Figure 4A Cells depleted of endogenous TFG were transfected with a variety of TFG transgenes ( including full ‐ length , untagged TFG , a truncation mutant encoding amino acids 1 ‐ 300 of untagged TFG , and a truncation mutant encoding amino acids 1 ‐ 350 of untagged TFG ) and stained using α ‐ TFG and α ‐ Sec31A antibodies . Scale bar , 5 μm . Inset scale bar , 1 μm . Images shown are representative of at least 15 individual cells analyzed for each condition . B A recombinant polyhistidine ‐ and SUMO ‐ tagged TFG fragment ( Sumo ‐ TFGc , amino acids 194 ‐ 400 ; total molecular mass of 42 kDa ) was expressed and purified from Escherichia coli extracts using nickel affinity resin . Coomassie ‐ stained gels of the peak elution fractions after separation of the recombinant protein on a S200 gel filtration column ( top ) or a 10 - 30 % glycerol gradient ( bottom ) are shown . To determine the native molecular weight of the protein , the following equation was used : M = 6πηNas / ( 1 − υρ ) , where M is the native molecular weight , η is the viscosity of the medium , N is Avogadro ' s number , a is the Stokes radius , s is the sedimentation value , υ is the partial specific volume , and ρ is the density of the medium ( Siegel Monty , ) . These data suggest that this fragment of TFG is capable of forming dimers in solution . C , D Control cells ( C ) or cells depleted of endogenous TFG ( D ) were transfected with a construct expressing TFG with a carboxyl ‐ terminal GFP tag and stained using α ‐ Sec31A antibodies . Scale bar , 5 μm . Images shown are representative of at least 20 individual cells analyzed for each condition .

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

Figure 4A Cells depleted of endogenous TFG were transfected with a variety of TFG transgenes (including full‐length, untagged TFG, a truncation mutant encoding amino acids 1‐300 of untagged TFG, and a truncation mutant encoding amino acids 1‐350 of untagged TFG) and stained using α‐TFG and α‐Sec31A antibodies. Scale bar, 5 μm. Inset scale bar, 1 μm. Images shown are representative of at least 15 individual cells analyzed for each condition.B A recombinant polyhistidine‐ and SUMO‐tagged TFG fragment (Sumo‐TFGc, amino acids 194‐400; total molecular mass of 42 kDa) was expressed and purified from Escherichia coli extracts using nickel affinity resin. Coomassie‐stained gels of the peak elution fractions after separation of the recombinant protein on a S200 gel filtration column (top) or a 10-30% glycerol gradient (bottom) are shown. To determine the native molecular weight of the protein, the following equation was used: M = 6πηNas/(1 − υρ), where M is the native molecular weight, η is the viscosity of the medium, N is Avogadro's number, a is the Stokes radius, s is the sedimentation value, υ is the partial specific volume, and ρ is the density of the medium (Siegel Monty, ). These data suggest that this fragment of TFG is capable of forming dimers in solution.C, D Control cells (C) or cells depleted of endogenous TFG (D) were transfected with a construct expressing TFG with a carboxyl‐terminal GFP tag and stained using α‐Sec31A antibodies. Scale bar, 5 μm. Images shown are representative of at least 20 individual cells analyzed for each condition.


Figure 5A HumanRPE ‐ 1 cells were mock ‐ transfected ( control ) or transfected with a TFG siRNA for 60 h . Cells were fixed and stained using α ‐ Sec16A and α ‐ Sec31A antibodies and imaged using confocal microscopy . Images shown are projections of 3D data sets ( 4 μm in z ) . Both individual and merged images with Sec16A in green and Sec31A in red are shown ( at least 15 cells for each condition ) . Scale bar , 5 μm . Higher magnification views of the indicated regions ( boxed ) are also shown in the lower or upper right portion of each image . Inset scale bar , 1 μm . B Quantification of the number of Sec31A ‐ labeled structures in a 4 μm2 region . For both conditions ( control or TFG siRNA ‐ treated ) , at least 50 distinct regions away from the peri ‐ nuclear Golgi were examined . Error bars represent mean ± SEM ; at least 15 different cells per condition . * * P 0 . 01 compared with control , calculated using a paired t ‐ test . C Quantification of the number of Sec16A ‐ labeled structures in a 4 μm2 region . For both conditions ( control or TFG siRNA ‐ treated ) , at least 50 distinct regions away from the peri ‐ nuclear Golgi were examined . Error bars represent mean ± SEM ; at least 15 different cells per condition . No statistically significant difference was observed , following analysis using a paired t ‐ test . D Cells stably expressing low levels of GFP ‐ ERGIC ‐ 53 and mRuby ‐ Sec23A were fixed and imaged using SR ‐ SIM ( top ) , or depleted of endogenous TFG prior to fixation and SR ‐ SIM imaging ( bottom ) . Images shown are representative of at least 15 individual cells analyzed for each condition . Scale bar , 5 μm . Inset scale bar , 1 μm . E HumanRPE ‐ 1 cells were mock ‐ transfected ( control ) or transfected with a TFG siRNA for 60 h . Cells were fixed and stained using α ‐ Sec16A and α ‐ ERGIC ‐ 53 antibodies and imaged using SR ‐ SIM . Images shown are projections of 3D data sets ( 4 μm in z ) . Both individual and merged images with Sec16A in green and ERGIC ‐ 53 in red are shown ( at least 15 cells for each condition ) . Scale bar , 5 μm . Higher magnification views of the indicated regions ( boxed ) are also shown in the lower or upper right portion of each image . Arrowheads highlight Sec16A ‐ labeled structures that do not exhibit juxtaposed ERGIC ‐ 53 staining . Inset scale bar , 1 μm . F Based on structured illumination microscopy , quantification of the number of Sec16A ‐ labeled structures juxtaposed ( within 500 nm ) to ERGIC ‐ 53 ‐ labeled structures is shown . For both conditions ( control or TFG siRNA ‐ treated ) , at least 1 , 000 unique Sec16A ‐ labeled structures were examined . Error bars represent mean ± SEM ; at least 15 different cells per condition . * * P 0 . 01 compared with control , calculated using a paired t ‐ test .

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

Figure 5A HumanRPE‐1 cells were mock‐transfected (control) or transfected with a TFG siRNA for 60 h. Cells were fixed and stained using α‐Sec16A and α‐Sec31A antibodies and imaged using confocal microscopy. Images shown are projections of 3D data sets (4 μm in z). Both individual and merged images with Sec16A in green and Sec31A in red are shown (at least 15 cells for each condition). Scale bar, 5 μm. Higher magnification views of the indicated regions (boxed) are also shown in the lower or upper right portion of each image. Inset scale bar, 1 μm.B Quantification of the number of Sec31A‐labeled structures in a 4 μm2 region. For both conditions (control or TFG siRNA‐treated), at least 50 distinct regions away from the peri‐nuclear Golgi were examined. Error bars represent mean ± SEM; at least 15 different cells per condition. **P 0.01 compared with control, calculated using a paired t‐test.C Quantification of the number of Sec16A‐labeled structures in a 4 μm2 region. For both conditions (control or TFG siRNA‐treated), at least 50 distinct regions away from the peri‐nuclear Golgi were examined. Error bars represent mean ± SEM; at least 15 different cells per condition. No statistically significant difference was observed, following analysis using a paired t‐test.D Cells stably expressing low levels of GFP‐ERGIC‐53 and mRuby‐Sec23A were fixed and imaged using SR‐SIM (top), or depleted of endogenous TFG prior to fixation and SR‐SIM imaging (bottom). Images shown are representative of at least 15 individual cells analyzed for each condition. Scale bar, 5 μm. Inset scale bar, 1 μm.E HumanRPE‐1 cells were mock‐transfected (control) or transfected with a TFG siRNA for 60 h. Cells were fixed and stained using α‐Sec16A and α‐ERGIC‐53 antibodies and imaged using SR‐SIM. Images shown are projections of 3D data sets (4 μm in z). Both individual and merged images with Sec16A in green and ERGIC‐53 in red are shown (at least 15 cells for each condition). Scale bar, 5 μm. Higher magnification views of the indicated regions (boxed) are also shown in the lower or upper right portion of each image. Arrowheads highlight Sec16A‐labeled structures that do not exhibit juxtaposed ERGIC‐53 staining. Inset scale bar, 1 μm.F Based on structured illumination microscopy, quantification of the number of Sec16A‐labeled structures juxtaposed (within 500 nm) to ERGIC‐53‐labeled structures is shown. For both conditions (control or TFG siRNA‐treated), at least 1,000 unique Sec16A‐labeled structures were examined. Error bars represent mean ± SEM; at least 15 different cells per condition. **P 0.01 compared with control, calculated using a paired t‐test.


Figure 6A HumanRPE ‐ 1 cells stably expressing low levels of mannosidase II ‐ mApple ( ManII ‐ mApple ) were mock ‐ transfected ( control ) or transfected with a TFG siRNA for 60 h , then fixed and stained using TFG antibodies and imaged using confocal microscopy . Alternatively , cells were either treated with DMSO ( top row ) or brefeldin A ( BFA ) for 1 h ( middle row ) , followed by fixation , or washed into fresh media following DMSO or BFA treatment for 30 min ( bottom row ) , prior to fixation . Images shown are projections of 3D data sets ( 4 μm in z ) . Merged images with TFG in green and ManII in red are shown . Scale bar , 5 μm . Images shown are representative of at least 10 individual cells analyzed for each condition . B Human RPE ‐ 1 cells were mock ‐ transfected ( control ) or transfected with a TFG siRNA for 60 h , then fixed and stained using TFG and GM130 antibodies and imaged using confocal microscopy . Alternatively , cells were either treated with DMSO ( top row ) or brefeldin A ( BFA ) for 1 h ( middle row ) , followed by fixation , or washed into fresh media following DMSO or BFA treatment for 30 min ( bottom row ) , prior to fixation . Images shown are projections of 3D data sets ( 4 μm in z ) . Merged images with TFG in green and GM130 in red are shown . Scale bar , 5 μm . Images shown are representative of at least 10 individual cells analyzed for each condition . C Higher magnification views of the indicated regions in ( B , boxed ) are shown ( GM130 distribution only ) . Scale bar , 2 μm .

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

Figure 6A HumanRPE‐1 cells stably expressing low levels of mannosidase II‐mApple (ManII‐mApple) were mock‐transfected (control) or transfected with a TFG siRNA for 60 h, then fixed and stained using TFG antibodies and imaged using confocal microscopy. Alternatively, cells were either treated with DMSO (top row) or brefeldin A (BFA) for 1 h (middle row), followed by fixation, or washed into fresh media following DMSO or BFA treatment for 30 min (bottom row), prior to fixation. Images shown are projections of 3D data sets (4 μm in z). Merged images with TFG in green and ManII in red are shown. Scale bar, 5 μm. Images shown are representative of at least 10 individual cells analyzed for each condition.B Human RPE‐1 cells were mock‐transfected (control) or transfected with a TFG siRNA for 60 h, then fixed and stained using TFG and GM130 antibodies and imaged using confocal microscopy. Alternatively, cells were either treated with DMSO (top row) or brefeldin A (BFA) for 1 h (middle row), followed by fixation, or washed into fresh media following DMSO or BFA treatment for 30 min (bottom row), prior to fixation. Images shown are projections of 3D data sets (4 μm in z). Merged images with TFG in green and GM130 in red are shown. Scale bar, 5 μm. Images shown are representative of at least 10 individual cells analyzed for each condition.C Higher magnification views of the indicated regions in (B, boxed) are shown (GM130 distribution only). Scale bar, 2 μm.


Figure 7A HumanRPE ‐ 1 cells stably expressing low levels of mannosidase II ‐ mApple were transfected with a TFG siRNA for 60 h . Cells were subsequently treated with brefeldin A ( BFA ) for 1 h , followed by a wash into fresh media and further incubation for 2 h in the absence of BFA , prior to fixation . Images shown are projections of 3D data sets ( 4 μm in z ) . Merged images with Sec31A in green and ManII in red are shown ( representative of at least 15 cells analyzed ) . Scale bar , 5 μm . B HumanRPE ‐ 1 cells stably expressing low levels of mannosidase II ‐ mApple were transfected with a TFG siRNA for 60 h . Cells were subsequently treated with BFA for 1 h , followed by a wash into fresh media and further incubation for 1 h in the absence of BFA , prior to fixation and staining using α ‐ Sec16A and α ‐ Sec31A antibodies . Images shown are projections of 3D data sets ( 4 μm in z ) . Merged images with Sec31A ( green ) , ManII ( red ) , and Sec16A ( blue ) are shown ( representative of at least 15 cells analyzed ) . Scale bar , 5 μm . C Higher magnification views of the indicated regions in ( B , boxed ) are shown . Arrows highlight COPII ‐ positive transport carriers that contain the cargo ManII , which are not juxtaposed to Sec16A ‐ labeled sites on the ER . Additionally , arrowheads point out distinct foci in which COPII continues to associate with Sec16A ‐ labeled sites , indicating that COPII vesicle formation continues in the absence of TFG . Scale bar , 1 μm .

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

Figure 7A HumanRPE‐1 cells stably expressing low levels of mannosidase II‐mApple were transfected with a TFG siRNA for 60 h. Cells were subsequently treated with brefeldin A (BFA) for 1 h, followed by a wash into fresh media and further incubation for 2 h in the absence of BFA, prior to fixation. Images shown are projections of 3D data sets (4 μm in z). Merged images with Sec31A in green and ManII in red are shown (representative of at least 15 cells analyzed). Scale bar, 5 μm.B HumanRPE‐1 cells stably expressing low levels of mannosidase II‐mApple were transfected with a TFG siRNA for 60 h. Cells were subsequently treated with BFA for 1 h, followed by a wash into fresh media and further incubation for 1 h in the absence of BFA, prior to fixation and staining using α‐Sec16A and α‐Sec31A antibodies. Images shown are projections of 3D data sets (4 μm in z). Merged images with Sec31A (green), ManII (red), and Sec16A (blue) are shown (representative of at least 15 cells analyzed). Scale bar, 5 μm.C Higher magnification views of the indicated regions in (B, boxed) are shown. Arrows highlight COPII‐positive transport carriers that contain the cargo ManII, which are not juxtaposed to Sec16A‐labeled sites on the ER. Additionally, arrowheads point out distinct foci in which COPII continues to associate with Sec16A‐labeled sites, indicating that COPII vesicle formation continues in the absence of TFG. Scale bar, 1 μm.


Figure 2Jurkat T cells were treated with either DMSO ( vehicle ) or with 100μM of Dieldrin for the indicated times . Abundance of mRNA for the indicated genes or HERVs was assessed by RT - qPCR . Jurkat T cells were treated with either DMSO ( vehicle ) or with 100μM of PMA for the indicated times . Abundance of mRNA for the indicated genes or HERVs was assessed by RT - qPCR . Jurkat T cells were treated with either DMSO ( vehicle ) or with 100μM of either Dieldrin or PMA for the indicated times . Abundance of mRNA for the indicated genes by RT - qPCR . Jurkat cells were exposed to either DMSO , PMA in DMSO , or dieldrin in DMSO for 30 min in triplicates . RNA - seq was then performed on cDNA libraries prepared with poly ( A ) selection : principal component analysis on the 485 genes the most affected by DMSO , PMA , or Dieldrin .

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Figure 2Jurkat T cells were treated with either DMSO (vehicle) or with 100μM of Dieldrin for the indicated times. Abundance of mRNA for the indicated genes or HERVs was assessed by RT-qPCR.Jurkat T cells were treated with either DMSO (vehicle) or with 100μM of PMA for the indicated times. Abundance of mRNA for the indicated genes or HERVs was assessed by RT-qPCR.Jurkat T cells were treated with either DMSO (vehicle) or with 100μM of either Dieldrin or PMA for the indicated times. Abundance of mRNA for the indicated genes by RT-qPCR.Jurkat cells were exposed to either DMSO, PMA in DMSO, or dieldrin in DMSO for 30 min in triplicates. RNA-seq was then performed on cDNA libraries prepared with poly(A) selection: principal component analysis on the 485 genes the most affected by DMSO, PMA, or Dieldrin.


Figure 3Jurkat cells were exposed to either DMSO , or dieldrin in DMSO for 30 min . RNA - seq was then performed on cDNA libraries prepared after depletion of ribosomal RNAs . Screen captures from IGV showing an example of a HERV becoming a site of divergent transcription upon exposure of the Jurkat cells to dieldrin . Top tracks show coverage from sequencing of poly ( A ) - enriched libraries for comparison . All coverage tracks are at the same scale and all the sequencing result files are of comparable size . Blue and red reads ( rds ) are in opposite orientation . CAGE peaks use the same color code . Reads mapping inside a series of 4686 HERVs located away ( 30Kb ) from protein - coding genes were quantified both in the poly ( A ) - enriched and the ribo - depleted data . Read counts from each replicate were then summed and plotted . Jurkat cells were exposed to either DMSO , or dieldrin in DMSO for 30 min . RNA - seq was then performed on cDNA libraries prepared after depletion of ribosomal RNAs . Distribution of regions annotated " LTR " in RepeatMasker relative to sites of divergent transcription in Jurkat cells treated with either DMSO or dieldrin in DMSO . Jurkat cells were exposed to either DMSO , or dieldrin in DMSO for 30 min . RNA - seq was then performed on cDNA libraries prepared after depletion of ribosomal RNAs . Jurkat T cells were treated with either DMSO ( vehicle ) or 100μM dieldrin for the indicated times . Abundance of IL2 mRNA was assessed by RT - qPCR . Data shown are means + / - SEM from four independent experiments . Significance ( p - value ) was estimated using the two - sided student t test appropriate for small sample numbers . * * * : p - value < 0 . 001 . Jurkat cells were exposed to either DMSO , or dieldrin in DMSO for 30 min . RNA - seq was then performed on cDNA libraries prepared after depletion of ribosomal RNAs . Screen captures from IGV showing increased divergent transcription inside a HERV sequence upon exposure of the Jurkat cells to dieldrin in the intergenic region between MUC21 and MUC22 . Color code as in ( A ) .

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

Figure 3Jurkat cells were exposed to either DMSO, or dieldrin in DMSO for 30 min. RNA-seq was then performed on cDNA libraries prepared after depletion of ribosomal RNAs. Screen captures from IGV showing an example of a HERV becoming a site of divergent transcription upon exposure of the Jurkat cells to dieldrin. Top tracks show coverage from sequencing of poly(A)-enriched libraries for comparison. All coverage tracks are at the same scale and all the sequencing result files are of comparable size. Blue and red reads (rds) are in opposite orientation. CAGE peaks use the same color code. Reads mapping inside a series of 4686 HERVs located away (30Kb) from protein-coding genes were quantified both in the poly(A)-enriched and the ribo-depleted data. Read counts from each replicate were then summed and plotted.Jurkat cells were exposed to either DMSO, or dieldrin in DMSO for 30 min. RNA-seq was then performed on cDNA libraries prepared after depletion of ribosomal RNAs. Distribution of regions annotated "LTR" in RepeatMasker relative to sites of divergent transcription in Jurkat cells treated with either DMSO or dieldrin in DMSO.Jurkat cells were exposed to either DMSO, or dieldrin in DMSO for 30 min. RNA-seq was then performed on cDNA libraries prepared after depletion of ribosomal RNAs. Jurkat T cells were treated with either DMSO (vehicle) or 100μM dieldrin for the indicated times. Abundance of IL2 mRNA was assessed by RT-qPCR. Data shown are means +/- SEM from four independent experiments. Significance (p-value) was estimated using the two-sided student t test appropriate for small sample numbers. ***: p-value<0.001.Jurkat cells were exposed to either DMSO, or dieldrin in DMSO for 30 min. RNA-seq was then performed on cDNA libraries prepared after depletion of ribosomal RNAs. Screen captures from IGV showing increased divergent transcription inside a HERV sequence upon exposure of the Jurkat cells to dieldrin in the intergenic region between MUC21 and MUC22. Color code as in (A).


Figure 4A human phospho - kinase antibody array was probed with extracts from Jurkat cells treated with either DMSO , dieldrin , or PMA for 30 min as indicated . The array processed as a western and revealed by ECL . Graphic shows selected antibodies . Values shown are means + / - SEM from two measures . Jurkat cells were treated with either DMSO ( vehicle ) , dieldrin , or PMA for the indicated times . Cell extracts were resolved by PAGE and western blotting was carried out with the indicated antibodies . Nearest size marker is indicated to the right ( kDa ) . Jurkat cells were cultured in the absence or in the presence of the indicated small molecule inhibitors for 1h then exposed to 100μM dieldrin for 30 min . Abundance of mRNA for the indicated genes or HERVs was assessed by RT - qPCR . Jurkat cells were cultured in the absence or in the presence of the indicated small molecule inhibitors for 1h then exposed to 100μM dieldrin for 30 min . Abundance of mRNA for the indicated genes or HERVs was assessed by RT - qPCR . Jurkat cells treated as in ( C ) were subject to fractionation as indicated in the Material and Method section to distinguish a fraction with mostly RNAPII S2p ( F1 ) , one with mostly histone H3 ( F3 ) , and one with both RNAPII S2p and histone H3 ( P ) . Fractions were resolved by PAGE , and western blots were carried out with the indicated antibodies .

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Figure 4A human phospho-kinase antibody array was probed with extracts from Jurkat cells treated with either DMSO, dieldrin, or PMA for 30 min as indicated. The array processed as a western and revealed by ECL. Graphic shows selected antibodies. Values shown are means +/- SEM from two measures.Jurkat cells were treated with either DMSO (vehicle), dieldrin, or PMA for the indicated times. Cell extracts were resolved by PAGE and western blotting was carried out with the indicated antibodies. Nearest size marker is indicated to the right (kDa).Jurkat cells were cultured in the absence or in the presence of the indicated small molecule inhibitors for 1h then exposed to 100μM dieldrin for 30 min. Abundance of mRNA for the indicated genes or HERVs was assessed by RT-qPCR.Jurkat cells were cultured in the absence or in the presence of the indicated small molecule inhibitors for 1h then exposed to 100μM dieldrin for 30 min. Abundance of mRNA for the indicated genes or HERVs was assessed by RT-qPCR.Jurkat cells treated as in (C) were subject to fractionation as indicated in the Material and Method section to distinguish a fraction with mostly RNAPII S2p (F1), one with mostly histone H3 (F3), and one with both RNAPII S2p and histone H3 (P). Fractions were resolved by PAGE, and western blots were carried out with the indicated antibodies.


Figure 1 . ( A ) Proteins detected in Vps34 - TAP immunoprecipitations by MS analysis are listed . The number of identified peptides of each protein and the peptide coverage are also shown . ( B ) Cells expressing TAP - tagged Atg38 and the indicated HA - tagged proteins were grown in YPD . The extracts were prepared and were immunoprecipitated by IgG - Dynabeads as described in Materials and methods . The whole - cell extract ( left , " input " ) and the precipitated proteins ( right ) were analyzed by immunoblotting with the indicated antibodies . ( C ) Relative abundances of proteins identified by LC - MS / MS of Atg14 - TAP and Atg38 - TAP purifications from YPD - grown cells . Relative abundances are defined as emPAIprey / emPAIbait and are shown as mean ± SD of two independent experiments . ( D ) Cell extracts were prepared from wild - type and atg14Δ cells expressing Atg38 - TAP as well as HA - tagged proteins of complex I components . The IgG Dynabead - precipitated proteins ( bottom ) , together with the whole - cell extracts ( top ) , were immunoblotted with the indicated antibodies . ( E ) ATG38 - 2GFP ATG17 - 2mCherry cells in wild - type and atg14Δ were cultured at 30 ° C with or without rapamycin . After 180 min , cells were analyzed by fluorescence microscopy . Bar , 5 µm .

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

Figure 1.(A) Proteins detected in Vps34-TAP immunoprecipitations by MS analysis are listed. The number of identified peptides of each protein and the peptide coverage are also shown.(B) Cells expressing TAP-tagged Atg38 and the indicated HA-tagged proteins were grown in YPD. The extracts were prepared and were immunoprecipitated by IgG-Dynabeads as described in Materials and methods. The whole-cell extract (left, "input") and the precipitated proteins (right) were analyzed by immunoblotting with the indicated antibodies.(C) Relative abundances of proteins identified by LC-MS/MS of Atg14-TAP and Atg38-TAP purifications from YPD-grown cells. Relative abundances are defined as emPAIprey/emPAIbait and are shown as mean ± SD of two independent experiments.(D) Cell extracts were prepared from wild-type and atg14Δ cells expressing Atg38-TAP as well as HA-tagged proteins of complex I components. The IgG Dynabead-precipitated proteins (bottom), together with the whole-cell extracts (top), were immunoblotted with the indicated antibodies.(E) ATG38-2GFP ATG17-2mCherry cells in wild-type and atg14Δ were cultured at 30°C with or without rapamycin. After 180 min, cells were analyzed by fluorescence microscopy. Bar, 5 µm.


Figure 2 . ( A ) Cells expressing Pho8Δ60 were grown in YPD and shifted to SD ( - N ) medium for 3 h at 30 ° C . Lysates from each group of cells were tested for ALP activity . ALP activity is shown as mean ± SD of three independent experiments with the activity of wild - type cells normalized to 1 . ( B ) Cells with the indicated genotypes were grown in YPD and treated with rapamycin for 3 h at 30 ° C . Total protein was separated by SDS - PAGE and detected by immunoblotting with anti - API . ( C ) Cells were grown in YPD medium for 1 h at 30 ° C and separated to the intracellular ( I ; cells ) and extracellular ( E ; medium ) fractions by centrifugation . The fractions were treated as detailed in Materials and methods and aliquots of each were separated by SDS - PAGE . The positions of the precursor ( p2 - CPY ) and mature ( m - CPY ) forms of CPY are indicated .

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

Figure 2.(A) Cells expressing Pho8Δ60 were grown in YPD and shifted to SD(-N) medium for 3 h at 30°C. Lysates from each group of cells were tested for ALP activity. ALP activity is shown as mean ± SD of three independent experiments with the activity of wild-type cells normalized to 1.(B) Cells with the indicated genotypes were grown in YPD and treated with rapamycin for 3 h at 30°C. Total protein was separated by SDS-PAGE and detected by immunoblotting with anti-API.(C) Cells were grown in YPD medium for 1 h at 30°C and separated to the intracellular (I; cells) and extracellular (E; medium) fractions by centrifugation. The fractions were treated as detailed in Materials and methods and aliquots of each were separated by SDS-PAGE. The positions of the precursor (p2-CPY) and mature (m-CPY) forms of CPY are indicated.


Figure 3 . ( A ) Wild - type and atg38Δ cells expressing the indicated GFP fusion proteins and Ape1 - mRFP were cultured at 30 ° C in the presence of rapamycin . After 60 min , cells were analyzed by fluorescence microscopy . Of the total pool of Ape1 - mRFP signal , the percentage of Ape1 - mRFP colocalizing with GFP was calculated . This percentage is shown as mean ± SD of two independent experiments , with the number of cells counted indicated at the bottom of the graph . ( B ) Examples of wild - type and atg38Δ cells expressing either Atg14 - 3 × GFP or Vps34 - 3 × GFP in A . Bar , 5 µm . ( C ) Cell extracts were prepared from the cells in A , then analyzed by immunoblotting with anti - GFP and anti - Pgk1 antibodies .

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

Figure 3.(A) Wild-type and atg38Δ cells expressing the indicated GFP fusion proteins and Ape1-mRFP were cultured at 30°C in the presence of rapamycin. After 60 min, cells were analyzed by fluorescence microscopy. Of the total pool of Ape1-mRFP signal, the percentage of Ape1-mRFP colocalizing with GFP was calculated. This percentage is shown as mean ± SD of two independent experiments, with the number of cells counted indicated at the bottom of the graph. (B) Examples of wild-type and atg38Δ cells expressing either Atg14-3 × GFP or Vps34-3 × GFP in A. Bar, 5 µm.(C) Cell extracts were prepared from the cells in A, then analyzed by immunoblotting with anti-GFP and anti-Pgk1 antibodies.


Figure 4 . ( A ) Cell extracts were prepared from wild - type and atg38Δ strains expressing TAP - tagged Atg38 and the indicated HA proteins . The extracts were immunoprecipitated by IgG - Dynabeads . The whole - cell extracts ( top , " input " ) and the precipitates ( bottom ) were analyzed by immunoblotting with the indicated antibodies . Relative binding efficiency was determined as described in Materials and methods . To compare results from multiple experiments , the value for the reference sample ( wild - type cells ) was normalized to 1 . 0 . Relative binding efficiency is shown as mean ± SD of three independent experiments . ( B ) Relative abundances of proteins identified in TAP purifications from the indicated strains were analyzed as described in Fig . 1 C and are shown as mean ± SD of two independent experiments . ( C ) Cells expressing Pho8Δ60 were grown in YPD and shifted to SD ( - N ) medium for 3 h at 30 ° C . Cell lysates from each group of cells were tested for ALP activity . ALP activity is shown as mean ± SD of three independent experiments with the activity of wild - type cells normalized to 1 . ( D ) Cells expressing Vps34 - 6HA , Vps30 - 9myc , Atg38 - 3FLAG , and Atg14 - TAP were lysed in a buffer containing 150 mM NaCl . The Atg14 - TAP immunoprecipitate with IgG - Dynabeads from this lysate was split into eight fractions and washed with lysis buffer containing the indicated concentrations of NaCl . The resulting precipitates were analyzed by immunoblotting with the indicated antibodies . Relative binding efficiency was determined as described in Materials and methods , although a 150 - mM NaCl sample was used as a control and normalized to 1 . 0 . Relative binding efficiency is shown as mean ± SD of three independent experiments .

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

Figure 4.(A) Cell extracts were prepared from wild-type and atg38Δ strains expressing TAP-tagged Atg38 and the indicated HA proteins. The extracts were immunoprecipitated by IgG-Dynabeads. The whole-cell extracts (top, "input") and the precipitates (bottom) were analyzed by immunoblotting with the indicated antibodies. Relative binding efficiency was determined as described in Materials and methods. To compare results from multiple experiments, the value for the reference sample (wild-type cells) was normalized to 1.0. Relative binding efficiency is shown as mean ± SD of three independent experiments.(B) Relative abundances of proteins identified in TAP purifications from the indicated strains were analyzed as described in Fig. 1 C and are shown as mean ± SD of two independent experiments.(C) Cells expressing Pho8Δ60 were grown in YPD and shifted to SD(-N) medium for 3 h at 30°C. Cell lysates from each group of cells were tested for ALP activity. ALP activity is shown as mean ± SD of three independent experiments with the activity of wild-type cells normalized to 1.(D) Cells expressing Vps34-6HA, Vps30-9myc, Atg38-3FLAG, and Atg14-TAP were lysed in a buffer containing 150 mM NaCl. The Atg14-TAP immunoprecipitate with IgG-Dynabeads from this lysate was split into eight fractions and washed with lysis buffer containing the indicated concentrations of NaCl. The resulting precipitates were analyzed by immunoblotting with the indicated antibodies. Relative binding efficiency was determined as described in Materials and methods, although a 150-mM NaCl sample was used as a control and normalized to 1.0. Relative binding efficiency is shown as mean ± SD of three independent experiments.


Figure 5 . ( A ) AH109 cells containing the indicated GAL4 activation domain fusions ( bait ) and GAL4 DNA - binding domain fusions ( prey ) were selected on SD - Leu - Trp plates at 30 ° C ( − LW , left ) . The interaction of fusion proteins was examined by plating cells on SD - Leu - Trp - His ( − LWH , middle ) and SD - Leu - Trp - Ade ( − LWA , right ) plates at 30 ° C for 3 d . ( B ) Interactions between the indicated fusion proteins were analyzed as in A . AH109 cells containing the indicated GAL4 activation domain fusions ( bait ) and GAL4 DNA - binding domain fusions ( prey ) were selected on SD - Leu - Trp plates at 30 ° C ( − LW , left ) . The interaction of fusion proteins was examined by plating cells on SD - Leu - Trp - His ( − LWH , middle ) and SD - Leu - Trp - Ade ( − LWA , right ) plates at 30 ° C for 3 d . ( C ) The TAP - tagged Vps34 protein complex was purified from the vps30Δ atg14Δ vps38Δ cells . The purified proteins were separated on SDS - PAGE and visualized by Coomassie staining . Recombinant GST - Atg38 ( WT ) , GST - Atg381 - 120 ( N ) , and GST - Atg38121 - 226 ( C ) were incubated with Vps15 - Vps34 - TAP complex bound to IgG - Dynabeads . The eluted proteins were analyzed by immunoblotting .

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Figure 5.(A) AH109 cells containing the indicated GAL4 activation domain fusions (bait) and GAL4 DNA-binding domain fusions (prey) were selected on SD-Leu-Trp plates at 30°C (−LW, left). The interaction of fusion proteins was examined by plating cells on SD-Leu-Trp-His (−LWH, middle) and SD-Leu-Trp-Ade (−LWA, right) plates at 30°C for 3 d.(B) Interactions between the indicated fusion proteins were analyzed as in A. AH109 cells containing the indicated GAL4 activation domain fusions (bait) and GAL4 DNA-binding domain fusions (prey) were selected on SD-Leu-Trp plates at 30°C (−LW, left). The interaction of fusion proteins was examined by plating cells on SD-Leu-Trp-His (−LWH, middle) and SD-Leu-Trp-Ade (−LWA, right) plates at 30°C for 3 d.(C) The TAP-tagged Vps34 protein complex was purified from the vps30Δ atg14Δ vps38Δ cells. The purified proteins were separated on SDS-PAGE and visualized by Coomassie staining. Recombinant GST-Atg38 (WT), GST-Atg381-120 (N), and GST-Atg38121-226 (C) were incubated with Vps15-Vps34-TAP complex bound to IgG-Dynabeads. The eluted proteins were analyzed by immunoblotting.


Figure 6 . ( A ) Interactions between the indicated fusion proteins were analyzed as in Fig . 4 A . AH109cells containing the indicated GAL4 activation domain fusions ( bait ) and GAL4 DNA - binding domain fusions ( prey ) were selected on SD - Leu - Trp plates at 30 ° C ( − LW , left ) . The interaction of fusion proteins was examined by plating cells on SD - Leu - Trp - His ( − LWH , middle ) and SD - Leu - Trp - Ade ( − LWA , right ) plates at 30 ° C for 3 d . ( B ) Analysis of Atg38 by gel filtration . Recombinant Atg38 protein was fractionated over a gel filtration column . Each fraction was subjected to SDS - PAGE and visualized by Coomassie staining .

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

Figure 6.(A) Interactions between the indicated fusion proteins were analyzed as in Fig. 4 A. AH109cells containing the indicated GAL4 activation domain fusions (bait) and GAL4 DNA-binding domain fusions (prey) were selected on SD-Leu-Trp plates at 30°C (−LW, left). The interaction of fusion proteins was examined by plating cells on SD-Leu-Trp-His (−LWH, middle) and SD-Leu-Trp-Ade (−LWA, right) plates at 30°C for 3 d.(B) Analysis of Atg38 by gel filtration. Recombinant Atg38 protein was fractionated over a gel filtration column. Each fraction was subjected to SDS-PAGE and visualized by Coomassie staining.


Figure 7 . ( A ) Cell extracts were prepared from atg38Δ strains expressing Atg14 - TAP and either Vps34 - 6HA or Vps15 - 6HA with Atg381 - 120 fused with GFP and either GBP or GBP3A . The cell extracts were immunoprecipitated with IgG - Dynabeads . The precipitated proteins , together with the whole - cell extracts , were immunoblotted with the indicated antibodies ( left ) . Relative binding efficiency was determined as described in Fig . 3 A and was shown as mean ± SD of three independent experiments .

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Figure 7.(A) Cell extracts were prepared from atg38Δ strains expressing Atg14-TAP and either Vps34-6HA or Vps15-6HA with Atg381-120 fused with GFP and either GBP or GBP3A. The cell extracts were immunoprecipitated with IgG-Dynabeads. The precipitated proteins, together with the whole-cell extracts, were immunoblotted with the indicated antibodies (left). Relative binding efficiency was determined as described in Fig. 3 A and was shown as mean ± SD of three independent experiments.


Figure 1Assay principle : RNAi was performed with luciferase - expressing cells that were challenged with or without CTLs and bi - specific antibody . Before readout , cell supernatant was removed and the remaining intact cells were lysed to measure the residual - cell - associated luciferase . To identify immune - checkpoint regulators , the difference between normalized luciferase measurements for conditions with CTLs and without CTLs was calculated . siRNAs enhancing CTL cytotoxicity would only reduce normalized luciferase levels under conditions with CTLs ; hence , the difference between luciferase measurements will be > 0 . Luc - CTL assay performed at different T cell to MCF7 cell ratio with PBMC - derived CD8 + T cells and anti - EpCAM x CD3 bi - specific antibody ( ○ ) . Anti - CD19 × CD3 bi - specific Ab ( ▪ ) was used as a specificity control since CD19 is a B - lymphocyte - specific antigen and therefore this bsAb fails to cross - link tumor to T cells . Lower luciferase intensity indicates higher lysis . Error bars denote ± SEM . Luc - CTL assay was performed with MCF7 cells transfected with control or PD - L1 - specific siRNAs and cocultured with or without CTLs and bsAb . For each condition , the luciferase activity of PD - L1 - siRNA - treated cells was normalized to that of the control siRNA ; n = 8 . Error bars denote ± SEM . Comparison between the Luc - CTL assay ( ▪ ) and the classical chromium ( Cr ) - release assay ( ○ ) with MCF7 as target cells and survivin - specific T cells as effector cells at varying effector to target ( E : T ) ratios . Error bars denote ± SEM .

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

Figure 1Assay principle: RNAi was performed with luciferase-expressing cells that were challenged with or without CTLs and bi-specific antibody. Before readout, cell supernatant was removed and the remaining intact cells were lysed to measure the residual-cell-associated luciferase. To identify immune-checkpoint regulators, the difference between normalized luciferase measurements for conditions with CTLs and without CTLs was calculated. siRNAs enhancing CTL cytotoxicity would only reduce normalized luciferase levels under conditions with CTLs; hence, the difference between luciferase measurements will be > 0.Luc-CTL assay performed at different T cell to MCF7 cell ratio with PBMC-derived CD8+ T cells and anti-EpCAM x CD3 bi-specific antibody (○). Anti-CD19 × CD3 bi-specific Ab (▪) was used as a specificity control since CD19 is a B-lymphocyte-specific antigen and therefore this bsAb fails to cross-link tumor to T cells. Lower luciferase intensity indicates higher lysis. Error bars denote ± SEM.Luc-CTL assay was performed with MCF7 cells transfected with control or PD-L1-specific siRNAs and cocultured with or without CTLs and bsAb. For each condition, the luciferase activity of PD-L1-siRNA-treated cells was normalized to that of the control siRNA; n = 8. Error bars denote ± SEM.Comparison between the Luc-CTL assay (▪) and the classical chromium (Cr)-release assay (○) with MCF7 as target cells and survivin-specific T cells as effector cells at varying effector to target (E:T) ratios. Error bars denote ± SEM.


Figure 2B Correlation between replicates for screen 2 for both viability and toxicity sets , as determined by the Pearson rank correlation test . C - E Graphical summary of gene function related to modification of T cell - mediated tumor lysis and cellviability for screens 1 , 2 , and 3 , respectively . Positive score = reduced cancer cellviability ; negative score = increased viability . X - axis : influence on cellviability without addition of T cells . Y - axis : influence on cellviability with addition of T cells . Appropriate immune - suppressive ( PD - L1 , CEACAM - 6 , GAL - 3 ) and lethality ( UBC , PLK - 1 ) controls , along with few positive ( GRM4 , GRK5 ) and negative ( CCR9 , IL8 ) candidate immune - modulatory hits are highlighted herein . C - E Graphical summary of gene function related to modification of T cell - mediated tumor lysis and cellviability for screens 1 , 2 , and 3 , respectively . Positive score = reduced cancer cellviability ; negative score = increased viability . X - axis : influence on cellviability without addition of T cells . Y - axis : influence on cellviability with addition of T cells . Appropriate immune - suppressive ( PD - L1 , CEACAM - 6 , GAL - 3 ) and lethality ( UBC , PLK - 1 ) controls , along with few positive ( GRM4 , GRK5 ) and negative ( CCR9 , IL8 ) candidate immune - modulatory hits are highlighted herein .

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

Figure 2B Correlation between replicates for screen 2 for both viability and toxicity sets, as determined by the Pearson rank correlation test.C-E Graphical summary of gene function related to modification of T cell-mediated tumor lysis and cellviability for screens 1, 2, and 3, respectively. Positive score = reduced cancer cellviability; negative score = increased viability. X-axis: influence on cellviability without addition of T cells. Y-axis: influence on cellviability with addition of T cells. Appropriate immune-suppressive (PD-L1, CEACAM-6, GAL-3) and lethality (UBC, PLK-1) controls, along with few positive (GRM4, GRK5) and negative (CCR9, IL8) candidate immune-modulatory hits are highlighted herein.C-E Graphical summary of gene function related to modification of T cell-mediated tumor lysis and cellviability for screens 1, 2, and 3, respectively. Positive score = reduced cancer cellviability; negative score = increased viability. X-axis: influence on cellviability without addition of T cells. Y-axis: influence on cellviability with addition of T cells. Appropriate immune-suppressive (PD-L1, CEACAM-6, GAL-3) and lethality (UBC, PLK-1) controls, along with few positive (GRM4, GRK5) and negative (CCR9, IL8) candidate immune-modulatory hits are highlighted herein.


Figure 4A MCF7 cells were transfected with the described siRNA sequences and harvested after 72 h for mRNA and protein estimation using RT - PCR ( upper ) and immunoblot ( lower ) analysis , respectively . GAPDH and beta - actin were used as controls for RNA and protein normalization , respectively . B Luc - CTL cytotoxicity assay with PBMC - derived CTLs and bi - specific Ab as effector population and MCF7 as target cells , which were transfected with individual ( s1 - s4 ) or pooled CCR9 siRNA sequences . PD - L1 and non - specific control siRNAs were used as positive and negative controls , respectively , for CTL - mediated cytotoxicity . C , D Cr - release assay showing % specific lysis of MCF7 cells by survivin - specific T cells at different ratios upon CCR9 knockdown ( C ) or overexpression ( D ) . MCF7 cells were transfected with either CCR9 siRNA s1 ( Δ ) , pooled siRNA sequences ( ○ ) , positive control PD - L1 ( □ ) , and non - specific control siRNA ( ▪ ) ( C ) or with pCMV6 - AC - His control vector ( ▪ ) and pCMV6 - AC - His - CCR9 expression construct ( ○ ) ( D ) 72 h prior to the assay . E Cr - release assay showing % specific lysis of MDA - MB - 231 breast tumor cell line by survivin - specific T cells at different ratios upon CCR9 knockdown ( ○ ) in comparison to the control knockdown ( ▪ ) . F , G Cr - release assay showing lysis of patient - derived melanoma cells ( M579 - A2 ) by tumor - infiltrating lymphocytes ( TIL 412 ) ( F ) or lysis of PANC - 1 pancreatic adenocarcinoma cells by patient - derived pancreatic TIL 53 ( G ) at different E : T ratios upon CCR9 ( ○ ) or control ( ▪ ) knockdown .

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Figure 4A MCF7 cells were transfected with the described siRNA sequences and harvested after 72 h for mRNA and protein estimation using RT-PCR (upper) and immunoblot (lower) analysis, respectively. GAPDH and beta-actin were used as controls for RNA and protein normalization, respectively.B Luc-CTL cytotoxicity assay with PBMC-derived CTLs and bi-specific Ab as effector population and MCF7 as target cells, which were transfected with individual (s1-s4) or pooled CCR9 siRNA sequences. PD-L1 and non-specific control siRNAs were used as positive and negative controls, respectively, for CTL-mediated cytotoxicity.C, D Cr-release assay showing % specific lysis of MCF7 cells by survivin-specific T cells at different ratios upon CCR9 knockdown (C) or overexpression (D). MCF7 cells were transfected with either CCR9 siRNA s1 (Δ), pooled siRNA sequences (○), positive control PD-L1 (□), and non-specific control siRNA (▪) (C) or with pCMV6-AC-His control vector (▪) and pCMV6-AC-His-CCR9 expression construct (○) (D) 72 h prior to the assay.E Cr-release assay showing % specific lysis of MDA-MB-231 breast tumor cell line by survivin-specific T cells at different ratios upon CCR9 knockdown (○) in comparison to the control knockdown (▪).F, G Cr-release assay showing lysis of patient-derived melanoma cells (M579-A2) by tumor-infiltrating lymphocytes (TIL 412) (F) or lysis of PANC-1 pancreatic adenocarcinoma cells by patient-derived pancreatic TIL 53 (G) at different E:T ratios upon CCR9 (○) or control (▪) knockdown.


Figure 5A , B ELISpot assay showing IFN - γ ( A ) and granzyme B ( B ) secretion by survivin - specific T cells , as spot numbers , upon CCR9 knockdown ( black bars ) in MCF7 cells compared to the control knockdown ( white bars ) . T cells ( TC ) alone ( grey bars ) were used as control for background spot numbers . C Luminex assay showing cytokine levels in the supernatant from the coculture of survivin - specific TC and either CCR9hi MCF7 ( transfected with CCR9 - specific siRNA ) or CCR9lo MCF7 ( transfected with control siRNA ) cells . D Phospho - plex analysis showing the phospho - STAT levels in survivin - specific TC upon encountering CCR9hi or CCR9lo MCF7 cells . Log2 ratio of mean fluorescent intensity ( MFI ) of the respective analytes to the unstimulated TC is plotted herein . E Immunoblot analysis showing the phospho - STAT1 levels in the CCR9hi - treated , CCR9lo - treated or unstimulated TC using the phospho - specific STAT1 ( pTyr701 ) antibody . Beta - actin was used as the loading control .

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Figure 5A, B ELISpot assay showing IFN-γ (A) and granzyme B (B) secretion by survivin-specific T cells, as spot numbers, upon CCR9 knockdown (black bars) in MCF7 cells compared to the control knockdown (white bars). T cells (TC) alone (grey bars) were used as control for background spot numbers.C Luminex assay showing cytokine levels in the supernatant from the coculture of survivin-specific TC and either CCR9hi MCF7 (transfected with CCR9-specific siRNA) or CCR9lo MCF7 (transfected with control siRNA) cells.D Phospho-plex analysis showing the phospho-STAT levels in survivin-specific TC upon encountering CCR9hi or CCR9lo MCF7 cells. Log2 ratio of mean fluorescent intensity (MFI) of the respective analytes to the unstimulated TC is plotted herein.E Immunoblot analysis showing the phospho-STAT1 levels in the CCR9hi-treated, CCR9lo-treated or unstimulated TC using the phospho-specific STAT1 (pTyr701) antibody. Beta-actin was used as the loading control.


Figure 6A ELISA showing CCL25 levels in cell lysates from indicated tumorcell lines . CCR9 knockdown ( k . d . ) in MCF7 cells was achieved using specific shRNA ( see Materials and Methods ) . B Cr - release assay showing % specific lysis of MCF7 cells by survivin TC upon CCL25 ( □ ) or CCR9 ( ○ ) inhibition using specific siRNAs in comparison to the control siRNA ( ▪ ) . Mean ± SEM are depicted herein . C MCF7 cells were transfected with control or CCR9 - specific siRNAs , and 48 h later , the supernatants ( CCR9lo or CCR9hi SSN , respectively ) were used to culture survivin TCs overnight . Supernatant - treated TCs were then used as effector cells against CCR9lo or CCR9hi MCF7 tumor cells in the Cr - release assay along with wild - type MCF7 cells . Mean ± SEM are depicted herein . D Cr - release assay showing % specific lysis of MCF7 cells that were pre - treated with or without pertussis toxin ( PTX ) , or knocked down for CCR9 using specific siRNA . Mean ± SEM are depicted herein . E , F MCF7 cells transfected with control siRNA ( CCR9hi ) or CCR9 siRNA ( CCR9lo ) were cocultured with survivin TCs for 12 h . Gene microarray was performed with the total RNA extracted from purified T cells after the coculture . Volcano plot ( E ) illustrating fold change ( FC ; log2 ) in gene expression intensities compared with P - value ( − log2 ) between CCR9hi - and CCR9lo - treated TCs . Horizontal bar at y = 4 . 32 represents a statistical significance of P = 0 . 05 ( genes in gray below this line did not reach significance ) . LogFC cutoff at ± 0 . 5 is represented by the vertical lines . Heatmap representation of the top upregulated ( LogFC > 0 . 5 ) and downregulated ( LogFC < − 0 . 85 ) genes ( F ) with P ≤ 0 . 05 . Individual replicates per sample group are shown herein .

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Figure 6A ELISA showing CCL25 levels in cell lysates from indicated tumorcell lines. CCR9 knockdown (k.d.) in MCF7 cells was achieved using specific shRNA (see Materials and Methods).B Cr-release assay showing % specific lysis of MCF7 cells by survivin TC upon CCL25 (□) or CCR9 (○) inhibition using specific siRNAs in comparison to the control siRNA (▪). Mean ± SEM are depicted herein.C MCF7 cells were transfected with control or CCR9-specific siRNAs, and 48 h later, the supernatants (CCR9lo or CCR9hi SSN, respectively) were used to culture survivin TCs overnight. Supernatant-treated TCs were then used as effector cells against CCR9lo or CCR9hi MCF7 tumor cells in the Cr-release assay along with wild-type MCF7 cells. Mean ± SEM are depicted herein.D Cr-release assay showing % specific lysis of MCF7 cells that were pre-treated with or without pertussis toxin (PTX), or knocked down for CCR9 using specific siRNA. Mean ± SEM are depicted herein.E, F MCF7 cells transfected with control siRNA (CCR9hi) or CCR9 siRNA (CCR9lo) were cocultured with survivin TCs for 12 h. Gene microarray was performed with the total RNA extracted from purified T cells after the coculture. Volcano plot (E) illustrating fold change (FC; log2) in gene expression intensities compared with P-value (−log2) between CCR9hi- and CCR9lo-treated TCs. Horizontal bar at y = 4.32 represents a statistical significance of P = 0.05 (genes in gray below this line did not reach significance). LogFC cutoff at ± 0.5 is represented by the vertical lines. Heatmap representation of the top upregulated (LogFC > 0.5) and downregulated (LogFC < −0.85) genes (F) with P ≤ 0.05. Individual replicates per sample group are shown herein.


Figure 7A Cr - release assay showing TIL 209 - mediated lysis of CCR9 + M579 - A2 ( transduced with control shRNA ) or CCR9 − M579 - A2 cells ( transduced with CCR9 - specific shRNA ) . Curves represent mean ± SEM . C , D Tumorgrowth curves showing mean ± SEMtumor volume of CCR9 + or CCR9 − M579 - A2tumors in TIL - treated mice ( C ) or the PBS alone group ( D ) . Statistical difference was calculated using the unpaired one - sided Mann - Whitney U - test .

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Figure 7A Cr-release assay showing TIL 209-mediated lysis of CCR9+ M579-A2 (transduced with control shRNA) or CCR9− M579-A2 cells (transduced with CCR9-specific shRNA). Curves represent mean ± SEM.C, D Tumorgrowth curves showing mean ± SEMtumor volume of CCR9+ or CCR9−M579-A2tumors in TIL-treated mice (C) or the PBS alone group (D). Statistical difference was calculated using the unpaired one-sided Mann-Whitney U-test.


Figure 1 ( A ) Confocal laser scanning micrographs showing the localization of a functional CDKA ; 1 : mVenus fusion protein in the wildtype ( WT ) and cartoons on top highlighting the changes in abundance of CDKA ; 1 : mVenus in the nucleus and cytoplasm during the course of meiosis . The region colored in beige represents the cytoplasm , in green the nucleoplasm , and in white the nucleolus . Scale bar : 10 μm . ( B ) Quantitative analysis of the signal distribution of the nuclear versus cytoplasmic fraction of CDKA ; 1 : mVenus during prophase I of meiosis as revealed by live cell imaging Twenty cells at each time point were used for the analysis . Error bars represent mean ± SD and two biological replicates were performed . ( C ) Immunolocalization of CDKA ; 1 ( green ) and ASY1 ( red ) on spread chromsomes in leptotene and zygotene of wild - type plants expressing a functional PROCDKA ; 1 : CDKA ; 1 : Strep construct . The last lane shows a magnification of the region marked by the red rectangle . Arrowheads indicate synapsed regions of homologous chromosomes where CDKA ; 1 is no longer present . Scale bar : 5 μm . ( D ) Chromosome spread analysis of the wildtype and the hypomorphic cdka ; 1 mutant CDKA ; 1T161D . ( a , h ) zygotene or zygotene - like stages ; ( b , i ) pachytene or pachytene - like stages ; ( c , j , k ) diakinesis or dikinesis - like stages ; ( d ) metaphase I ; ( e , i , m , n ) end of meiosis I with two ( e , m ) or three ( i ) pools of chromosomes ; ( f ) metaphase II ; ( g ) tetrad . Red arrowheads indicate the initiated formation of a phragmoplast . White arrowheads depict mitochondria . Scale bars : 10 μm .

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Figure 1(A) Confocal laser scanning micrographs showing the localization of a functional CDKA;1:mVenus fusion protein in the wildtype (WT) and cartoons on top highlighting the changes in abundance of CDKA;1:mVenus in the nucleus and cytoplasm during the course of meiosis. The region colored in beige represents the cytoplasm, in green the nucleoplasm, and in white the nucleolus. Scale bar: 10 μm.(B) Quantitative analysis of the signal distribution of the nuclear versus cytoplasmic fraction of CDKA;1:mVenus during prophase I of meiosis as revealed by live cell imaging Twenty cells at each time point were used for the analysis. Error bars represent mean ± SD and two biological replicates were performed.(C) Immunolocalization of CDKA;1 (green) and ASY1 (red) on spread chromsomes in leptotene and zygotene of wild-type plants expressing a functional PROCDKA;1:CDKA;1:Strep construct. The last lane shows a magnification of the region marked by the red rectangle. Arrowheads indicate synapsed regions of homologous chromosomes where CDKA;1 is no longer present. Scale bar: 5 μm.(D) Chromosome spread analysis of the wildtype and the hypomorphic cdka;1 mutant CDKA;1T161D. (a, h) zygotene or zygotene-like stages; (b, i) pachytene or pachytene-like stages; (c, j, k) diakinesis or dikinesis-like stages; (d) metaphase I; (e, i, m, n) end of meiosis I with two (e, m) or three (i) pools of chromosomes; (f) metaphase II; (g) tetrad. Red arrowheads indicate the initiated formation of a phragmoplast. White arrowheads depict mitochondria. Scale bars: 10 μm.


Figure 2 ( A ) ASY1 : GFP and ASY1T142D : GFP localization in late G2 and leptotene of male meiocytes of the wildtype and CDKA ; 1T161D mutants . ASY3 : RFP , highlighting chromsomes , was used as a marker for the staging of meiosis . Scale bar : 5 μm . ( B ) Kinase assays of CDKA ; 1 - SDS , - TAM , and - CYCA3 ; 1 complexes using ASY1 puried from baculovirus - infected insect cells as a substrate . The upper panel shows the autoradiograph . The control reaction without CDKA ; 1 - cyclin complex indicates a background activity co - purified from insect cells . The lower panel indicates protein loading by coomassie briliant blue ( CBB ) staining . Arrowheads indicate ASY1 proteins and asterisks depict the relevant cyclin used which also gets phosphorylated in the assay . Numbers indicate the relative intensities of ASY1 bands . ( C ) The upper panel shows a phos - tag gel analysis of ASY11 - 300 and ASY11 - 300 / T142V ; T184V with and without CDKA ; 1 - SDS kinase complexes using an anti - MBP antibody . The lower panel denotes loading of CDKA ; 1 using an anti - Strep antibody . Arrowheads represent the proteins as indicated .

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Figure 2(A) ASY1:GFP and ASY1T142D:GFP localization in late G2 and leptotene of male meiocytes of the wildtype and CDKA;1T161D mutants. ASY3:RFP, highlighting chromsomes, was used as a marker for the staging of meiosis. Scale bar: 5 μm.(B) Kinase assays of CDKA;1-SDS, -TAM, and -CYCA3;1 complexes using ASY1 puried from baculovirus-infected insect cells as a substrate. The upper panel shows the autoradiograph. The control reaction without CDKA;1-cyclin complex indicates a background activity co-purified from insect cells. The lower panel indicates protein loading by coomassie briliant blue (CBB) staining. Arrowheads indicate ASY1 proteins and asterisks depict the relevant cyclin used which also gets phosphorylated in the assay. Numbers indicate the relative intensities of ASY1 bands.(C) The upper panel shows a phos-tag gel analysis of ASY11-300 and ASY11-300/T142V;T184V with and without CDKA;1-SDS kinase complexes using an anti-MBP antibody. The lower panel denotes loading of CDKA;1 using an anti-Strep antibody. Arrowheads represent the proteins as indicated.


Figure 3 ( B ) Localization patterns of different ASY1 : GFP variants together with ASY3 : RFP ( for staging of zygotene and pachytene ) in a asy1 mutant background during prophase I . Bar : 5 μ ( C ) Signal distribution profiles of ASY1 : GFP and ASY1T142V : GFP at leptotene The many small peaks with low amplitude in ASY1T142V : GFP indicate diffused localization as opposed to the clear peaks seen in the wildtype . ( D , E ) Immunolocalization of ASY1 ( D ) and ZYP1 ( E ) in ASY1 : GFP ( asy1 ) and ASY1T142V : GFP ( asy1 ) plants using anti - GFP and anti - ZYP1 antibodies , respectively . DNA was stained with DAPI ( blue ) . Scale bars : 5 μm .

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Figure 3(B) Localization patterns of different ASY1:GFP variants together with ASY3:RFP (for staging of zygotene and pachytene) in a asy1 mutant background during prophase I. Bar: 5 μ(C) Signal distribution profiles of ASY1:GFP and ASY1T142V:GFP at leptotene The many small peaks with low amplitude in ASY1T142V:GFP indicate diffused localization as opposed to the clear peaks seen in the wildtype.(D, E) Immunolocalization of ASY1 (D) and ZYP1 (E) in ASY1:GFP (asy1) and ASY1T142V:GFP (asy1) plants using anti-GFP and anti-ZYP1 antibodies, respectively. DNA was stained with DAPI (blue). Scale bars: 5 μm.


Figure 4 ( A ) Yeast two - hybrid interaction assays of ASY3 with different ASY1 variants . Monomeric GFP ( mGFP ) fused with AD ( activating domain ) and BD ( binding domain ) were used as controls . Yeast cells harboring both the AD and BD plasmids were grown on synthetic medium supplied with glucose in the absence of Leu and Trp ( - L / T , left panel ) , on Synthetic Drop - out ( SD ) medium in the absence of Leu , Trp , and His ( - L / T / H , middle panel ) , and on SD medium in the absence of Leu , Trp , His and Ade ( - L / T / H / A , right panel ) . Yeast cells were incubated until OD600 = 1 and then diluted 10 - , 100 - and 1000 - fold for the assays . ( B ) GST pull down of ASY3 with different ASY1 variants . The numbers above the bands show the relative intensity of the bands . The input and pull down fractions were analyzed by immuno - blotting with the anti - GST ( ASY3 ) and anti - MBP ( ASY1 ) antibodies . ( C ) Quantification of the pull down fractions of ASY1 as shown in ( B ) . The band intensity in the pull down of ASY11 - 300 / T142V ; T184V at a Triton X - 100 concentration of 0 . 5 % was defined as 1 . The relative amount of ASY1 in the pull down fractions was normalized by the band intensity of the pulled - down ASY3 fraction . The average band intensity of ASY1 at different concentrations of Triton X - 100 used was plotted . Asteriks indicate significant difference ( two - tailed t - test , p < 0 . 05 ) . Error bars represent mean ± SD and two biological replicates were performed . .

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Figure 4(A) Yeast two-hybrid interaction assays of ASY3 with different ASY1 variants. Monomeric GFP (mGFP) fused with AD (activating domain) and BD (binding domain) were used as controls. Yeast cells harboring both the AD and BD plasmids were grown on synthetic medium supplied with glucose in the absence of Leu and Trp (-L/T, left panel), on Synthetic Drop-out (SD) medium in the absence of Leu, Trp, and His (-L/T/H, middle panel), and on SD medium in the absence of Leu,Trp, His and Ade (-L/T/H/A, right panel). Yeast cells were incubated until OD600 = 1 and then diluted 10-, 100- and 1000-fold for the assays.(B) GST pull down of ASY3 with different ASY1 variants. The numbers above the bands show the relative intensity of the bands. The input and pull down fractions were analyzed by immuno-blotting with the anti-GST (ASY3) and anti-MBP (ASY1) antibodies. (C) Quantification of the pull down fractions of ASY1 as shown in (B). The band intensity in the pull down of ASY11-300/T142V;T184V at a Triton X-100 concentration of 0.5% was defined as 1. The relative amount of ASY1 in the pull down fractions was normalized by the band intensity of the pulled-down ASY3 fraction. The average band intensity of ASY1 at different concentrations of Triton X-100 used was plotted. Asteriks indicate significant difference (two-tailed t-test, p < 0.05). Error bars represent mean ± SD and two biological replicates were performed..


Figure 5 ( A ) Localization patterns of ASY1T142V ; T184V : GFP together with ASY3 - RFP in the wildtype and pch2 mutants . Please note that images of ASY1T142V ; T184V : GFP in pch2 mutants were taken with increased sensitivity for a better visibility . Scale bar : 5 μm . ( B ) Seed sets ( mean ± SD , n = 5 ) of WT , asy1 , pch2 , asy1 pch2 , ASY1T142V ; T184V : GFP ( asy1 ) and ASY1T142V ; T184V : GFP ( asy1 pch2 ) plants . Asteriks indicate significant differences ( two - tailed t - test , P < 0 . 01 ) and ns depicts no significant difference . Scale bar : 2 mm . ( C ) Localization patterns of ASY1 : GFP , ASY1T142V ; T184V : GFP , and ASY11 - 570 : GFP in the wildtype ( WT ) and / or in pch2 mutant plants at early prophase I . Scale bars : 5 μm . ( D ) Localization pattern of PCH2 : GFP together with ASY1 : RFP in the male meiocytes of wildtype . Arrowheads indicate the chromosomal regions where the ASY1 removal was concomitant with the localization of PCH2 . Scale bar : 10 μm .

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Figure 5(A) Localization patterns of ASY1T142V;T184V:GFP together with ASY3-RFP in the wildtype and pch2 mutants. Please note that images of ASY1T142V;T184V:GFP in pch2 mutants were taken with increased sensitivity for a better visibility. Scale bar: 5 μm.(B) Seed sets (mean ± SD, n = 5) of WT, asy1, pch2, asy1 pch2, ASY1T142V;T184V:GFP (asy1) and ASY1T142V;T184V:GFP (asy1 pch2) plants. Asteriks indicate significant differences (two-tailed t-test, P < 0.01) and ns depicts no significant difference. Scale bar: 2 mm.(C) Localization patterns of ASY1:GFP, ASY1T142V;T184V:GFP, and ASY11-570:GFP in the wildtype (WT) and/or in pch2 mutant plants at early prophase I. Scale bars: 5 μm.(D) Localization pattern of PCH2:GFP together with ASY1:RFP in the male meiocytes of wildtype. Arrowheads indicate the chromosomal regions where the ASY1 removal was concomitant with the localization of PCH2. Scale bar: 10 μm.


Figure 6 ( B ) Interaction assays of different ASY1 fragments ( with and without the closure motif ) . ( C ) Interaction analysis of the ASY1 closure motif ( ASY1571 - 596 ) with different ASY1 HORMA domain variants .

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Figure 6(B) Interaction assays of different ASY1 fragments (with and without the closure motif).(C) Interaction analysis of the ASY1 closure motif (ASY1571-596) with different ASY1 HORMA domain variants.


Figure 1PI ( 5 ) P Regulates Autophagosome Biogenesis ( A and B ) Western blot analysis of LC3 - II and tubulin levels and quantification of LC3 - II / tubulin ratio in HeLa cells treated with carrier alone , or in combination with PI ( 5 ) P di - C16 at indicated concentrations for 1 hr , in the absence and presence of 400 nM BAF ( treated in combination with lipids ) . Note that LC3 - I is often very faint compared with LC3 - II in HeLa cells under the protein extraction conditions we used ( see Figure S1C ) . However , this was not a problem because one should relate LC3 - II to tubulin ( mean ± SEM ) . PI ( 5 ) P Regulates Autophagosome Biogenesis ( C and D ) HeLa cells stably expressing GFP - LC3 were treated as in ( A ) and then left in complete media ( basal ) or starvation media ( HBSS ) for 2 hr , then fixed and analyzed on a Cellomics ArrayScan system . Quantification of numbers of GFP - LC3 vesicles per cell in the different conditions is shown in ( D ) ( mean ± SEM ) . ( E ) HeLa cells stably expressing GFP - mRFP - LC3 were treated as in ( A ) and analyzed on a Cellomics ArrayScan system . Quantification of numbers of autophagic vesicles ( AV ) or autolysosomes ( AL ) per cell in the different conditions is shown in the graph ( mean ± SEM ) . ( F and G ) Western blot analysis of free ATG12 and ATG5ATG12 complex levels with anti - HA antibody in HeLa cells transfected with HA - ATG12 and ATG5 and loaded with exogenous lipids . We quantified the ratio of ATG5ATG12 versus free ATG12 by direct infrared fluorescence detection on an Odyssey Infrared Imaging System to assess ATG5 - ATG12 conjugation efficiency ( mean ± SEM ) . ( H ) HeLa cells transfected with GFP - PHD3X or GFP - PHD3X Znmut and RFP - LC3 for 16 hr were left in complete media ( basal ) or starvation media ( HBSS ) for 1 hr , then fixed and imaged on a confocal microscope . Empty arrowheads indicate PHD3X single - labeled structures , while filled arrowheads indicate PHD3X - LC3 - and PHD3X - ATG16 - positive structures . Bar , 10 μm . ( I ) HeLa cells transfected with GFP - PHD3X and RFP - LC3 for 16 hr were left in HBSS for 1 hr , then fixed and imaged on Elyra superresolution microscope . Final visualization was performed in Volocity 6 . 3 software using isosurface rendering of selected cropped regions of the data sets . Note that this rendering means that vesicles positive for green and red do not look yellow but have green and red on the surface . Representative cropped regions from different cells are shown . Bar , 1 . 13 μm . See also Figure S1 .

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

Figure 1PI(5)P Regulates Autophagosome Biogenesis(A and B) Western blot analysis of LC3-II and tubulin levels and quantification of LC3-II/tubulin ratio in HeLa cells treated with carrier alone, or in combination with PI(5)P di-C16 at indicated concentrations for 1 hr, in the absence and presence of 400 nM BAF (treated in combination with lipids). Note that LC3-I is often very faint compared with LC3-II in HeLa cells under the protein extraction conditions we used (see Figure S1C). However, this was not a problem because one should relate LC3-II to tubulin (mean ± SEM).PI(5)P Regulates Autophagosome Biogenesis(C and D) HeLa cells stably expressing GFP-LC3 were treated as in (A) and then left in complete media (basal) or starvation media (HBSS) for 2 hr, then fixed and analyzed on a Cellomics ArrayScan system. Quantification of numbers of GFP-LC3 vesicles per cell in the different conditions is shown in (D) (mean ± SEM).(E) HeLa cells stably expressing GFP-mRFP-LC3 were treated as in (A) and analyzed on a Cellomics ArrayScan system. Quantification of numbers of autophagic vesicles (AV) or autolysosomes (AL) per cell in the different conditions is shown in the graph (mean ± SEM).(F and G) Western blot analysis of free ATG12 and ATG5ATG12 complex levels with anti-HA antibody in HeLa cells transfected with HA-ATG12 and ATG5 and loaded with exogenous lipids. We quantified the ratio of ATG5ATG12 versus free ATG12 by direct infrared fluorescence detection on an Odyssey Infrared Imaging System to assess ATG5-ATG12 conjugation efficiency (mean ± SEM).(H) HeLa cells transfected with GFP-PHD3X or GFP-PHD3X Znmut and RFP-LC3 for 16 hr were left in complete media (basal) or starvation media (HBSS) for 1 hr, then fixed and imaged on a confocal microscope. Empty arrowheads indicate PHD3X single-labeled structures, while filled arrowheads indicate PHD3X-LC3- and PHD3X-ATG16-positive structures. Bar, 10 μm.(I) HeLa cells transfected with GFP-PHD3X and RFP-LC3 for 16 hr were left in HBSS for 1 hr, then fixed and imaged on Elyra superresolution microscope. Final visualization was performed in Volocity 6.3 software using isosurface rendering of selected cropped regions of the data sets. Note that this rendering means that vesicles positive for green and red do not look yellow but have green and red on the surface. Representative cropped regions from different cells are shown. Bar, 1.13 μm. See also Figure S1.


Figure 2 ( B and C ) Western blot analysis of LC3 - II and tubulin levels and quantification of LC3 - II / tubulin ratio in HeLa cells treated with DMSO or increasing concentrations of YM - 201636 for 2 hr in the presence or absence of BAF ( mean ± SEM ) . ( D and E ) HeLa cells transiently transfected with Strawberry - ATG16L1 were treated with YM - 201636 ( 100 nM , 2 hr ) in HBSS , then fixed and stained for endogenous WIPI - 2 . Bar , 10 μm . ( E ) Quantification of ATG16L1 and WIPI2 structures per cell , n = 20 cells ( mean ± SEM ; n = 3 independent experiments ; ∗ ∗ ∗ p < 0 . 001 , t test ) . ( F ) Western blot analysis of free ATG12 and ATG5 - ATG12 complex levels with anti - HA antibody in HeLa cells transfected with HA - ATG12 and ATG5 and treated with YM - 201636 ( 100 nM , 2 hr ) ( mean ± SEM ) . ( G ) HeLa cells transfected with GFP - PHD3X and Strawberry - ATG16L1 for 16 hr were left in HBSS for 1 hr , then fixed and imaged on Elyra superresolution microscope . Final visualization was performed as previously described . Bar , 1 . 13 μm . ( H and I ) Western blot analysis of LC3 - II and tubulin levels and quantification of LC3 - II / tubulin ratio in cells incubated with YM - 201636 ( 100 nM , 2 hr ) and loaded with exogenous PI ( 5 ) P for the last 1 hr in the presence or absence of BAF ( mean ± SEM ) . See also Figure S2 .

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

Figure 2(B and C) Western blot analysis of LC3-II and tubulin levels and quantification of LC3-II/tubulin ratio in HeLa cells treated with DMSO or increasing concentrations of YM-201636 for 2 hr in the presence or absence of BAF (mean ± SEM).(D and E) HeLa cells transiently transfected with Strawberry-ATG16L1 were treated with YM-201636 (100 nM, 2 hr) in HBSS, then fixed and stained for endogenous WIPI-2. Bar, 10 μm. (E) Quantification of ATG16L1 and WIPI2 structures per cell, n = 20 cells (mean ± SEM; n = 3 independent experiments; ∗∗∗p < 0.001, t test).(F) Western blot analysis of free ATG12 and ATG5-ATG12 complex levels with anti-HA antibody in HeLa cells transfected with HA-ATG12 and ATG5 and treated with YM-201636 (100 nM, 2 hr) (mean ± SEM).(G) HeLa cells transfected with GFP-PHD3X and Strawberry-ATG16L1 for 16 hr were left in HBSS for 1 hr, then fixed and imaged on Elyra superresolution microscope. Final visualization was performed as previously described. Bar, 1.13 μm.(H and I) Western blot analysis of LC3-II and tubulin levels and quantification of LC3-II/tubulin ratio in cells incubated with YM-201636 (100 nM, 2 hr) and loaded with exogenous PI(5)P for the last 1 hr in the presence or absence of BAF (mean ± SEM). See also Figure S2.


Figure 3 ( A and B ) Western blot analysis of LC3 - II and tubulin levels and quantification of LC3 - II / tubulin ratio in HeLa cells transfected for 5 days with two rounds of control , PI5P4K2A , 2B , or 2C siRNA either left untreated or treated with BAF ( 200 nM , 16 hr ) ( mean ± SEM ) . ( C ) HeLa cells stably expressing GFP - mRFP - LC3 treated as in ( A ) were analyzed on a Cellomics ArrayScan system as previously described ( mean ± SEM ) . ( D ) Western blot analysis of free ATG12 and ATG5 - ATG12 complex levels with anti - HA antibody in HeLa cells treated with control , PI5P4K2A , 2B , and 2C siRNA and transfected with HA - ATG12 and ATG5 for the last 16 hr ( mean ± SEM ) . ( E and F ) HeLa cells transfected with GFP - PI5P4K2A , 2B , 2C , or catalytic - dead PI5P4K2C along with RFP - LC3 for 16 hr ( E ) or 30 hr ( F ) were fixed and imaged on a confocal microscope . Bar , 10 μm . ( G ) Quantification of cells ( percentage of total ) showing more than ten autophagic vesicles ( RFP - LC3 vesicles ) in the different conditions from ( F ) is shown in the graph ; n = 200 cells ( mean ± SEM ) . See also Figure S3 .

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

Figure 3(A and B) Western blot analysis of LC3-II and tubulin levels and quantification of LC3-II/tubulin ratio in HeLa cells transfected for 5 days with two rounds of control, PI5P4K2A, 2B, or 2C siRNA either left untreated or treated with BAF (200 nM, 16 hr) (mean ± SEM).(C) HeLa cells stably expressing GFP-mRFP-LC3 treated as in (A) were analyzed on a Cellomics ArrayScan system as previously described (mean ± SEM).(D) Western blot analysis of free ATG12 and ATG5-ATG12 complex levels with anti-HA antibody in HeLa cells treated with control, PI5P4K2A, 2B, and 2C siRNA and transfected with HA-ATG12 and ATG5 for the last 16 hr (mean ± SEM).(E and F) HeLa cells transfected with GFP-PI5P4K2A, 2B, 2C, or catalytic-dead PI5P4K2C along with RFP-LC3 for 16 hr (E) or 30 hr (F) were fixed and imaged on a confocal microscope. Bar, 10 μm.(G) Quantification of cells (percentage of total) showing more than ten autophagic vesicles (RFP-LC3 vesicles) in the different conditions from (F) is shown in the graph; n = 200 cells (mean ± SEM). See also Figure S3.


Figure 4 ( A and B ) Western blot analysis of LC3 - II and tubulin levels and quantification of LC3 - II / tubulin ratio in SKNSH and MEFs treated for 2 hr with carrier alone or in combination with PI ( 5 ) P di - C16 ( 10 μM ) in the absence and presence of BAF ( mean ± SEM ) . ( C ) HeLa cells treated with PI5P4K2A or PI5P4K2C siRNA for 5 days or with pCMV - PI5P4K2C overexpression construct for 48 hr were transfected with EGFP - httQ74 for the last 48 hr . Percentage of cells with EGFP - positive aggregates is shown in the graph ; n = 500 cells ( mean ± SEM ) . Cells expressing control vector typically have 25 % of aggregates and we set the control at 100 % to enable comparison and statistics from independent experiments . ( D and E ) Percentage of cells with EGFP - positive aggregates was scored in HEK293 cells treated with PI5P4K2C siRNA for 5 days and cotransfected with EGFP - httQ74 for the last 48 hr ( D ) , or in COS7 and SKNSH cells ( E ) , transfected with PI5P4K2C plasmid together with EGFP - httQ74 for the last 48 hr . ( F and G ) WT ( Atg5 + / + ) or autophagy - deficient cells lacking the key autophagy gene Atg5 ( Atg5 − / − ) MEFs were treated with PI5P4K2C siRNA ( 5 days ) or PI5P4K2C plasmid ( 48 hr ) together with EGFP - httQ74 for 48 hr . The percentages of transfected cells with EGFP - httQ74 aggregates were assessed and shown in the graph ; n = 500 cells ( mean ± SEM ) . Representative confocal images are shown in ( G ) . See also Figures S3 and S4 .

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

Figure 4(A and B) Western blot analysis of LC3-II and tubulin levels and quantification of LC3-II/tubulin ratio in SKNSH and MEFs treated for 2 hr with carrier alone or in combination with PI(5)P di-C16 (10 μM) in the absence and presence of BAF (mean ± SEM).(C) HeLa cells treated with PI5P4K2A or PI5P4K2C siRNA for 5 days or with pCMV-PI5P4K2C overexpression construct for 48 hr were transfected with EGFP-httQ74 for the last 48 hr. Percentage of cells with EGFP-positive aggregates is shown in the graph; n = 500 cells (mean ± SEM). Cells expressing control vector typically have 25% of aggregates and we set the control at 100% to enable comparison and statistics from independent experiments.(D and E) Percentage of cells with EGFP-positive aggregates was scored in HEK293 cells treated with PI5P4K2C siRNA for 5 days and cotransfected with EGFP-httQ74 for the last 48 hr (D), or in COS7 and SKNSH cells (E), transfected with PI5P4K2C plasmid together with EGFP-httQ74 for the last 48 hr.(F and G) WT (Atg5+/+) or autophagy-deficient cells lacking the key autophagy gene Atg5 (Atg5−/−) MEFs were treated with PI5P4K2C siRNA (5 days) or PI5P4K2C plasmid (48 hr) together with EGFP-httQ74 for 48 hr. The percentages of transfected cells with EGFP-httQ74 aggregates were assessed and shown in the graph; n = 500 cells (mean ± SEM). Representative confocal images are shown in (G). See also Figures S3 and S4.