run for flow cytometry to measure phagocytosis . The ratio of KO cells with beads / WT cells with beads was then obtained . Graphs show three independent experiments and p values were calculated using two - tailed ratio - paired Student ' s t - test . Error bars show SD between 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

CD11b+ CD11c+ dendritic cells were enriched from WT or Rab39a KO spleens via magnetic negative selection. Cells were fed with 3 µm magnetic beads conjugated to Alexa fluor dye. After 4 hours, cells were harvested and resuspended in 1 mg/ml Trypan blue to quench fluorescence of uneaten beads. Cells were run for flow cytometry to measure phagocytosis. The ratio of KO cells with beads / WT cells with beads was then obtained. Graphs show three independent experiments and p values were calculated using two-tailed ratio-paired Student's t-test. Error bars show SD between experiments.


Rab39a KO and WT mice were injected intravenously with 1 x 105 A20 - Ova cells . The next day , CFSE labeled OTI - splenocytes were injected i . v . . 3 days post OT - I transfer , spleens were harvested and assayed for OT - I proliferation . Three independent experiments ( WT vs KO ) were performed . To combine the data , the OT - I proliferation in each individual mouse was normalized to the average proliferation of the WT controls in that experiment . Error bars show SD between individual mice and p value was derived from two - tailed student ' s t - test . n = 14 ( WT ) or 9 ( KO ) .

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Rab39a KO and WT mice were injected intravenously with 1 x 105 A20-Ova cells. The next day, CFSE labeled OTI-splenocytes were injected i.v.. 3 days post OT-I transfer, spleens were harvested and assayed for OT-I proliferation. Three independent experiments (WT vs KO) were performed. To combine the data, the OT-I proliferation in each individual mouse was normalized to the average proliferation of the WT controls in that experiment. Error bars show SD between individual mice and p value was derived from two-tailed student's t-test. n = 14 (WT) or 9 (KO).


C Co - crystal structure of gephyrin E - domain ( blue ) and bound GlyR ß - loop ( red - PDB code 2FTS ( Kim et al . , 2006 ) ) . The mutated gephyrin Gly375 residue is shown in orange .

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C Co-crystal structure of gephyrin E-domain (blue) and bound GlyR ß-loop (red - PDB code 2FTS (Kim et al., 2006)). The mutated gephyrin Gly375 residue is shown in orange.


A Representative images of GFP - gephyrin and GFP - G375D , expressed in hippocampal neurons at 9 + 4 days in vitro . Inset shows higher magnification . Scale bar = 20 µm . B Relative distribution of cluster density of gephyrin variants on dendrites from transfected neurons . Number of gephyrin clusters in each dendrite is normalized to 20 µm segments .

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A Representative images of GFP-gephyrin and GFP-G375D, expressed in hippocampal neurons at 9 + 4 days in vitro. Inset shows higher magnification. Scale bar = 20 µm.B Relative distribution of cluster density of gephyrin variants on dendrites from transfected neurons. Number of gephyrin clusters in each dendrite is normalized to 20 µm segments.


C Representative segments of dendrites expressing GFP - gephyrin or GFP - G375D and immunostained with the presynaptic marker VGAT . Arrowheads show co - localization of gephyrin and VGAT clusters . Scale bar = 10 µm . D Quantification of co - localization between gephyrin and VGAT clusters ( gephyrin - VGAT 77 . 4 ± 2 . 9 % , G375D - VGAT 30 ± 4 . 4 % ) . 31 GFP - gephyrin and 30 GFP - G375D neurons from three independent cultures were used for quantifications . Results are expressed as mean ±

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C Representative segments of dendrites expressing GFP-gephyrin or GFP-G375D and immunostained with the presynaptic marker VGAT. Arrowheads show co-localization of gephyrin and VGAT clusters. Scale bar = 10 µm.D Quantification of co-localization between gephyrin and VGAT clusters (gephyrin-VGAT 77.4±2.9 %, G375D-VGAT 30±4.4 %). 31 GFP-gephyrin and 30 GFP-G375D neurons from three independent cultures were used for quantifications. Results are expressed as mean ± SEM


A Representative dendritic segment of a neuron expressing GFP - G375D and 3B11 - immunostained endogenous gephyrin ( red ) . B Quantification of density and ( C ) size of endogenous gephyrin clusters in neurons expressing GFP , GFP - G375D or non - transfected neighboring neurons . 20 GFP - G375 and non - transfected neurons and 15 GFP - expressing neurons from three independent cultures were used for quantifications . Results are expressed as mean ± SEM and data were analyzed by t - test .

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A Representative dendritic segment of a neuron expressing GFP-G375D and 3B11-immunostained endogenous gephyrin (red).B Quantification of density and (C) size of endogenous gephyrin clusters in neurons expressing GFP, GFP-G375D or non-transfected neighboring neurons. 20 GFP-G375 and non-transfected neurons and 15 GFP-expressing neurons from three independent cultures were used for quantifications. Results are expressed as mean ± SEM and data were analyzed by t-test.


A Representative neurons expressing myc - tagged gephyrin or G375D and pHluorin - tagged GABAAR γ2 . Surface - expressed γ2 - subunits were immunolabelled with GFP - antibodies in red , and the pHluorin - fluorescence ( total γ2 levels ) are shown in green . B Size and C fluorescence ( fl . ) intensity of surface - expressed γ2 clusters in myc - gephyrin and myc - G375D - expressing dendrites ( n = 20 myc - gephyrin and 21 myc - G375D neurons in B and 18 myc - gephyrin and myc - G375D neurons in C ) D Mean γ2 - pHluorin fluorescence in myc - gephyrin and myc - G375D - expressing dendrites ( n = 20 and 18 myc - gephyrin and myc - G375D expressing neurons ) . E Number of surface γ2 clusters on dendrites normalized to 20 µm segments ( n = 18 myc - gephyrin and myc - G375D expressing neurons ) . At least three independent cultures were used for each experiment . Results are expressed as mean ± SEM ; Data were analyzed by t - test .

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A Representative neurons expressing myc-tagged gephyrin or G375D and pHluorin-tagged GABAAR γ2. Surface-expressed γ2-subunits were immunolabelled with GFP-antibodies in red, and the pHluorin-fluorescence (total γ2 levels) are shown in green.B Size and C fluorescence (fl.) intensity of surface-expressed γ2 clusters in myc-gephyrin and myc-G375D-expressing dendrites (n = 20 myc-gephyrin and 21 myc-G375D neurons in B and 18 myc-gephyrin and myc-G375D neurons in C)D Mean γ2-pHluorin fluorescence in myc-gephyrin and myc-G375D-expressing dendrites (n = 20 and 18 myc-gephyrin and myc-G375D expressing neurons).E Number of surface γ2 clusters on dendrites normalized to 20 µm segments (n = 18 myc-gephyrin and myc-G375D expressing neurons). At least three independent cultures were used for each experiment. Results are expressed as mean ± SEM; Data were analyzed by t-test.


F Representative traces of miniature recordings on cultured hippocampal neurons transfected either with GFP - gephyrin or GFP - G375D . G mIPSC amplitudes from neurons expressing GFP - gephyrin or GFP - G375D . Numbers show analyzed cell - numbers . Data were analyzed by one - way ANOVA and post - hoc Tukey test . H Frequency of the mIPSCs from neurons expressing GFP - gephyrin or GFP - G375D . The horizontal line within the boxes indicates the median frequency for the represented condition ( bottom / top boundary indicates the 25th / 75th percentile ) . Top and bottom error bars indicate the 90th and 10th percentiles , respectively . Analysis by Kruskal - Wallis test followed by post - hoc Mann - Whitney test .

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F Representative traces of miniature recordings on cultured hippocampal neurons transfected either with GFP-gephyrin or GFP-G375D.G mIPSC amplitudes from neurons expressing GFP-gephyrin or GFP-G375D. Numbers show analyzed cell-numbers. Data were analyzed by one-way ANOVA and post-hoc Tukey test.H Frequency of the mIPSCs from neurons expressing GFP-gephyrin or GFP-G375D. The horizontal line within the boxes indicates the median frequency for the represented condition (bottom/top boundary indicates the 25th /75th percentile). Top and bottom error bars indicate the 90th and 10th percentiles, respectively. Analysis by Kruskal-Wallis test followed by post-hoc Mann-Whitney test.


A Western blot of HEK293 cells expressing GFP - tagged gephyrin or G375D using GFP - specific and polyclonal gephyrin antibodies . Note that both gephyrin variants are stably expressed and show no degradation in the cells . Asterisk in the poly - gephyrin blot shows endogenous gephyrin .

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A Western blot of HEK293 cells expressing GFP-tagged gephyrin or G375D using GFP-specific and polyclonal gephyrin antibodies. Note that both gephyrin variants are stably expressed and show no degradation in the cells. Asterisk in the poly-gephyrin blot shows endogenous gephyrin.


B SDS - PAGE of E . coli expressed and purified 6His - gephyrins and CD spectra . Mean residue ellipticity of gephyrin and G375D show profiles with minima at 208 nm and 222 nm .

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B SDS-PAGE of E. coli expressed and purified 6His-gephyrins and CD spectra. Mean residue ellipticity of gephyrin and G375D show profiles with minima at 208 nm and 222 nm.


C Size - exclusion chromatography of purified 6His - gephyrin and isolated E - domain variants . Full - length gephyrins and isolated E - domains were loaded onto a Superose 6 gel filtration column and elution of the protein was monitored by measuring absorbance at 280 nm ( mAU , milliabsorbance units ) .

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C Size-exclusion chromatography of purified 6His-gephyrin and isolated E-domain variants. Full-length gephyrins and isolated E-domains were loaded onto a Superose 6 gel filtration column and elution of the protein was monitored by measuring absorbance at 280 nm (mAU, milliabsorbance units).


D Co - immunoprecipitation of HEK293 cell lysates expressing myc - tagged gephyrin together with GFP - tagged gephyrin or G375D using myc - specific antibodies . Control IgG were used to show specificity of the assay .

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D Co-immunoprecipitation of HEK293 cell lysates expressing myc-tagged gephyrin together with GFP-tagged gephyrin or G375D using myc-specific antibodies. Control IgG were used to show specificity of the assay.


B Representative pulldown - experiment and quantification of 6His - tagged gephyrin and G375D using immobilized α3 - peptide . 6His - tagged gephyrin variants were expressed in E . coli cells , purified to homogeneity and incubated with α3 - peptides immobilized to NeutrAvidin beads . n = 3 , normalized to 6His - gephyrin . 38 . 9 ± 1 . 5 % G375D were pulled down .

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B Representative pulldown-experiment and quantification of 6His-tagged gephyrin and G375D using immobilized α3-peptide. 6His-tagged gephyrin variants were expressed in E. coli cells, purified to homogeneity and incubated with α3-peptides immobilized to NeutrAvidin beads. n = 3, normalized to 6His-gephyrin. 38.9±1.5% G375D were pulled down.


C Representative SPR sensograms of 6His - gephyrin and 6His - G375D binding to immobilized GABAAR α3 - peptide . Response units are plotted against time . Injected gephyrin concentrations were 0 . 5 , 2 , 10 , 25 , 40 µM .

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C Representative SPR sensograms of 6His-gephyrin and 6His-G375D binding to immobilized GABAAR α3-peptide. Response units are plotted against time. Injected gephyrin concentrations were 0.5, 2, 10, 25, 40 µM.


D SPR binding isotherms of a representative experiment as in C . Calculated KD values of 3 . 2 and 10 µM for gephyrin and G375D , respectively , are indicated by vertical lines . The experiment was repeated three times with two independently purified protein batches and two sensor chips .

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D SPR binding isotherms of a representative experiment as in C. Calculated KD values of 3.2 and 10 µM for gephyrin and G375D, respectively, are indicated by vertical lines. The experiment was repeated three times with two independently purified protein batches and two sensor chips.


E Representative ITC binding isotherms of the cytoplasmic loop of the GlyR ß - subunit ( GlyR - ßL , residue 378 - 426 ) and 6His - tagged gephyrin or G375D . The GlyR - ßL - gephyrin binding data ( black ) were fitted in a two - side model , whereas G375D binding could only be fitted to a single binding site . Dissociation constants KD and stoichiometry ( N = number of binding sites ) are given for each binding component . n = 5 from two independently purified protein batches . Results are expressed as mean ± SEM .

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E Representative ITC binding isotherms of the cytoplasmic loop of the GlyR ß-subunit (GlyR-ßL, residue 378-426) and 6His-tagged gephyrin or G375D. The GlyR-ßL-gephyrin binding data (black) were fitted in a two-side model, whereas G375D binding could only be fitted to a single binding site. Dissociation constants KD and stoichiometry (N = number of binding sites) are given for each binding component. n = 5 from two independently purified protein batches. Results are expressed as mean ± SEM.


A Biosynthesis pathway of Moco with all stable intermediates . Gephyrin G - and E - domain catalyze the last two steps as depicted . B In vitro Moco - synthesis assay using 150 pmol purified 6His - tagged gephyrins . D580A is an activity - deficient gephyrin mutation previously identified in Moco - deficient patients ( Reiss et al . , 2011 ) . Assays without the addition of gephyrin ( - gephyrin ) or molybdenum ( - Mo ) served as internal controls of the assay ( see also ( Belaidi Schwarz , 2013 ) ) . C Experiment as in B but with pre - incubation of 6His - gephyrin / G375D in a 1 : 1 ratio to simulate the heterozygous mutation . n = at least 3 with two independently purified protein batches . Results are expressed as mean ± SEM .

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 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 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

A Biosynthesis pathway of Moco with all stable intermediates. Gephyrin G- and E-domain catalyze the last two steps as depicted.B In vitro Moco-synthesis assay using 150 pmol purified 6His-tagged gephyrins. D580A is an activity-deficient gephyrin mutation previously identified in Moco-deficient patients (Reiss et al., 2011). Assays without the addition of gephyrin (-gephyrin) or molybdenum (-Mo) served as internal controls of the assay (see also (Belaidi Schwarz, 2013)).C Experiment as in B but with pre-incubation of 6His-gephyrin/G375D in a 1:1 ratio to simulate the heterozygous mutation. n = at least 3 with two independently purified protein batches. Results are expressed as mean ± SEM.


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

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


( A ) STK38 interacts with XPO1 . HekRasV12 cells were transiently transfected with myc - STK38 ( wt ) together with either Flag - XPO1 ( wt ) plasmid , Flag - control ( ctrl = Sirt3 ) plasmid or without DNA . 24h later , cells were incubated with Okadaic Acid ( OA ) , ( final concentration = 1µM ) for 1 hour or with DMSO . Flag fusions were pulled - down and co - immunoprecipited proteins were analyzed by western blotting ( WB ) . Upper panel displays whole cell lysates ( WCL ) and lower panel represents immunoprecipitated proteins .

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

(A) STK38 interacts with XPO1. HekRasV12 cells were transiently transfected with myc-STK38(wt) together with either Flag-XPO1(wt) plasmid, Flag-control (ctrl = Sirt3) plasmid or without DNA. 24h later, cells were incubated with Okadaic Acid (OA), (final concentration = 1µM) for 1 hour or with DMSO. Flag fusions were pulled-down and co-immunoprecipited proteins were analyzed by western blotting (WB). Upper panel displays whole cell lysates (WCL) and lower panel represents immunoprecipitated proteins.


STK38 accumulates in the cytoplasm upon nutrient starvation in a XPO1 - dependant manner . ( B ) HeLa cells were transfected with myc - STK38 ( wt ) plasmid . The next day , cells were incubated with DMEM or EBSS in the presence of XPO1 inhibitors KPT - 185 or KPT - 330 as indicated ( final concentration = 1 µM ) or DMSO for 4 hours . Cells were then fixed and stained for myc - tag . Representative images are shown and scale bars are 40 µm .

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STK38 accumulates in the cytoplasm upon nutrient starvation in a XPO1-dependant manner. (B) HeLa cells were transfected with myc-STK38(wt) plasmid. The next day, cells were incubated with DMEM or EBSS in the presence of XPO1 inhibitors KPT-185 or KPT-330 as indicated (final concentration = 1 µM) or DMSO for 4 hours. Cells were then fixed and stained for myc-tag. Representative images are shown and scale bars are 40 µm.


STK38 accumulates in the cytoplasm upon nutrient starvation in a XPO1 - dependant manner . ( C ) Quantification of myc - STK38 ( wt ) nuclear / cytoplasmic staining ( n > 30 cells from 3 independent experiments , mean ± SEM ; * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 , Mann - Whitney test ) .

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STK38 accumulates in the cytoplasm upon nutrient starvation in a XPO1-dependant manner. (C) Quantification of myc-STK38(wt) nuclear/cytoplasmic staining (n > 30 cells from 3 independent experiments, mean ± SEM; *p<0.05, **p<0.01, ***p<0.001, Mann-Whitney test).


XPO1 activity is required for nutrient starvation - induced autophagy . ( D ) Immunoblotting with indicated antibodies of whole cell lysates of cells and its graphical representation ( n = 3 independent experiments , mean ± SEM ; * * * p < 0 . 001 , ns , not significant , Student ' s t test ) . As expected , p62 degradation in starvation conditions was inhibited in presence of XPO1 inhibitors .

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XPO1 activity is required for nutrient starvation-induced autophagy. (D) Immunoblotting with indicated antibodies of whole cell lysates of cells and its graphical representation (n = 3 independent experiments, mean ± SEM; ***p<0.001, ns, not significant, Student's t test). As expected, p62 degradation in starvation conditions was inhibited in presence of XPO1 inhibitors.


XPO1 activity is required for nutrient starvation - induced autophagy . ( E ) HeLa cells stably expressing the GFP - LC3 - RFP - LC3ΔG reporter autophagic probe [ 26 ] were incubated with DMEM or EBSS in the presence of XPO1 inhibitors KPT - 185 or KPT - 330 ( final concentration = 1 µM ) or DMSO for 4 hours . The GFP - LC3 ( degraded upon autophagy ) and RFP - LC3ΔG ( non - degraded upon autophagy ) signals were recorded by FACS analysis and then shown as a ratio recapitulating overall LC3 level ( n = 3 independent experiments , mean ± SEM ; * p < 0 . 05 , * * p < 0 . 01 , ns , not significant , Mann - Whitney test ) . Here again , incubation with XPO1 inhibitors significantly impaired the autophagy

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

XPO1 activity is required for nutrient starvation-induced autophagy. (E) HeLa cells stably expressing the GFP-LC3-RFP-LC3ΔG reporter autophagic probe [26] were incubated with DMEM or EBSS in the presence of XPO1 inhibitors KPT-185 or KPT-330 (final concentration = 1 µM) or DMSO for 4 hours. The GFP-LC3 (degraded upon autophagy) and RFP-LC3ΔG (non-degraded upon autophagy) signals were recorded by FACS analysis and then shown as a ratio recapitulating overall LC3 level (n=3 independent experiments, mean ± SEM; *p<0.05, **p<0.01, ns, not significant, Mann-Whitney test). Here again, incubation with XPO1 inhibitors significantly impaired the autophagy process


STK38 kinase activity is required and sufficient for its cytoplasmic accumulation upon nutrient starvation . ( A ) HeLa cells silenced for endogenous STK38 were transfected with myc - STK38 ( wt ) expressing plasmid , myc - STK38 ( K118R ) ( STK38 kinase - dead version ) plasmid or with HA - STK38 ( PIF ) ( STK38 constitutively active version ) plasmid . 24 hours later , cells were incubated with DMEM or EBSS for 4 hours , fixed and stained for myc - tag or HA - tag . Representative images are shown and scale bars are 40 µm .

STK38 kinase activity is required and sufficient for its cytoplasmic accumulation upon nutrient starvation. (A) HeLa cells silenced for endogenous STK38 were transfected with myc-STK38(wt) expressing plasmid, myc-STK38(K118R) (STK38 kinase-dead version) plasmid or with HA-STK38(PIF) (STK38 constitutively active version) plasmid. 24 hours later, cells were incubated with DMEM or EBSS for 4 hours, fixed and stained for myc-tag or HA-tag. Representative images are shown and scale bars are 40 µm.


STK38 kinase activity is required and sufficient for its cytoplasmic accumulation upon nutrient starvation . ( B ) Graphical representation of tag - STK38 variants nuclear staining / cytoplasmic staining ( n > 30 cells from 3 independent experiments , mean ± SEM ; * * p < 0 . 01 , * * * p < 0 . 001 , ns , not significant , Mann - Whitney test ) .

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STK38 kinase activity is required and sufficient for its cytoplasmic accumulation upon nutrient starvation. (B) Graphical representation of tag-STK38 variants nuclear staining/cytoplasmic staining (n > 30 cells from 3 independent experiments, mean ± SEM; **p<0.01, ***p<0.001, ns, not significant, Mann-Whitney test).


STK38 kinase activity is required and sufficient for nutrient - starvation induced autophagy . ( C ) HeLa cells stably expressing the GFP - LC3 - RFP - LC3ΔG autophagic probe [ 26 ] were transiently transfected with siRNA targeting the 3 ' UTR region of endogenous STK38 ( or with non - targeting siRNA ( siNT ) ) . The next day , cells were transiently transfected with the indicated STK38 mutants expressing plasmids 24 hours after plasmid transfection , cells were incubated with DMEM or EBSS for 4 hours . The GFP - LC3 ( degraded upon autophagy ) and RFP - LC3ΔG ( non - degraded upon autophagy ) signals were recorded by FACS analysis and then shown as a ratio recapitulating overall LC3 level ( n = 4 independent experiments , mean ± SEM ; * p < 0 . 05 , * * * p < 0 . 001 , ns , not significant , Mann - Whitney test ) . depleting STK38 prevents autophagy to take place . This effect was partially reversed by expressing the wt ORF as well as constitutively active STK38 whereas kinase - dead version failed to reproduce endogenous STK38 effect . Expression of constitutively active STK38 is sufficient to induce a substantial change in the autophagic flux upon nutrient rich

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

STK38 kinase activity is required and sufficient for nutrient-starvation induced autophagy. (C) HeLa cells stably expressing the GFP-LC3-RFP-LC3ΔG autophagic probe [26] were transiently transfected with siRNA targeting the 3'UTR region of endogenous STK38 (or with non-targeting siRNA (siNT)). The next day, cells were transiently transfected with the indicated STK38 mutants expressing plasmids 24 hours after plasmid transfection, cells were incubated with DMEM or EBSS for 4 hours. The GFP-LC3 (degraded upon autophagy) and RFP-LC3ΔG (non-degraded upon autophagy) signals were recorded by FACS analysis and then shown as a ratio recapitulating overall LC3 level (n=4 independent experiments, mean ± SEM; *p<0.05, ***p<0.001, ns, not significant, Mann-Whitney test). depleting STK38 prevents autophagy to take place. This effect was partially reversed by expressing the wt ORF as well as constitutively active STK38 whereas kinase-dead version failed to reproduce endogenous STK38 effect. Expression of constitutively active STK38 is sufficient to induce a substantial change in the autophagic flux upon nutrient rich conditions


STK38 kinase activity is required and sufficient for nutrient - starvation induced autophagy . ( D ) HeLa cells were transiently transfected with siRNA targeting the 3 ' UTR region of endogenous STK38 ( or with non - targeting siRNA ( siNT ) ) and then transiently transfected with the indicated STK38 mutants expressing plasmids 48h after . The next day , the cells were incubated with DMEM or EBSS for 4 hours and overall p62 level was quantified for autophagy estimation ( n = 3 independent experiments , mean ± SEM ; * * * p < 0 . 001 , ns , not significant , Student ' s t test ) .

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STK38 kinase activity is required and sufficient for nutrient-starvation induced autophagy. (D) HeLa cells were transiently transfected with siRNA targeting the 3'UTR region of endogenous STK38 (or with non-targeting siRNA (siNT)) and then transiently transfected with the indicated STK38 mutants expressing plasmids 48h after. The next day, the cells were incubated with DMEM or EBSS for 4 hours and overall p62 level was quantified for autophagy estimation (n = 3 independent experiments, mean ± SEM; ***p<0.001, ns, not significant, Student's t test).


( A - B ) STK38 is required for XPO1 _ S1055 phosphorylation . ( A ) HeLa cells were transiently transfected with the indicated siRNA and subjected to Flag - XPO1 ( wt ) transient transfection the following day . 48 hours later , cells were incubated with Okadaic Acid ( OA , final concentration = 1 µM ) for 1 hour or with DMSO . Immunoblotting was performed on whole cell lysates with indicated antibodies . ( B ) Graphical representation of the phospho S1055 - XPO1 signal on total XPO1 ( n = 3 independent experiments , mean ± SEM ; * p < 0 . 05 , ns , not significant , Mann - Whitney test ) . As expected , XPO1 is phosphorylated on its Ser1055 upon OA treatment but not when STK38 is

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

(A-B) STK38 is required for XPO1_S1055 phosphorylation. (A) HeLa cells were transiently transfected with the indicated siRNA and subjected to Flag-XPO1(wt) transient transfection the following day. 48 hours later, cells were incubated with Okadaic Acid (OA, final concentration = 1 µM) for 1 hour or with DMSO. Immunoblotting was performed on whole cell lysates with indicated antibodies. (B) Graphical representation of the phospho S1055-XPO1 signal on total XPO1 (n = 3 independent experiments, mean ± SEM; *p<0.05, ns, not significant, Mann-Whitney test). As expected, XPO1 is phosphorylated on its Ser1055 upon OA treatment but not when STK38 is silenced


( C ) STK38 kinase activity is required for XPO1 _ S1055 phosphorylation . HekRasV12 cells silenced ( or not ) for endogenous STK38 were transfected with Flag - XPO1 ( wt ) expression plasmid in addition to myc - STK38 ( wt ) or myc - STK38 ( K118R ) ( STK38 kinase - dead version ) plasmids . The next day , cells were treated with 1 µM OA or with vehicle ( DMSO ) for 1 hour . Flag fusions were immunoprecipited and pulled - down proteins were analyzed by western blotting . Upper panel displays immunoprecipited proteins and lower panel represents whole cell lysates .

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

(C) STK38 kinase activity is required for XPO1_S1055 phosphorylation. HekRasV12 cells silenced (or not) for endogenous STK38 were transfected with Flag-XPO1(wt) expression plasmid in addition to myc-STK38(wt) or myc-STK38(K118R) (STK38 kinase-dead version) plasmids. The next day, cells were treated with 1 µM OA or with vehicle (DMSO) for 1 hour. Flag fusions were immunoprecipited and pulled-down proteins were analyzed by western blotting. Upper panel displays immunoprecipited proteins and lower panel represents whole cell lysates.


( A ) Graphical representation of XPO1 protein . C528 to S amino acid mutation confers XPO1 resistance to both KPT - 185 and KPT - 330 chemical inhibitors [ 24 ] . S1055 amino acid correspond to the phosphorylation target of the STK38 . Finally , the ΔCter region is also highlighted corresponding to a XPO1 construct lacking the 39 C - terminal residues ( B - C ) XPO1 _ S1055 phosphorylation is required and sufficient for STK38 nuclear exit upon nutrient starvation . ( B ) HeLa cells were transiently transfected with myc - STK38 ( wt ) in addition to indicated Flag - XPO1 mutant plasmids . 24 hours later , cells were incubated with DMEM or EBSS for 4 hours both supplemented with KPT - 185 ( final concentration = 1 µM ) in order to inhibit endogenous XPO1 activity . Cells were then fixed and stained for Flag and myc tags . Only cells positives for both Flag - XPO1 and myc - STK38 ( wt ) were captured . Representative images are shown and scale bars are 40 µm . ( C ) Graphical representation of myc - STK38 ( wt ) nuclear staining / cytoplasmic staining ( n > 30 cells from 3 independent experiments , mean ± SEM ; * * * p < 0 . 001 , ns , not significant , Mann - Whitney test ) . Expression of wt XPO1 failed to induce cytoplasmic localization of STK38 upon nutrient starvation in presence of KPT - 185 where C528S mutant recapitulates results described in Figure 2B - C . Expression of phosphonegative XPO1 ( S1055A ) failed to induces STK38 cytoplasmic localization upon EBSS treatment while phosphomimetics XPO1 ( S1055D and S1055E ) were sufficient to promote STK38 cytoplasmic localization without autophagic stimuli . Finally , expression of XPO1 lacking in its 39 C - terminal residues mimicked phosphomimetics variants .

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 O O O O O O O O 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Graphical representation of XPO1 protein. C528 to S amino acid mutation confers XPO1 resistance to both KPT-185 and KPT-330 chemical inhibitors [24]. S1055 amino acid correspond to the phosphorylation target of the STK38. Finally, the ΔCter region is also highlighted corresponding to a XPO1 construct lacking the 39 C-terminal residues (B-C) XPO1_S1055 phosphorylation is required and sufficient for STK38 nuclear exit upon nutrient starvation. (B) HeLa cells were transiently transfected with myc-STK38(wt) in addition to indicated Flag-XPO1 mutant plasmids. 24 hours later, cells were incubated with DMEM or EBSS for 4 hours both supplemented with KPT-185 (final concentration = 1 µM) in order to inhibit endogenous XPO1 activity. Cells were then fixed and stained for Flag and myc tags. Only cells positives for both Flag-XPO1 and myc-STK38(wt) were captured. Representative images are shown and scale bars are 40 µm. (C) Graphical representation of myc-STK38(wt) nuclear staining/cytoplasmic staining (n > 30 cells from 3 independent experiments, mean ± SEM; ***p<0.001, ns, not significant, Mann-Whitney test). Expression of wt XPO1 failed to induce cytoplasmic localization of STK38 upon nutrient starvation in presence of KPT-185 where C528S mutant recapitulates results described in Figure 2B-C. Expression of phosphonegative XPO1 (S1055A) failed to induces STK38 cytoplasmic localization upon EBSS treatment while phosphomimetics XPO1 (S1055D and S1055E) were sufficient to promote STK38 cytoplasmic localization without autophagic stimuli. Finally, expression of XPO1 lacking in its 39 C-terminal residues mimicked phosphomimetics variants.


( D ) HAP1 cells carrying genomic XPO1 mutations of S1055 were subjected for IMDM ( nutrient rich medium ) or EBSS ( starvation ) incubation for 4 hours followed by western - blot analysis for p62 level measurement . This result indicates that p62 degradation in starvation conditions was inhibited in XPO1 _ S1055A HAP1 cells whereas p62 degradation was potentiated in both phosphomimetics variant ( S1055D & S1055E ) ( n = 4 independent experiments , mean ± SEM ; * p < 0 . 05 , ns , not significant , Mann - Whitney

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(D) HAP1 cells carrying genomic XPO1 mutations of S1055 were subjected for IMDM (nutrient rich medium) or EBSS (starvation) incubation for 4 hours followed by western-blot analysis for p62 level measurement. This result indicates that p62 degradation in starvation conditions was inhibited in XPO1_S1055A HAP1 cells whereas p62 degradation was potentiated in both phosphomimetics variant (S1055D & S1055E) (n = 4 independent experiments, mean ± SEM; *p<0.05, ns, not significant, Mann-Whitney test


( E ) Hek - HT - iRFP - LC3 cells were transiently transfected with siRNA targeting endogenous STK38 ( or with non - targeting siRNA ( siNT ) ) . Two days after , cells were transiently transfected with indicated Flag - XPO1 mutant plasmids . 24 hours later , cells were incubated with DMEM or EBSS for 4 hours both supplemented with KPT - 185 ( final concentration = 1 µM ) in order to inhibit endogenous XPO1 activity and with chloroquine ( final concentration = 10 µM ) . Cells were then fixed and the number of iRFP - LC3 dots per cell was recorded only in cells positive for Flag - XPO1 mutants As expected , silencing of endogenous STK38 prevented the formation of LC3 dots upon starvation where introduction of phosphonegative XPO1 ( S1055A ) failed also to increase LC3 dots upon starvation . Interestingly , phosphomimetic XPO1 ( S1055D ) was sufficient to increase the number of LC3 dots even in complete and also STK38 - depleted conditions .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Hek-HT-iRFP-LC3 cells were transiently transfected with siRNA targeting endogenous STK38 (or with non-targeting siRNA (siNT)). Two days after, cells were transiently transfected with indicated Flag-XPO1 mutant plasmids. 24 hours later, cells were incubated with DMEM or EBSS for 4 hours both supplemented with KPT-185 (final concentration = 1 µM) in order to inhibit endogenous XPO1 activity and with chloroquine (final concentration = 10 µM). Cells were then fixed and the number of iRFP-LC3 dots per cell was recorded only in cells positive for Flag-XPO1 mutants As expected, silencing of endogenous STK38 prevented the formation of LC3 dots upon starvation where introduction of phosphonegative XPO1 (S1055A) failed also to increase LC3 dots upon starvation. Interestingly, phosphomimetic XPO1 (S1055D) was sufficient to increase the number of LC3 dots even in complete and also STK38-depleted conditions.


( A ) XPO1 and STK38 are required for Beclin1 cytoplasmic accumulation upon nutrient starvation - induced autophagy . HeLa cells were transiently transfected with the indicated siRNA ( control and KPT conditions = siNT ) . 72 hours later , cells were incubated with XPO1 inhibitors KPT - 185 or KPT - 330 as indicated ( final concentration = 1 µM ) or with DMSO for all other conditions for 2 hours prior incubation with DMEM or EBSS supplemented ( or not ) with inhibitors for 2 hours . Cells were fixed and stained for endogenous Beclin1 . Graphical representation of endogenous Beclin1 nuclear staining / cytoplasmic staining ( n > 30 cells from 3 independent experiments , mean ± SEM ; * p < 0 . 05 , * * * p < 0 . 001 , ns , not significant , Mann - Whitney test ) . XPO1 activity inhibition by both KPT - 185 and KPT - 330 as well as STK38 silencing failed to induce Beclin1 cytoplasmic accumulation upon nutrient

O O O O O O O O O O O O O O O O O O O O O O O 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 O O O O O O O O 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 Beclin1 cytoplasmic accumulation upon nutrient starvation-induced autophagy. HeLa cells were transiently transfected with the indicated siRNA (control and KPT conditions = siNT). 72 hours later, cells were incubated with XPO1 inhibitors KPT-185 or KPT-330 as indicated (final concentration = 1 µM) or with DMSO for all other conditions for 2 hours prior incubation with DMEM or EBSS supplemented (or not) with inhibitors for 2 hours. Cells were fixed and stained for endogenous Beclin1. Graphical representation of endogenous Beclin1 nuclear staining/cytoplasmic staining (n > 30 cells from 3 independent experiments, mean ± SEM; *p<0.05, ***p<0.001, ns, not significant, Mann-Whitney test). XPO1 activity inhibition by both KPT-185 and KPT-330 as well as STK38 silencing failed to induce Beclin1 cytoplasmic accumulation upon nutrient starvation


( B ) XPO1 _ S1055 phosphorylation is required and sufficient for Beclin1 nuclear exit upon nutrient starvation . Genome edited XPO1 mutant HAP1 cells were incubated with IMDM ( complete ) or EBSS ( starvation ) for 2 hours . Cells were then fixed and stained for endogenous Beclin1 . Graphical representation of endogenous Beclin1 nuclear staining / cytoplasmic staining ( n > 30 cells from 3 independent experiments , mean ± SEM ; * * * p < 0 . 001 , ns , not significant , Mann - Whitney test ) . Here , phosphonegative XPO1 failed to induce Beclin1 cytoplasmic accumulation upon nutrient starvation whereas phosphomimetics variant ( S1055D & S1055E ) potentiated Beclin1 cytoplasmic

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