( A ) WT ( SCY62 ) , tag ∆ ste ∆ ( H1246 ) , tag ∆ ( H1226 ) and ste ∆ ( H1112 ) cells expressing GFP − Atg8 were grown to mid - log phase in YPD and shifted to SD - N for the indicated time periods . Cells were lysed and subjected to SDS − PAGE , followed by western blot analysis using anti - GFP antibodies . ( B ) Quantification of the GFP / GFP − Atg8 ratio . Error bars represent the s . e . m . of three independent experiments . * P < 0 . 05 ( Student ' s t - test ) . (

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

(A) WT (SCY62), tag∆ste∆ (H1246), tag∆ (H1226) and ste∆ (H1112) cells expressing GFP−Atg8 were grown to mid-log phase in YPD and shifted to SD-N for the indicated time periods. Cells were lysed and subjected to SDS−PAGE, followed by western blot analysis using anti-GFP antibodies. (B) Quantification of the GFP/GFP−Atg8 ratio. Error bars represent the s.e.m. of three independent experiments. *P < 0.05 (Student's t-test). (


. ( C ) WT ( SCY62 ) , tag ∆ ste ∆ ( H1246 ) , tag ∆ ( H1226 ) and ste ∆ ( H1112 ) cells expressing GFP - Atg8 were grown to mid - log phase in YPD and shifted to SD - N for 2 h . GFP − Atg8 was visualized by fluorescence microscopy . Scale bar , 5 μm . (

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

. (C) WT (SCY62), tag∆ste∆ (H1246), tag∆ (H1226) and ste∆ (H1112) cells expressing GFP-Atg8 were grown to mid-log phase in YPD and shifted to SD-N for 2 h. GFP−Atg8 was visualized by fluorescence microscopy. Scale bar, 5 μm. (


( D ) WT ( SCY62 ) , tag ∆ ste ∆ ( H1246 ) , tag ∆ ( H1226 ) and ste ∆ ( H1112 ) cells were grown as in ( A ) . Lysates were subjected to SDS − PAGE in urea gel , followed by western blot analysis using anti - Atg8 and anti - Pgk1 antibodies . Atg8 I , non - lipidated Atg8 ; Atg8 II , lipidated Atg8 . DIC , Differential interference contrast ; SD - N , nitrogen - starvation medium ; WT , wild type .

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

(D) WT (SCY62), tag∆ste∆ (H1246), tag∆ (H1226) and ste∆ (H1112) cells were grown as in (A). Lysates were subjected to SDS−PAGE in urea gel, followed by western blot analysis using anti-Atg8 and anti-Pgk1 antibodies. Atg8 I, non-lipidated Atg8; Atg8 II, lipidated Atg8. DIC, Differential interference contrast; SD-N, nitrogen-starvation medium; WT, wild type.


Fig . 6 Lack of lipid droplets inhibits starvation - induced formation of autophagosomes . ( A , H ) WT pep4 ∆ ( pep4 ∆ ) ( A , B ) , tag ∆ ste ∆ pep4 ∆ ( tag ∆ ste ∆ pep4 ∆ ) ( C , D ) , ste ∆ pep4 ∆ ( ste ∆ pep4 ∆ ) ( E , F ) and tag ∆ pep4 ∆ ( tag ∆ pep4 ∆ ) ( G , H ) cells were grown to an exponential phase in YPD before being starved in SD - N for 2 h , and were then processed for electron microscopy . H is a magnification of the box in panel G . Magnification of proliferating ER in a tag ∆ ste ∆ ( H1246 ) cell is shown in ( I ) Asterisks indicate autophagic bodies . N , nucleus ; PM , plasma membrane ; CW , cell wall ; ER , endoplasmic reticulum ; M , mitochondrion ; V , vacuole ; # , trehalose ; F , ER proliferation . Scale bars : 1 µm ( A - H ) and 200 nm ( I ) . ( J ) Average number of autophagic bodies per cell section was determined by counting 100 randomly selected cell profiles . Error bars represent standard deviations fromcounting of the three grids . * * * P < 0 . 001 ( Student ' s t - test ) . SD - N , nitrogen - starvation medium ; WT , wild type YPD , complete medium .

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

Fig. 6 Lack of lipid droplets inhibits starvation-induced formation of autophagosomes. (A, H) WT pep4∆ (pep4∆) (A,B), tag∆ste∆ pep4∆ (tag∆ste∆pep4∆)(C,D), ste∆ pep4∆ (ste∆pep4∆)(E,F) and tag∆ pep4∆ (tag∆pep4∆) (G,H) cells were grown to an exponential phase in YPD before being starved in SD-N for 2 h, and were then processed for electron microscopy. H is a magnification of the box in panel G. Magnification of proliferating ER in a tag∆ste∆ (H1246) cell is shown in (I) Asterisks indicate autophagic bodies. N, nucleus; PM, plasma membrane; CW, cell wall; ER, endoplasmic reticulum; M, mitochondrion; V, vacuole; #, trehalose; F, ER proliferation. Scale bars: 1 µm (A-H) and 200 nm (I). (J) Average number of autophagic bodies per cell section was determined by counting 100 randomly selected cell profiles. Error bars represent standard deviations fromcounting of the three grids. ***P < 0.001 (Student's t-test). SD-N,nitrogen-starvation medium; WT, wild type YPD, complete medium.


( A ) WT ( BY4741 ) , tgl1 ∆ , yeh1 ∆ , and yeh2 ∆ cells expressing GFP − Atg8 were grown to mid - log phase in YPD and shifted to SD - N for the indicated time periods . Cells were lysed and subjected to SDS − PAGE , followed by western blot analysis using anti - GFP antibodies . Quantification of the GFP / GFP − Atg8 ratio is presented on the right . Error bars represent the s . e . m . of three independent experiments . * P < 0 . 05 ( Student ' s t - test ) .

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

(A) WT (BY4741), tgl1∆, yeh1∆ ,and yeh2∆ cells expressing GFP−Atg8 were grown to mid-log phase in YPD and shifted to SD-N for the indicated time periods. Cells were lysed and subjected to SDS−PAGE, followed by western blot analysis using anti-GFP antibodies. Quantification of the GFP/GFP−Atg8 ratio is presented on the right. Error bars represent the s.e.m. of three independent experiments. *P < 0.05 (Student's t-test).


( B ) WT ( BY4741 ) and tgl3 ∆ tgl4 ∆ tgl5 ∆ cells expressing GFP − Atg8 were grown , treated , lysed , and western blotted as in ( A ) . Quantification of the GFP / GFP − Atg8 ratio is presented on the right . Error bars represent the s . e . m . of three independent experiments . * P < 0 . 05 ( Student ' s t - test ) . (

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

(B) WT (BY4741) and tgl3∆tgl4∆tgl5∆ cells expressing GFP−Atg8 were grown, treated, lysed, and western blotted as in (A). Quantification of the GFP/GFP−Atg8 ratio is presented on the right. Error bars represent the s.e.m. of three independent experiments. *P < 0.05 (Student's t-test). (


( C ) WT ( BY4741 ) , ayr1 ∆ and ldh1 ∆ cells expressing GFP − Atg8 were grown , treated , lysed , and western blotted as in ( A ) . Quantification of the GFP / GFP − Atg8 ratio is presented on the right . Error bars represent the s . e . m . of three independent experiments . * P < 0 . 05 ( Student ' s t - test ) .

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

(C) WT (BY4741), ayr1∆ and ldh1∆ cells expressing GFP−Atg8 were grown, treated, lysed, and western blotted as in (A). Quantification of the GFP/GFP−Atg8 ratio is presented on the right. Error bars represent the s.e.m. of three independent experiments. *P < 0.05 (Student's t-test).


( D ) WT ( BY4741 ) , ldh1 ∆ ayr1 ∆ cells expressing GFP − Atg8 were grown , treated , lysed , and western blotted as in ( A ) . Quantification of the GFP / GFP − Atg8 ratio is presented on the right . Error bars represent the s . e . m . of three independent experiments . * * P < 0 . 01 ( Student ' s - t test ) .

(D) WT (BY4741), ldh1∆ayr1∆ cells expressing GFP−Atg8 were grown, treated, lysed, and western blotted as in (A). Quantification of the GFP/GFP−Atg8 ratio is presented on the right. Error bars represent the s.e.m. of three independent experiments. **P < 0.01 (Student's-t test).


( E ) WT ( BY4741 ) , ice2 ∆ and ldb16 ∆ cells expressing GFP − Atg8 were grown , treated , lysed , and western blotted as in ( A ) . Quantification of the GFP / GFP − Atg8 ratio is presented on the right . Error bars represent the s . e . m . of three independent experiments . * P < 0 . 05 , * * P < 0 . 01 * * * P < 0 . 001 ( 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

(E) WT (BY4741), ice2∆ and ldb16∆ cells expressing GFP−Atg8 were grown, treated, lysed, and western blotted as in (A). Quantification of the GFP/GFP−Atg8 ratio is presented on the right. Error bars represent the s.e.m. of three independent experiments. *P < 0.05, **P < 0.01 ***P < 0.001 (Student's t-test).


( F ) WT ( BY4741 ) , ice2 ∆ and yeh1 ∆ cells expressing GFP − Atg8 were grown to mid - log phase in YPD and shifted to SD - N for 4 h . GFP − Atg8 was visualized by fluorescence microscopy . Scale bar , 5 μm . DIC , Differential interference contrast ; SD - N , nitrogen - starvation medium ; WT , wild type .

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

(F) WT (BY4741), ice2∆ and yeh1∆ cells expressing GFP−Atg8 were grown to mid-log phase in YPD and shifted to SD-N for 4 h. GFP−Atg8 was visualized by fluorescence microscopy. Scale bar, 5 μm. DIC, Differential interference contrast; SD-N, nitrogen-starvation medium; WT, wild type.


( A ) Total lysates of different mouse organs underwent immunoblotting analysis using anti - Pdia4 and anti - actin antibodies . Data information : Data from 3 experiments are presented as the mean ± SD .

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

(A) Total lysates of different mouse organs underwent immunoblotting analysis using anti-Pdia4 and anti-actin antibodies. Data information: Data from 3 experiments are presented as the mean ± SD.


( C ) Min6 cells were treated with glucose and palmitate at the indicated dosages . Total lysate underwent immunoblotting analysis using the indicated antibodies . ( D ) Human islets were treated with glucose and palmitate at the indicated dosages , followed by immunoblotting analysis . ( Data information : Data from 3 experiments are presented as the mean ± SD .

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

(C) Min6 cells were treated with glucose and palmitate at the indicated dosages. Total lysate underwent immunoblotting analysis using the indicated antibodies. (D) Human islets were treated with glucose and palmitate at the indicated dosages, followed by immunoblotting analysis. ( Data information: Data from 3 experiments are presented as the mean ± SD.


( G ) Sera from wild - type ( WT ) BKS and Leprdb / db mice , aged 4 , 8 , and 18 weeks , B6 mice , fed with a normal diet ( ND ) and a high fat diet ( HFD ) for 4 weeks , and humans , healthy volunteers and diabetic patients ( T2D ) , were quantified for Pdia4 level using an anti - Pdia4 ELISA kit . Data information : Data from 3 experiments more are presented as the mean ± SD . One - way ANOVA test was used for statistical analysis of differences between groups , and P ( * ) < 0 . 05 ; P ( * * ) < 0 . 01 and P ( * * * ) < 0 . 001 are considered statistically significant .

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

(G) Sera from wild-type (WT) BKS and Leprdb/db mice, aged 4, 8, and 18 weeks, B6 mice, fed with a normal diet (ND) and a high fat diet (HFD) for 4 weeks, and humans, healthy volunteers and diabetic patients (T2D), were quantified for Pdia4 level using an anti-Pdia4 ELISA kit. Data information: Data from 3 experiments more are presented as the mean ± SD. One-way ANOVA test was used for statistical analysis of differences between groups, and P (*) < 0.05; P (**) < 0.01 and P (***) < 0.001 are considered statistically significant.


( H ) Immunoblotting analysis of Pdia4 and markers in the cytosolic ( Cyto ) , nuclear ( Nuc ) , membrane ( Mem ) , mitochondrial ( Mito ) , and ER compartments of Ming 6 cells using the indicated antibodies . Data information : Data from 3 experiments are presented as the mean ± SD .

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

(H) Immunoblotting analysis of Pdia4 and markers in the cytosolic (Cyto), nuclear (Nuc), membrane (Mem), mitochondrial (Mito), and ER compartments of Ming 6 cells using the indicated antibodies. Data information: Data from 3 experiments are presented as the mean ± SD.


( A ) Survival rate and life span of WT , Pdia4 - / - ( KO ) , and Pdia4tg / tg ( TG ) mice on Leprdb / db background from birth to 120 weeks Data information : The number of mice ( n ) is indicated in parentheses . Data from over 3 experiments are presented as the mean ± SD . Log rank test were used for statistical analysis of differences between groups , and P ( * ) < 0 . 05 ; P ( * * ) < 0 . 01 and P ( * * * ) < 0 . 001 are considered statistically significant .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Survival rate and life span of WT, Pdia4-/-(KO), and Pdia4tg/tg (TG) mice on Leprdb/db background from birth to 120 weeks Data information: The number of mice (n) is indicated in parentheses. Data from over 3 experiments are presented as the mean ± SD. Log rank test were used for statistical analysis of differences between groups, and P (*) < 0.05; P (**) < 0.01 and P (***) < 0.001 are considered statistically significant.


( B ) Pancreata of 18 - week - old WT and Pdia4 - / - ( KO ) mice on BKS or Leprdb / db background were stained with anti - insulin ( αIns ) antibody and dihydroethidium ( DHE ) ( left ) . Islet area ( μm2 ) and relative fluorescence intensity ( RFI ) were quantified ( right ) . Scale bar : 100 μm . The dash circles indicate islet regions . Data information : Data from over 3 experiments are presented as the mean ± SD . one - way ANOVA test were used for statistical analysis of differences between groups , and P ( * ) < 0 . 05 ; P ( * * ) < 0 . 01 and P ( * * * ) < 0 . 001 are considered statistically significant .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Pancreata of 18-week-old WT and Pdia4-/- (KO) mice on BKS or Leprdb/db background were stained with anti-insulin (αIns) antibody and dihydroethidium (DHE) (left). Islet area (μm2) and relative fluorescence intensity (RFI) were quantified (right). Scale bar: 100 μm. The dash circles indicate islet regions. Data information: Data from over 3 experiments are presented as the mean ± SD. one-way ANOVA test were used for statistical analysis of differences between groups, and P (*) < 0.05; P (**) < 0.01 and P (***) < 0.001 are considered statistically significant.


( D ) The same batch mice as in ( B ) were given water containing BrdU . The BrdU + cells of the islets were visualized and quantified ( BrdU + labeling ) . TUNEL - positive cells in the islets of the mice ( B ) were visualized and quantified ( TUNEL assay ) . BrdU + cells per islet area ( 0 . 01 mm2 ) and TUNEL + cells per islet area ( 0 . 05 mm2 ) are expressed in arbitrary units ( AU ) . The dash circles indicate islet regions and the black arrowheads indicate TUNEL + cells . Data information : Data from over 3 experiments are presented as the mean ± SD . one - way ANOVA test were used for statistical analysis of differences between groups , and P ( * ) < 0 . 05 ; P ( * * ) < 0 . 01 and P ( * * * ) < 0 . 001 are considered statistically significant .

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

(D) The same batch mice as in (B) were given water containing BrdU. The BrdU+ cells of the islets were visualized and quantified (BrdU+ labeling). TUNEL-positive cells in the islets of the mice (B) were visualized and quantified (TUNEL assay). BrdU+ cells per islet area (0.01 mm2) and TUNEL+ cells per islet area (0.05 mm2) are expressed in arbitrary units (AU). The dash circles indicate islet regions and the black arrowheads indicate TUNEL+ cells. Data information: Data from over 3 experiments are presented as the mean ± SD. one-way ANOVA test were used for statistical analysis of differences between groups, and P (*) < 0.05; P (**) < 0.01 and P (***) < 0.001 are considered statistically significant.


F ) insulin in supernatants of the mouse islets ( F ) in GSIS assays . High glucose ( HG , 16 . 7 mM ) or low glucose ( LG , 3 . 3 ) were used in GSIS assays . Data information : Data from over 3 experiments are presented as the mean ± SD . one - way ANOVA test were used for statistical analysis of differences between groups , and P ( * ) < 0 . 05 ; P ( * * ) < 0 . 01 and P ( * * * ) < 0 . 001 are considered statistically significant .

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

F) insulin in supernatants of the mouse islets (F) in GSIS assays. High glucose (HG, 16.7 mM) or low glucose (LG, 3.3) were used in GSIS assays. Data information: Data from over 3 experiments are presented as the mean ± SD. one-way ANOVA test were used for statistical analysis of differences between groups, and P (*) < 0.05; P (**) < 0.01 and P (***) < 0.001 are considered statistically significant.


( B ) Pancreata of the same batch of mice as in ( A ) , at the age of 14 weeks , were stained with anti - insulin ( αIns ) antibody and DHE ( left ) . Islet area ( μm2 ) and relative fluorescence intensity ( RFI ) were quantified ( right ) . Scale bar = 100 μm . The dash circles indicate islet regions . Data information : Data from 3 experiments more are presented as the mean ± SD . One - way ANOVA test was used for statistical analysis of differences between groups , and P ( * ) < 0 . 05 ; P ( * * ) < 0 . 01 and P ( * * * ) < 0 . 001 are considered statistically significant .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Pancreata of the same batch of mice as in (A), at the age of 14 weeks, were stained with anti-insulin (αIns) antibody and DHE (left). Islet area (μm2) and relative fluorescence intensity (RFI) were quantified (right). Scale bar = 100 μm. The dash circles indicate islet regions. Data information: Data from 3 experiments more are presented as the mean ± SD. One-way ANOVA test was used for statistical analysis of differences between groups, and P (*) < 0.05; P (**) < 0.01 and P (***) < 0.001 are considered statistically significant.


( D ) The islets of the mice from ( C ) were tested for GSIS . High glucose ( HG , 16 . 7 mM ) or low glucose ( LG , 3 . 3 mM ) were used in GSIS assays . Data information : Data from 3 experiments more are presented as the mean ± SD . One - way ANOVA test was used for statistical analysis of differences between groups , and P ( * ) < 0 . 05 ; P ( * * ) < 0 . 01 and P ( * * * ) < 0 . 001 are considered statistically significant .

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

(D) The islets of the mice from (C) were tested for GSIS. High glucose (HG, 16.7 mM) or low glucose (LG, 3.3 mM) were used in GSIS assays. Data information: Data from 3 experiments more are presented as the mean ± SD. One-way ANOVA test was used for statistical analysis of differences between groups, and P (*) < 0.05; P (**) < 0.01 and P (***) < 0.001 are considered statistically significant.


( A ) The islets of WT , Pdia4 - / - ( KO ) and Pdia4tg / tg ( TG ) BKS mice were isolated and grown in complete DMEM medium containing 3 . 3 mM ( LG ) and 30 mM glucose ( HG ) for 12 h . The islets stained with propidium iodide ( PI ) , photographed , and quantified . Scale bar = 50 μm . Data information : Data from 3 experiments are presented as the mean ± SD . One - way ANOVA test was used for statistical analysis of differences between groups , and P ( * ) < 0 . 05 ; P ( * * ) < 0 . 01 and P ( * * * ) < 0 . 001 are considered statistically significant .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) The islets of WT, Pdia4-/- (KO) and Pdia4tg/tg (TG) BKS mice were isolated and grown in complete DMEM medium containing 3.3 mM (LG) and 30 mM glucose (HG) for 12 h. The islets stained with propidium iodide (PI), photographed, and quantified. Scale bar = 50 μm. Data information: Data from 3 experiments are presented as the mean ± SD. One-way ANOVA test was used for statistical analysis of differences between groups, and P (*) < 0.05; P (**) < 0.01 and P (***) < 0.001 are considered statistically significant.


The islets from the mice ( A ) were incubated with MitoGreen plus MitoSOX in the presence of glucose at 3 . 3 mM ( LG ) and 16 . 7 mM ( HG ) . Mitochondrial ROS in the islets were visualized and quantified . Scale bar = 100 μm . Data information : Data from 3 experiments are presented as the mean ± SD . One - way ANOVA test was used for statistical analysis of differences between groups , and P ( * ) < 0 . 05 ; P ( * * ) < 0 . 01 and P ( * * * ) < 0 . 001 are considered statistically significant .

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

The islets from the mice (A) were incubated with MitoGreen plus MitoSOX in the presence of glucose at 3.3 mM (LG) and 16.7 mM (HG). Mitochondrial ROS in the islets were visualized and quantified. Scale bar = 100 μm. Data information: Data from 3 experiments are presented as the mean ± SD. One-way ANOVA test was used for statistical analysis of differences between groups, and P (*) < 0.05; P (**) < 0.01 and P (***) < 0.001 are considered statistically significant.


The islets from the mice ( A ) were incubated with Hoechst 33342 ( Ho ) plus CM - H2DCFDA in the presence of glucose at 3 . 3 mM ( LG ) and 16 . 7 mM ( HG ) . cytosolic ROS in the islets were visualized and quantified . Scale bar = 100 μm . Data information : Data from 3 experiments are presented as the mean ± SD . One - way ANOVA test was used for statistical analysis of differences between groups , and P ( * ) < 0 . 05 ; P ( * * ) < 0 . 01 and P ( * * * ) < 0 . 001 are considered statistically significant .

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

The islets from the mice (A) were incubated with Hoechst 33342 (Ho) plus CM-H2DCFDA in the presence of glucose at 3.3 mM (LG) and 16.7 mM (HG). cytosolic ROS in the islets were visualized and quantified. Scale bar = 100 μm. Data information: Data from 3 experiments are presented as the mean ± SD. One-way ANOVA test was used for statistical analysis of differences between groups, and P (*) < 0.05; P (**) < 0.01 and P (***) < 0.001 are considered statistically significant.


( E ) Mitochondria isolated from the mouse islets ( A ) were measured for the activity of ETC CI ( left ) , CII / III ( middle ) and CIV ( right ) using MitoCheck assays . Data information : Data from 3 experiments are presented as the mean ± SD . One - way ANOVA test was used for statistical analysis of differences between groups , and P ( * ) < 0 . 05 ; P ( * * ) < 0 . 01 and P ( * * * ) < 0 . 001 are considered statistically significant .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Mitochondria isolated from the mouse islets (A) were measured for the activity of ETC CI (left), CII/III (middle) and CIV (right) using MitoCheck assays. Data information: Data from 3 experiments are presented as the mean ± SD. One-way ANOVA test was used for statistical analysis of differences between groups, and P (*) < 0.05; P (**) < 0.01 and P (***) < 0.001 are considered statistically significant.


( A ) Ultrastructure of mitochondria and insulin granules in typical β - cells of 3 mouse lines on BKS and Leprdb / db backgrounds . Arrows indicate the mitochondria whereas arrowheads indicate insulin granules . Scale bar = 1 μm . Data information : A total of 15 - 23 images per group were analyzed . Data from each group are presented as the mean ± SD .

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

(A) Ultrastructure of mitochondria and insulin granules in typical β-cells of 3 mouse lines on BKS and Leprdb/db backgrounds. Arrows indicate the mitochondria whereas arrowheads indicate insulin granules. Scale bar = 1 μm. Data information: A total of 15-23 images per group were analyzed. Data from each group are presented as the mean ± SD.


( E ) The construct encoding Flag - Pdia4 or its mutants and that expressing Myc / Flag - tagged Ndufs3 ( left ) or p22phox ( right ) were co - transfected into 293T cells . Total lysates ( TL ) and anti - Myc immunoprecipitates ( Myc IP ) underwent immunoblotting analysis . N - terminal Flag tag , catalytic domains ( a , a ' and a ' ' ) , non - catalytic domains ( b and b ' ) , and C - terminal KEEL are indicated . Pdia4 * is a mutant of Pdia4 whose 3 CGHC motifs were changed into 3 SGHS motifs ( a * , a ' * and a ' ' * ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) The construct encoding Flag-Pdia4 or its mutants and that expressing Myc/Flag-tagged Ndufs3 (left) or p22phox (right) were co-transfected into 293T cells. Total lysates (TL) and anti-Myc immunoprecipitates (Myc IP) underwent immunoblotting analysis. N-terminal Flag tag, catalytic domains (a, a' and a''), non-catalytic domains (b and b'), and C-terminal KEEL are indicated. Pdia4* is a mutant of Pdia4 whose 3 CGHC motifs were changed into 3 SGHS motifs (a*, a'* and a''*).


( F ) The construct encoding Myc / Flag - tagged p22phox , Ndufs3 or their mutants and that expressing Flag - tagged Pdia4 were co - transfected into 293T cells . Total lysates ( TL ) and anti - Myc immunoprecipitates ( Myc IP ) underwent immunoblotting analysis using anti - Flag antibody .

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

(F) The construct encoding Myc/Flag-tagged p22phox, Ndufs3 or their mutants and that expressing Flag-tagged Pdia4 were co-transfected into 293T cells. Total lysates (TL) and anti-Myc immunoprecipitates (Myc IP) underwent immunoblotting analysis using anti-Flag antibody.


( A ) Min6 cells infected with a lentivirus expressing a scramble RNAi ( GK ) , a Pdia4 RNAi ( KD ) and a Pdia4 cDNA ( OVE ) were sorted and tested for the Pdia4 protein level ( left ) . The cells were incubated with NAC ( 1 mM ) and then stained with MitoSOX or CellROX in response to 0 . 5 mM ( LG ) and 25 mM ( HG ) glucose for an additional 30 min . Signal from MitoSOX ( middle ) and CellROX ( right ) was re - plotted into histograms . Data information : Data from 3 experiments are presented as the mean ± SD . One - way ANOVA test was used for statistical analysis of differences between groups , and P ( * ) < 0 . 05 ; P ( * * ) < 0 . 01 and P ( * * * ) < 0 . 001 are considered statistically significant .

O O O O O O O O O O O O O O O O O O O O O O O O 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Min6 cells infected with a lentivirus expressing a scramble RNAi (GK), a Pdia4 RNAi (KD) and a Pdia4 cDNA (OVE) were sorted and tested for the Pdia4 protein level (left). The cells were incubated with NAC (1 mM) and then stained with MitoSOX or CellROX in response to 0.5 mM (LG) and 25 mM (HG) glucose for an additional 30 min. Signal from MitoSOX (middle) and CellROX (right) was re-plotted into histograms. Data information: Data from 3 experiments are presented as the mean ± SD. One-way ANOVA test was used for statistical analysis of differences between groups, and P (*) < 0.05; P (**) < 0.01 and P (***) < 0.001 are considered statistically significant.


( B ) Min6 cells infected with a lentivirus expressing a scramble RNAi ( GK ) and an RNAi of Ndufs3 ( Ndufs3 KD ) or p22phox ( p22phox KD ) were selected and tested for levels of Ndufs3 ( 1st column ) , mitochondrial ROS ( 2nd column , MitoSOX ) , p22phox ( 3rd column ) , and cytosolic ROS ( 4th column , CellROX ) . The cells were grown in the presence of 0 . 5 mM ( LG ) and 25 mM ( HG ) glucose . ( C ) The same experiments as ( B ) were conducted except that Min6 cells were infected with a lentivirus expressing a cDNA of truncated Ndufs3 ( tNdufs3 ) and p22phox ( tp22phox ) . ( Data information : Data from 3 experiments are presented as the mean ± SD . One - way ANOVA test was used for statistical analysis of differences between groups , and P ( * ) < 0 . 05 ; P ( * * ) < 0 . 01 and P ( * * * ) < 0 . 001 are considered statistically significant .

O O O O O O O O O O O O O O O O O O O O O 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-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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Min6 cells infected with a lentivirus expressing a scramble RNAi (GK) and an RNAi of Ndufs3 (Ndufs3 KD) or p22phox (p22phox KD) were selected and tested for levels of Ndufs3 (1st column), mitochondrial ROS (2nd column, MitoSOX), p22phox (3rd column), and cytosolic ROS (4th column, CellROX). The cells were grown in the presence of 0.5 mM (LG) and 25 mM (HG) glucose. (C) The same experiments as (B) were conducted except that Min6 cells were infected with a lentivirus expressing a cDNA of truncated Ndufs3 (tNdufs3) and p22phox (tp22phox). ( Data information: Data from 3 experiments are presented as the mean ± SD. One-way ANOVA test was used for statistical analysis of differences between groups, and P (*) < 0.05; P (**) < 0.01 and P (***) < 0.001 are considered statistically significant.


( D ) 293T cells , which were transfected with the construct encoding Flag - Pdia4 and that expressing Myc / Flag - tagged p22phox or Ndufs3 , were treated with GHTT ( 28 μM ) for 30 min . The cells were lysed and incubated with anti - Pdia4 antibodies plus protein G beads . Their total lysates ( TL ) and immunoprecipitates ( IP ) underwent immunoblotting analysis with anti - Flag antibody .

O O O O O O O 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

(D) 293T cells, which were transfected with the construct encoding Flag-Pdia4 and that expressing Myc/Flag-tagged p22phox or Ndufs3, were treated with GHTT (28 μM) for 30 min. The cells were lysed and incubated with anti-Pdia4 antibodies plus protein G beads. Their total lysates (TL) and immunoprecipitates (IP) underwent immunoblotting analysis with anti-Flag antibody.


( F ) The sections of pancreata of the mice ( E ) were stained with anti - insulin ( αIns ) antibody and dihydroethidium ( DHE ) ( left ) . Islet area ( μm2 ) and relative fluorescence intensity ( RFI ) were quantified ( right ) . Scale bar : 100 μm . The dash circles indicate islet regions . Data information : Data from 3 experiments are presented as the mean ± SD . one - way ANOVA test were used for statistical analysis of differences between groups , and P ( * ) < 0 . 05 ; P ( * * ) < 0 . 01 and P ( * * * ) < 0 . 001 are considered statistically significant .

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

(F) The sections of pancreata of the mice (E) were stained with anti-insulin (αIns) antibody and dihydroethidium (DHE) (left). Islet area (μm2) and relative fluorescence intensity (RFI) were quantified (right). Scale bar: 100 μm. The dash circles indicate islet regions. Data information: Data from 3 experiments are presented as the mean ± SD. one-way ANOVA test were used for statistical analysis of differences between groups, and P (*) < 0.05; P (**) < 0.01 and P (***) < 0.001 are considered statistically significant.


C , Microscale thermophoresis ( MST ) with purified ts5 dDATGAT construct displaying affinity of 10 µM for NO711 . D , dDATmfc did not show any interactions with NO711 in the MST experiments . Data information : All measurements of inhibition potency were the result of two independent experiments performed in triplicate ( n = 6 ) and all the points were used for calculating the values where the error bars represent s . e . m .

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-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

C, Microscale thermophoresis (MST) with purified ts5 dDATGAT construct displaying affinity of 10 µM for NO711. D, dDATmfc did not show any interactions with NO711 in the MST experiments. Data information: All measurements of inhibition potency were the result of two independent experiments performed in triplicate (n=6) and all the points were used for calculating the values where the error bars represent s.e.m.


E , SKF89976a interacts with purified ts5 dDATGAT construct with an affinity of 34 µM . F , MST experiments display no interactions of purified dDATmfc with SKF89976a . The MST profiles shown are one of two independent measurements . Data information : All measurements of inhibition potency were the result of two independent experiments performed in triplicate ( n = 6 ) and all the points were used for calculating the values where the error bars represent s . e . m .

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 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

E, SKF89976a interacts with purified ts5 dDATGAT construct with an affinity of 34 µM. F, MST experiments display no interactions of purified dDATmfc with SKF89976a. The MST profiles shown are one of two independent measurements. Data information: All measurements of inhibition potency were the result of two independent experiments performed in triplicate (n=6) and all the points were used for calculating the values where the error bars represent s.e.m.


G , Michaelis - Menten uptake kinetics of 3H - GABA by the GAT1WT used in the study displaying a KM value of 11 . 4 μM .

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

G, Michaelis-Menten uptake kinetics of 3H-GABA by the GAT1WT used in the study displaying a KM value of 11.4 μM.


H , Inhibition of 3H - GABA uptake by the substrate analogue nipecotic acid displaying a Ki value of 14 . 4 μM . Data information : All measurements of inhibition potency were the result of two independent experiments performed in triplicate ( n = 6 ) and all the points were used for calculating the values where the error bars represent s . e . m .

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

H, Inhibition of 3H-GABA uptake by the substrate analogue nipecotic acid displaying a Ki value of 14.4 μM. Data information: All measurements of inhibition potency were the result of two independent experiments performed in triplicate (n=6) and all the points were used for calculating the values where the error bars represent s.e.m.


I , Inhibition of 3H - GABA uptake through GAT1WT by tiagabine ( Ki = 725 nM ) , NO711 ( 1 . 07 μM ) and SKF89976a ( 7 . 3 μM ) . Data information : All measurements of inhibition potency were the result of two independent experiments performed in triplicate ( n = 6 ) and all the points were used for calculating the values where the error bars represent s . e . m .

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

I, Inhibition of 3H-GABA uptake through GAT1WT by tiagabine (Ki= 725 nM), NO711 (1.07 μM) and SKF89976a (7.3 μM). Data information: All measurements of inhibition potency were the result of two independent experiments performed in triplicate (n=6) and all the points were used for calculating the values where the error bars represent s.e.m.


B , Substitution of L300 to other hydrophobic side chains and the consequences on transport activity . L300W has the maximal effect on the transport activity . The histogram represents mean of a total 6 measurements ( n = 6 ) done as two independent measurements . Individual datapoints are represented as black dots around the error bar that represents s . e . m .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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, Substitution of L300 to other hydrophobic side chains and the consequences on transport activity. L300W has the maximal effect on the transport activity. The histogram represents mean of a total 6 measurements (n=6) done as two independent measurements. Individual datapoints are represented as black dots around the error bar that represents s.e.m.


Changes to Michaelis - Menten kinetics for L300F mutant that displays weakened 3H - GABA uptake activity with KM = 20 . 9 μM . A near 10 - fold loss of nipecotic acid inhibition potency was observed at a value of 134 . 3 μM . The inhibition potencies of the inhibitors display lowered value suggesting an improved ability to compete for the GABA uptake . Data information : All data plots were performed as two independent experiments each time done in triplicate ( n = 6 ) . Error bars in the data display s . e . m . Statistical significance of inhibition potencies were measured between the mutant and GAT1WT transporter measured with an unpaired t - test display p values as follows ; nipecotic acid ,

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

Changes to Michaelis-Menten kinetics for L300F mutant that displays weakened 3H-GABA uptake activity with KM = 20.9 μM. A near 10-fold loss of nipecotic acid inhibition potency was observed at a value of 134.3 μM. The inhibition potencies of the inhibitors display lowered value suggesting an improved ability to compete for the GABA uptake. Data information: All data plots were performed as two independent experiments each time done in triplicate (n=6). Error bars in the data display s.e.m. Statistical significance of inhibition potencies were measured between the mutant and GAT1WT transporter measured with an unpaired t-test display p values as follows; nipecotic acid,


Changes to Michaelis - Menten kinetics for L300F mutant that displays weakened 3H - GABA uptake activity with KM = 20 . 9 μM . The inhibition potencies of the inhibitors display lowered value suggesting an improved ability to compete for the GABA uptake . Data information : All data plots were performed as two independent experiments each time done in triplicate ( n = 6 ) . Error bars in the data display s . e . m . Statistical significance of inhibition potencies were measured between the mutant and GAT1WT transporter measured with an unpaired t - test display p values as follows panel E - 0 . 0001 ; tiagabine , panel F - 0 . 0017 ; NO711 , panel G - 0 . 018 ;

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

Changes to Michaelis-Menten kinetics for L300F mutant that displays weakened 3H-GABA uptake activity with KM = 20.9 μM. The inhibition potencies of the inhibitors display lowered value suggesting an improved ability to compete for the GABA uptake. Data information: All data plots were performed as two independent experiments each time done in triplicate (n=6). Error bars in the data display s.e.m. Statistical significance of inhibition potencies were measured between the mutant and GAT1WT transporter measured with an unpaired t-test display p values as follows panel E-0.0001; tiagabine, panel F-0.0017; NO711, panel G-0.018; SKF89976a


E , F , Eadie - Hofstee plots of E , tiagabine and F , NO711 display competitive mode of inhibition of 3H GABA uptake in GAT1WT . G , Eadie - Hofstee plot of SKF89976a displaying mixed ( competitive + non - competitive ) inhibition of 3H - GABA uptake in GAT1WT . Data information : ( E - G ) Concentrations of the inhibitors were decided based on the Ki values calculated for individual inhibitors . Each data point was plotted using 6 measurements carried out in two independent measurements in triplicates . Error bars represent s . e . m .

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

E, F, Eadie-Hofstee plots of E, tiagabine and F, NO711 display competitive mode of inhibition of 3H GABA uptake in GAT1WT. G, Eadie-Hofstee plot of SKF89976a displaying mixed (competitive+non-competitive) inhibition of 3H-GABA uptake in GAT1WT. Data information: (E-G) Concentrations of the inhibitors were decided based on the Ki values calculated for individual inhibitors. Each data point was plotted using 6 measurements carried out in two independent measurements in triplicates. Error bars represent s.e.m.


C , Michaelis - Menten kinetics of GAT1WT and GAT1 S359E displays a weakened KM value ( 28 . 54 ± 2 . 3 μM ) . The enhanced Vmax is a consequence of the enhanced expression of the mutant transporter in the assayed cells monitored by FSEC . Data information : The kinetic plots were plotted as a mean of 6 measurements carried out in two independent measurements in triplicate . The error bars represent s . e . m .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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, Michaelis-Menten kinetics of GAT1WT and GAT1 S359E displays a weakened KM value (28.54 ± 2.3 μM). The enhanced Vmax is a consequence of the enhanced expression of the mutant transporter in the assayed cells monitored by FSEC. Data information: The kinetic plots were plotted as a mean of 6 measurements carried out in two independent measurements in triplicate. The error bars represent s.e.m.


D , 3H - GABA uptake inhibition by the substrate analogue nipecotic acid for GAT1 S359E displaying a Ki value of 30 . 1 μM Data information : The kinetic plots were plotted as a mean of 6 measurements carried out in two independent measurements in triplicate . The error bars represent s . e . m .

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

D, 3H-GABA uptake inhibition by the substrate analogue nipecotic acid for GAT1 S359E displaying a Ki value of 30.1 μM Data information: The kinetic plots were plotted as a mean of 6 measurements carried out in two independent measurements in triplicate. The error bars represent s.e.m.


E , The equivalent mutation in dDATWT E384S causes a complete ablation of transport activity despite retaining similar expression levels as dDATWT . Data information : The kinetic plots were plotted as a mean of 6 measurements carried out in two independent measurements in triplicate . The error bars represent s . e . m .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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, The equivalent mutation in dDATWT E384S causes a complete ablation of transport activity despite retaining similar expression levels as dDATWT. Data information: The kinetic plots were plotted as a mean of 6 measurements carried out in two independent measurements in triplicate. The error bars represent s.e.m.


HEp - 2 cells remained untreated or were incubated with 20 µg / ml of whole cell extract of Y . pseudotuberculosis expressing full - length CNF1 , CNFY or the N - or C - terminally deleted toxin variants at 37 ° C for 4 h . The cells were thoroughly washed , pelleted , lysed and the toxin variants bound to the cells were identified by western blotting using an anti - Flag antibody .

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

HEp-2 cells remained untreated or were incubated with 20 µg/ml of whole cell extract of Y. pseudotuberculosis expressing full-length CNF1, CNFY or the N- or C-terminally deleted toxin variants at 37°C for 4 h. The cells were thoroughly washed, pelleted, lysed and the toxin variants bound to the cells were identified by western blotting using an anti-Flag antibody.


Left upper and right panel : HEp - 2 cells re mained untreated or were incubated with 20 µg / ml of whole cell extract of Y . pseudotuberculosis expressing full - length CNFY or the N - or C - terminally deleted toxin variants for 4 h . Cells were lysed and the de amidation of RhoA was analyzed by the shift of the modified Rho GTPase band in SDS PAGE gels ; left lower panel : HEp - 2 cells were lysed and the cell extracts were incubated with full - length CNFY or the N - ter minally deleted toxin variants for 4 h . The de amidation of RhoA in the cell extracts was analyzed by the mobility shift of the modified Rho GTPase on SDS PAGE after detection with anti - RhoA antibodies .

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

Left upper and right panel: HEp-2 cells remained untreated or were incubated with 20 µg/ml of whole cell extract of Y. pseudotuberculosis expressing full-length CNFY or the N- or C-terminally deleted toxin variants for 4 h. Cells were lysed and the deamidation of RhoA was analyzed by the shift of the modified Rho GTPase band in SDS PAGE gels; left lower panel: HEp-2 cells were lysed and the cell extracts were incubated with full-length CNFY or the N-terminally deleted toxin variants for 4 h. The deamidation of RhoA in the cell extracts was analyzed by the mobility shift of the modified Rho GTPase on SDS PAGE after detection with anti-RhoA antibodies.


HEp - 2 cells were incubated with 20 µg / ml of whole cell extract of Y . pseudotuberculosis expressing full - length CNFY or the N - or C - terminally deleted toxin variants for 24 h . The cell nuclei were strained with DAPI ( blue ) and the actin cytoskeleton was stained using FITC - phalloidin ( green ) . The formation of large , multinuclear cells was ob served by fluorescence micro scopy and the formation of thick actin stress fibers and membrane actin folding were only observed with CNFY - treated cells . The white scale bar is 40 µm . Cells incubated with extracts of YP147 ( ΔcnfY ) harboring the empty expression vector were used as negative controls .

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HEp-2 cells were incubated with 20 µg/ml of whole cell extract of Y. pseudotuberculosis expressing full-length CNFY or the N- or C-terminally deleted toxin variants for 24 h. The cell nuclei were strained with DAPI (blue) and the actin cytoskeleton was stained using FITC-phalloidin (green). The formation of large, multinuclear cells was observed by fluorescence microscopy and the formation of thick actin stress fibers and membrane actin folding were only observed with CNFY-treated cells. The white scale bar is 40 µm. Cells incubated with extracts of YP147 (ΔcnfY) harboring the empty expression vector were used as negative controls.


HEp - 2 cells were incubated with 20 µg / ml of whole cell extract of Y . pseudotuberculosis expressing full - length CNFY , N - or C - terminal deletion variants fused to GFP ( green ) for 90 or 180 min . Cells were fixed and processed for fluorescence microscopy . The red fluorescent signal represents late endosomes ( CellLight Late Endosomes - RFP ( Rab7a ) ) . Nuclei were stained with DAPI ( blue ) . A merged image of the different channels is shown , and smaller images are magnified views of boxed areas . White scale bar is 10 µm .

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HEp-2 cells were incubated with 20 µg/ml of whole cell extract of Y. pseudotuberculosis expressing full-length CNFY, N- or C-terminal deletion variants fused to GFP (green) for 90 or 180 min. Cells were fixed and processed for fluorescence microscopy. The red fluorescent signal represents late endosomes (CellLight Late Endosomes-RFP (Rab7a)). Nuclei were stained with DAPI (blue). A merged image of the different channels is shown, and smaller images are magnified views of boxed areas. White scale bar is 10 µm.


Nitrocefin ( 2 mM ) was added to the supernatant from 25 ° C overnight Yersinia cultures expressing the indicated CNFY derivatives to determine β - lactamase activity by measuring changes in absorbance at 390 nm ( yellow ) and 486 nm ( red ) . The data represent the mean ± SD of three independent experiments , carried out in triplicates .

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Nitrocefin (2 mM) was added to the supernatant from 25°C overnight Yersinia cultures expressing the indicated CNFY derivatives to determine β-lactamase activity by measuring changes in absorbance at 390 nm (yellow) and 486 nm (red). The data represent the mean ± SD of three independent experiments, carried out in triplicates.


The viability of Y . pseudotuberculosis YPIII expressing the indicated CNFY derivatives was assessed in equalized bacterial cultures using the BacTiter - Glo Microbial Cell Viability Assay kit ( Promega ) . The data represent the mean ± SD of three independent experiments , carried out in triplicates .

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The viability of Y. pseudotuberculosis YPIII expressing the indicated CNFY derivatives was assessed in equalized bacterial cultures using the BacTiter-Glo Microbial Cell Viability Assay kit (Promega). The data represent the mean ± SD of three independent experiments, carried out in triplicates.


Comparative analysis of RhoA activation in HEp - 2 cell lysate by CNFY and the recombinant D4 - 5 protein . Purified CNFY or the D4 - 5 fragment ( 1 µM ) was added to extracts of HEp - 2 cells and incubated for 10 , 20 , 30 or 60 min . De amidation of RhoA was analyzed by the shift of the modified Rho GTPase band in SDS PAGE gels after detection with anti - RhoA antibodies .

Comparative analysis of RhoA activation in HEp-2 cell lysate by CNFY and the recombinant D4-5 protein. Purified CNFY or the D4-5 fragment (1 µM) was added to extracts of HEp-2 cells and incubated for 10, 20, 30 or 60 min. Deamidation of RhoA was analyzed by the shift of the modified Rho GTPase band in SDS PAGE gels after detection with anti-RhoA antibodies.


Comparative analysis of recombinant RhoA deamidation by CNFY and the D4 - 5 fragment . Recombinant RhoA was incubated with purified CNFY or the D4 - 5 fragment and samples were separated by SDS - PAGE after the indicated times before subjecting to trypsin digestion and quantification of deamidation of Q63 by mass spectrometry . Error bars represent standard deviations of triplicate measurements .

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Comparative analysis of recombinant RhoA deamidation by CNFY and the D4-5 fragment. Recombinant RhoA was incubated with purified CNFY or the D4-5 fragment and samples were separated by SDS-PAGE after the indicated times before subjecting to trypsin digestion and quantification of deamidation of Q63 by mass spectrometry. Error bars represent standard deviations of triplicate measurements.


( B ) Dot - plot showing the fold change ( log2 ) in number of reads between vehicle and AZD8186 - treated conditions vs the p - value of the difference between the two treatment conditions for each shRNA . 9 out of 18 shRNAs targeting EGFR showed a p - value < 0 . 2 and are highlighted in the plot . The plot was generated considering the results from biological triplicate of the experiment .

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(B) Dot-plot showing the fold change (log2) in number of reads between vehicle and AZD8186-treated conditions vs the p-value of the difference between the two treatment conditions for each shRNA. 9 out of 18 shRNAs targeting EGFR showed a p-value <0.2 and are highlighted in the plot. The plot was generated considering the results from biological triplicate of the experiment.


( C MDA - MB - 468 were infected with the indicated shRNAs targeting EGFR and selected by puromycin . EGFR mRNA was then measured by RT - qPCR ( C ) Average ± SD of triplicates and representative of three independent experiments .

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(C MDA-MB-468 were infected with the indicated shRNAs targeting EGFR and selected by puromycin. EGFR mRNA was then measured by RT-qPCR (C) Average ± SD of triplicates and representative of three independent experiments.


D ) MDA - MB - 468 were infected with the indicated shRNAs targeting EGFR and selected by puromycin . cell viability was measured after 4 days of treatment with serial dilutions of AZD8186 ( D ) . Average ± SD of triplicates and representative of three independent experiments .

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D) MDA-MB-468 were infected with the indicated shRNAs targeting EGFR and selected by puromycin. cell viability was measured after 4 days of treatment with serial dilutions of AZD8186 (D). Average ± SD of triplicates and representative of three independent experiments.


( E MDA - MB - 468 were treated with serial dilutions of gefitinib ( E ) in the presence of vehicle , AZD8186 ( 0 . 25 μM ) , GDC0941 ( 0 . 25 μM ) or MK2206 ( 0 . 45 μM ) , as indicated . Cell viability was measured after 4 days and normalised within each of the PI3K pathway inhibitor - treated condition to the viability in the absence of gefitinib Average ± SD of triplicates and representative of two independent experiments .

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(E MDA-MB-468 were treated with serial dilutions of gefitinib (E) in the presence of vehicle, AZD8186 (0.25 μM), GDC0941 (0.25 μM) or MK2206 (0.45 μM), as indicated. Cell viability was measured after 4 days and normalised within each of the PI3K pathway inhibitor-treated condition to the viability in the absence of gefitinib Average ± SD of triplicates and representative of two independent experiments.


F ) MDA - MB - 468 were treated with serial dilutions of lapatinib ( F ) in the presence of vehicle , AZD8186 ( 0 . 25 μM ) , GDC0941 ( 0 . 25 μM ) or MK2206 ( 0 . 45 μM ) , as indicated . Cell viability was measured after 4 days and normalised within each of the PI3K pathway inhibitor - treated condition to the viability in the absence of lapatinib . Average ± SD of triplicates and representative of two independent experiments .

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F) MDA-MB-468 were treated with serial dilutions of lapatinib (F) in the presence of vehicle, AZD8186 (0.25 μM), GDC0941 (0.25 μM) or MK2206 (0.45 μM), as indicated. Cell viability was measured after 4 days and normalised within each of the PI3K pathway inhibitor-treated condition to the viability in the absence of lapatinib. Average ± SD of triplicates and representative of two independent experiments.


( G ) Viability of six PTEN - null VS five PTEN - WT TNBC cell lines - not carrying other known mutations in PIK3CA , PIK3CB or PIK3R1 genes - treated with PI3Kβi ( AZD8186 90 nM ) , EGFRi ( gefitinib 3 μM ) alone or in combination for 6 days . Mean of 3 independent experiments ± SD . Statistical significance of two - tailed unpaired student t - test in PTEN - null PI3Kbi versus PI3Kbi + EGFRi * * P = 0 . 0059 , PTEN - null EGFRi versus PI3Kbi + EGFRi * * P = 0 . 0047 , PTEN - null PI3Kbi + EGFRi versus PTEN - WT PI3Kbi + EGFRi * P = 0 . 0459 , PTEN - WT PI3Kbi versus PI3Kbi + EGFRi * P = 0 . 0108 , PTEN - WT EGFRi versus PI3Kbi + EGFRi n . s . P = 0 . 1926 . PTEN - null cell lines used in the experiments were : MDA - MB - 468 , HCC70 , HCC1937 , HCC38 , HCC1395 and BT - 549 ; PTEN - WT cell lines were : MDA - MB - 157 , MDA - MB - 231 , HCC1187 , HCC1428 and HCC1806 .

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(G) Viability of six PTEN-null VS five PTEN-WT TNBC cell lines - not carrying other known mutations in PIK3CA, PIK3CB or PIK3R1 genes - treated with PI3Kβi (AZD8186 90 nM), EGFRi (gefitinib 3 μM) alone or in combination for 6 days. Mean of 3 independent experiments ± SD. Statistical significance of two-tailed unpaired student t-test in PTEN-null PI3Kbi versus PI3Kbi+EGFRi **P=0.0059, PTEN-null EGFRi versus PI3Kbi+EGFRi **P=0.0047, PTEN-null PI3Kbi+EGFRi versus PTEN-WT PI3Kbi+EGFRi *P=0.0459, PTEN-WT PI3Kbi versus PI3Kbi+EGFRi *P=0.0108, PTEN-WT EGFRi versus PI3Kbi+EGFRi n.s. P=0.1926. PTEN-null cell lines used in the experiments were: MDA-MB-468, HCC70, HCC1937, HCC38, HCC1395 and BT-549; PTEN-WT cell lines were: MDA-MB-157, MDA-MB-231, HCC1187, HCC1428 and HCC1806.


( H ) Synergy score for combinations of serial dilutions of PI3Kβi ( AZD8186 ) plus EGFRi ( gefitinib ) tested on six PTEN - null and five PTEN - WT TNBC cell lines for 6 days in three independent experiments . The score was obtained analysing the viability data through the software Chalice Analyser . Mean of the synergy scores ± SD . Statistical significance of Mann Whitney two - tailed test * P = 0 . 0303 .

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(H) Synergy score for combinations of serial dilutions of PI3Kβi (AZD8186) plus EGFRi (gefitinib) tested on six PTEN-null and five PTEN-WT TNBC cell lines for 6 days in three independent experiments. The score was obtained analysing the viability data through the software Chalice Analyser. Mean of the synergy scores ± SD. Statistical significance of Mann Whitney two-tailed test *P=0.0303.


( I ) Patient samples from METABRIC dataset classified as TNBCs were assigned to the groups " PTEN - low " when falling in the lower quartile for PTEN expression , " PTEN - mut " when harbouring a non - synonymous mutation on PTEN gene , " PTEN - WT " in all other cases . Comparison of the expression of EGFR between the PTEN low or mut and the PTEN - WT groups . Data presented in a box and whisker plot with the central band indicating the median , the upper and lower extremes of the box or hinge being the third and first quartiles respectively and the whiskers extending to the most extreme data values Mean ± SD . P value calculated by unpaired t - test .

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(I) Patient samples from METABRIC dataset classified as TNBCs were assigned to the groups "PTEN-low" when falling in the lower quartile for PTEN expression, "PTEN-mut" when harbouring a non-synonymous mutation on PTEN gene, "PTEN-WT" in all other cases. Comparison of the expression of EGFR between the PTEN low or mut and the PTEN-WT groups. Data presented in a box and whisker plot with the central band indicating the median, the upper and lower extremes of the box or hinge being the third and first quartiles respectively and the whiskers extending to the most extreme data values Mean ± SD. P value calculated by unpaired t-test.


( A ) Tumor volume of MDA - MB - 468 mammary fat - pad xenografts treated with vehicle , AZD8186 ( 50 mg / kg , og twice / day ) , erlotinib ( 50 mg / kg IP once / day ) alone or in combination ( 5 - 6 mice per group , mean ± SEM ) . Statistical significance of two - way ANOVA statistical test * * P = 0 . 0028 and * * * P < 0 . 0001 . ( B ) Tumor volume of HCC - 70 mammary fat - pad xenografts treated with vehicle , AZD8186 ( 150 mg / kg , og once / day ) , erlotinib ( 50 mg / kg IP once / day ) alone or in combination ( 6 - 7 mice per group , mean ± SEM ) . Growth curves were compared using two - way ANOVA statistical test . Statistical significance of two - way ANOVA statistical test * * * P = 0 . 0006 and * * * * P < 0 . 0001 .

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(A) Tumor volume of MDA-MB-468 mammary fat-pad xenografts treated with vehicle, AZD8186 (50 mg/kg, og twice/day), erlotinib (50 mg/kg IP once/day) alone or in combination (5-6 mice per group, mean±SEM). Statistical significance of two-way ANOVA statistical test **P=0.0028 and ***P<0.0001. (B) Tumor volume of HCC-70 mammary fat-pad xenografts treated with vehicle, AZD8186 (150 mg/kg, og once/day), erlotinib (50 mg/kg IP once/day) alone or in combination (6-7 mice per group, mean±SEM). Growth curves were compared using two-way ANOVA statistical test. Statistical significance of two-way ANOVA statistical test ***P=0.0006 and ****P<0.0001.


( C ) Waterfall representation of changes in the volume of individual HCC - 70 tumors during the treatment . Statistical comparison between different treatment groups by Mann - Whitney test * * P = 0 . 0023 and * * * P = 0 . 0006 .

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(C) Waterfall representation of changes in the volume of individual HCC-70 tumors during the treatment. Statistical comparison between different treatment groups by Mann-Whitney test **P=0.0023 and ***P=0.0006.


( D ) Change in the body weight of mice harbouring HCC70 xenograft tumors ( 6 - 7 mice per group , mean ± SEM ) during 21 days of treatment .

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(D) Change in the body weight of mice harbouring HCC70 xenograft tumors (6-7 mice per group, mean±SEM) during 21 days of treatment.


( E ) A cell line derived from a mammary tumor spontaneously developed in a Wap - cre : Ptenfl / fl : Tp53fl / fl mouse was cloned and injected in the mammary fat pad of syngeneic C57BL6 / J recipient mice . Tumours grew in 12 out of 35 transplanted mice and only these tumours were selected for the treatments described in the figure . When tumours reached an average volume of 100mm3 , they were treated with vehicle or a combination of AZD8186 ( 150mg / kg , og once / day ) and erlotinib ( 50mg / kg IP once / day ) . Tumours were then measured during the treatment ( 6 mice per group , mean ± SEM ) . Statistical significance of two - way ANOVA statistical test * * * * P < 0 . 0001 .

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(E) A cell line derived from a mammary tumor spontaneously developed in a Wap-cre:Ptenfl/fl:Tp53fl/fl mouse was cloned and injected in the mammary fat pad of syngeneic C57BL6/J recipient mice. Tumours grew in 12 out of 35 transplanted mice and only these tumours were selected for the treatments described in the figure. When tumours reached an average volume of 100mm3, they were treated with vehicle or a combination of AZD8186 (150mg/kg, og once/day) and erlotinib (50mg/kg IP once/day). Tumours were then measured during the treatment (6 mice per group, mean±SEM). Statistical significance of two-way ANOVA statistical test ****P<0.0001.


( A ) MDA - MB - 468 cells were treated for 24h with vehicle , PI3Kβi ( AZD8186 250 nM ) , EGFRi ( gefitinib 3 μM ) or cetuximab ( 100 μg / ml ) , alone or in combination . The cell lysates were probed with the indicated antibodies .

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(A) MDA-MB-468 cells were treated for 24h with vehicle, PI3Kβi (AZD8186 250 nM), EGFRi (gefitinib 3 μM) or cetuximab (100 μg/ml), alone or in combination. The cell lysates were probed with the indicated antibodies.


( B ) MDA - MB - 468 cells were treated for 24h with vehicle , AKTi ( MK2206 450 nM ) , gefitinib ( 3 μM ) or cetuximab ( 100 μg / ml ) , alone or in the indicated combinations . The cell lysates were probed with the indicated antibodies .

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(B) MDA-MB-468 cells were treated for 24h with vehicle, AKTi (MK2206 450 nM), gefitinib (3 μM) or cetuximab (100 μg/ml), alone or in the indicated combinations. The cell lysates were probed with the indicated antibodies.


( C , D ) HCC70 ( C ) and ZR - 75 - 1 ( D ) parental cells or PI3Kβi - Res ( derivative cells with acquired resistance to AZD8186 ) , or AKTi - Res ( acquired resistance models to MK2206 ) were treated with vehicle , PI3Kβi ( AZD8186 250 nM ) or AKTi ( MK2206 1 μM ) for 24 hours . The whole cell lysates were then probed with the indicated antibodies . Spliced images of parental and resistant paired samples were taken from the same original blot and blots showing P - S6 and S6 tot in ( D ) have been cut and reassembled for figure purposes .

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(C, D) HCC70 (C) and ZR-75-1 (D) parental cells or PI3Kβi-Res (derivative cells with acquired resistance to AZD8186), or AKTi-Res (acquired resistance models to MK2206) were treated with vehicle, PI3Kβi (AZD8186 250 nM) or AKTi (MK2206 1 μM) for 24 hours. The whole cell lysates were then probed with the indicated antibodies. Spliced images of parental and resistant paired samples were taken from the same original blot and blots showing P-S6 and S6 tot in (D) have been cut and reassembled for figure purposes.


( E ) HCC70 parental , PI3Kβi - Res or AKTi - Res cells were treated with vehicle , AKTi ( MK2206 250 nM ) or gefitinib ( 3 μM ) , alone or in the indicated combinations . The cell lysates were probed with the indicated antibodies .

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(E) HCC70 parental, PI3Kβi-Res or AKTi-Res cells were treated with vehicle, AKTi (MK2206 250 nM) or gefitinib (3 μM), alone or in the indicated combinations. The cell lysates were probed with the indicated antibodies.


( F ) HCC70 MK res ( acquired resistance models to MK2206 ) were treated with serial dilutions of gefitinib , alone or in combination with MK2206 ( 810 nM ) , as indicated , and viability measured after 4 days of treatment . Average ± SD of triplicates and representative of two independent experiments .

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(F) HCC70 MK res (acquired resistance models to MK2206) were treated with serial dilutions of gefitinib, alone or in combination with MK2206 (810 nM), as indicated, and viability measured after 4 days of treatment. Average ± SD of triplicates and representative of two independent experiments.


( G ) HCC70 AZD res ( acquired resistance models to AZD8186 ) were treated with serial dilutions of gefitinib , alone or in combination with AZD8186 ( 270 nM ) , as indicated , and viability measured after 4 days . Average ± SD of triplicates and representative of two independent experiments .

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(G) HCC70 AZD res (acquired resistance models to AZD8186) were treated with serial dilutions of gefitinib, alone or in combination with AZD8186 (270 nM), as indicated, and viability measured after 4 days. Average ± SD of triplicates and representative of two independent experiments.


( H , I ) p110β co - immunoprecipitates with EGFR in MDA - MB - 468 ( H ) and in HCC70 ( I ) . Cells were pre - treated with different inhibitors , including vehicle , pan - PI3Ki GDC0941 ( 1 μM for MDA - MB - 468 or 0 . 5 μM for HCC70 ) , EGFRi ( gefitinib 3 μM ) , PI3Kβi ( AZD8186 250 nM ) or a combination of EGFRi and PI3Kβi . Cell lysates were incubated with IgG control or anti - EGFR antibody and the immuno - complexes or the total lysates were immune - blotted with the indicated antibodies .

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(H,I) p110β co-immunoprecipitates with EGFR in MDA-MB-468 (H) and in HCC70 (I). Cells were pre-treated with different inhibitors, including vehicle, pan-PI3Ki GDC0941 (1 μM for MDA-MB-468 or 0.5 μM for HCC70), EGFRi (gefitinib 3 μM), PI3Kβi (AZD8186 250 nM) or a combination of EGFRi and PI3Kβi. Cell lysates were incubated with IgG control or anti-EGFR antibody and the immuno-complexes or the total lysates were immune-blotted with the indicated antibodies.


( J ) EGF - induced increase in phospho - AKT is dependent on p110β kinase activity . MDA - MB - 468 were starved in 0 % FBS and pre - treated with vehicle or PI3Kβi ( AZD8186 250 nM ) for 1h . Cell lysates were probed with the indicated antibodies . Phospho - AKT and pan - AKT bands were quantified by the use of ImageLite software : the ratio of phospho - AKT to pan - AKT normalised to the control ( left hand lane ) is shown .

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(J) EGF-induced increase in phospho-AKT is dependent on p110β kinase activity. MDA-MB-468 were starved in 0% FBS and pre-treated with vehicle or PI3Kβi (AZD8186 250 nM) for 1h. Cell lysates were probed with the indicated antibodies. Phospho-AKT and pan-AKT bands were quantified by the use of ImageLite software: the ratio of phospho-AKT to pan-AKT normalised to the control (left hand lane) is shown.


( A Results of CRISPR - Cas9 screening in combination with AZD8186 100nM ( A ) The dot - plots show for each gene knocked - out by sgRNAs the fold change ( log2 ) between treated conditions ( AZD8186 and vehicle in the fluorescence signal ( anti - phosphoS6 immunofluorescence ) versus the p - value of the difference calculated by two sided t - test . The plots represent means of biological triplicates . Genes for which it was calculated a p < 0 . 0001 and Log2 ( fold change ) > 0 . 25 are reported and highlighted in green ; genes having a 0 . 0001 0 . 25 are reported and shown in red . Gefitinib combined with AZD8186 represents the positive control of the experiment ( A ) . < 0 . 05 >

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(A Results of CRISPR-Cas9 screening in combination with AZD8186 100nM (A) The dot-plots show for each gene knocked-out by sgRNAs the fold change (log2) between treated conditions (AZD8186 and vehicle in the fluorescence signal (anti-phosphoS6 immunofluorescence) versus the p-value of the difference calculated by two sided t-test. The plots represent means of biological triplicates. Genes for which it was calculated a p<0.0001 and Log2 (fold change)>0.25 are reported and highlighted in green; genes having a 0.0001 0.25 are reported and shown in red. Gefitinib combined with AZD8186 represents the positive control of the experiment (A). <0.05>


B ) Results of CRISPR - Cas9 screening in combination with GDC0941 400nM ( B ) . The dot - plots show for each gene knocked - out by sgRNAs the fold change ( log2 ) between treated conditions GDC0941 , and vehicle in the fluorescence signal ( anti - phosphoS6 immunofluorescence ) versus the p - value of the difference calculated by two sided t - test . The plots represent means of biological triplicates . Genes for which it was calculated a p < 0 . 0001 and Log2 ( fold change ) > 0 . 25 are reported and highlighted in green ; genes having a 0 . 0001 0 . 25 are reported and shown in red . < 0 . 05 >

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B) Results of CRISPR-Cas9 screening in combination with GDC0941 400nM (B). The dot-plots show for each gene knocked-out by sgRNAs the fold change (log2) between treated conditions GDC0941, and vehicle in the fluorescence signal (anti-phosphoS6 immunofluorescence) versus the p-value of the difference calculated by two sided t-test. The plots represent means of biological triplicates. Genes for which it was calculated a p<0.0001 and Log2 (fold change)>0.25 are reported and highlighted in green; genes having a 0.0001 0.25 are reported and shown in red. <0.05>


( C ) Box & Whisker plot showing the fold change in fluorescence signal between vehicle and GDC0941 - treated conditions for MDA - MB - 468 cells transduced with non - target Control , GNB2 or GNG5 sgRNAs ( N = 2 or 3 ) . Data presented in a box and whisker plot with the central band indicating the median , the upper and lower extremes of the box or hinge being the third and first quartiles respectively and the whiskers extending to the most extreme data values within 1 . 5 times the inter - quartile range . Statistical significance of unpaired t - test * * * * P < 0 . 0001 .

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(C) Box & Whisker plot showing the fold change in fluorescence signal between vehicle and GDC0941-treated conditions for MDA-MB-468 cells transduced with non-target Control, GNB2 or GNG5 sgRNAs (N=2 or 3). Data presented in a box and whisker plot with the central band indicating the median, the upper and lower extremes of the box or hinge being the third and first quartiles respectively and the whiskers extending to the most extreme data values within 1.5 times the inter-quartile range. Statistical significance of unpaired t-test ****P<0.0001.


( D ) Biochemical analysis of GNB2 KO cells . Cell lysates of MDA - MB - 468 parental cells and three MDA - MB - 468 GNB2 KO clones were probed with the indicated antibodies . Quantification of the bands was performed by ImageLite software .

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(D) Biochemical analysis of GNB2 KO cells. Cell lysates of MDA-MB-468 parental cells and three MDA-MB-468 GNB2 KO clones were probed with the indicated antibodies. Quantification of the bands was performed by ImageLite software.


( A ) MDA - MB - 468 parental cells and three MDA - MB - 468 GNB2 KO clones were treated with vehicle , GDC0941 1 μM , gefitinib 3 μM or lapatinib 1 μM for 24 hours . The cell lysates were probed with the indicated antibodies .

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(A) MDA-MB-468 parental cells and three MDA-MB-468 GNB2 KO clones were treated with vehicle, GDC0941 1 μM, gefitinib 3 μM or lapatinib 1 μM for 24 hours. The cell lysates were probed with the indicated antibodies.


( B - D ) Viability assays of MDA - MB - 468 parental cells and three MDA - MB - 468 GNB2 KO clones treated with serial dilutions of the indicated drugs for six days . Mean ± SD of triplicates and representative of two or three independent experiments .

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(B-D) Viability assays of MDA-MB-468 parental cells and three MDA-MB-468 GNB2 KO clones treated with serial dilutions of the indicated drugs for six days. Mean ± SD of triplicates and representative of two or three independent experiments.


( E ) Patient samples from METABRIC dataset classified as TNBCs ( N = 299 ) were assigned to the groups " PTEN - low " when falling in the lower quartile for PTEN expression , " PTEN - mut " when harbouring a non - synonymous mutation on PTEN gene , " PTEN - WT " in all other cases . Comparison of the expression of GNB2 between the PTEN low or mut and the PTEN - WT groups . Box & Whisker plot ( Median / IQR / 1 . 5 * IQR Whiskers ) . P value calculated by unpaired t - test .

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(E) Patient samples from METABRIC dataset classified as TNBCs (N=299) were assigned to the groups "PTEN-low" when falling in the lower quartile for PTEN expression, "PTEN-mut" when harbouring a non-synonymous mutation on PTEN gene, "PTEN-WT" in all other cases. Comparison of the expression of GNB2 between the PTEN low or mut and the PTEN-WT groups. Box & Whisker plot (Median/IQR/1.5*IQR Whiskers). P value calculated by unpaired t-test.


( A ) Schematic of the results from the drug screening with compounds targeting GPCR signaling . MDA - MB - 468 cells were treated with the compounds of the GPCR - targeted library at three different concentrations ( 0 . 1 , 1 or 10 μM ) in combination with vehicle , GDC0941 450 nM or lapatinib 1 μM . The pS6 signal was measured by IF after 24 hours of treatment and normalized to DAPI . Z scores of the normalized fluorescence values for each drug measured in the presence of vehicle , GDC0941 or lapatinib are reported on the y axis or on the left or right x axis of the dot plot , respectively . Readings acquired following treatment with the three different concentrations of the GPCR - targeted drugs are reported in 3 different colours . Dots corresponding to vorapaxar treatments at the three different concentrations are highlighted in the plot . The values reported are mean of a biological triplicate of the experiment .

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(A) Schematic of the results from the drug screening with compounds targeting GPCR signaling. MDA-MB-468 cells were treated with the compounds of the GPCR-targeted library at three different concentrations (0.1, 1 or 10 μM) in combination with vehicle, GDC0941 450 nM or lapatinib 1 μM. The pS6 signal was measured by IF after 24 hours of treatment and normalized to DAPI. Z scores of the normalized fluorescence values for each drug measured in the presence of vehicle, GDC0941 or lapatinib are reported on the y axis or on the left or right x axis of the dot plot, respectively. Readings acquired following treatment with the three different concentrations of the GPCR-targeted drugs are reported in 3 different colours. Dots corresponding to vorapaxar treatments at the three different concentrations are highlighted in the plot. The values reported are mean of a biological triplicate of the experiment.


( B ) MDA - MB - 468 were treated for 24 h with vehicle , vorapaxar ( 10 μM ) , GDC0941 ( 1 μM ) or lapatinib ( 1 μM ) alone or in the indicated combinations . The cell lysates were probed with the indicated antibodies .

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(B) MDA-MB-468 were treated for 24 h with vehicle, vorapaxar (10 μM), GDC0941 (1 μM) or lapatinib (1 μM) alone or in the indicated combinations. The cell lysates were probed with the indicated antibodies.


( C ) MDA - MB - 468 parental cells or GNB2 KO clones were starved and treated with scramble or PAR1 activating peptide for 5 minutes , alone or in combination with vorapaxar . The cell lysates were probed with the indicated antibodies . Phospho - AKT , pan - AKT , phospho - ERK1 / 2 and pan - ERK1 / 2 bands were quantified by the use of ImageLite software : the ratio of phospho - AKT to pan - AKT and phospho - ERK1 / 2 to pan - ERK1 / 2 normalised to the control peptide - treated conditions for WT and GNB2 KO cells ( first and forth lanes , respectively ) is shown .

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(C) MDA-MB-468 parental cells or GNB2 KO clones were starved and treated with scramble or PAR1 activating peptide for 5 minutes, alone or in combination with vorapaxar. The cell lysates were probed with the indicated antibodies. Phospho-AKT, pan-AKT, phospho-ERK1/2 and pan-ERK1/2 bands were quantified by the use of ImageLite software: the ratio of phospho-AKT to pan-AKT and phospho-ERK1/2 to pan-ERK1/2 normalised to the control peptide-treated conditions for WT and GNB2 KO cells (first and forth lanes, respectively) is shown.


( D ) p - S6 / loading control signal from western blots experiments in which a panel of six TNBC PTEN - null cell lines and HCC70 cells that acquired resistance to AZD8186 or MK2206 were treated with vehicle , vorapaxar ( 10 μM ) , GDC0941 ( 1 μM ) or lapatinib ( 1 μM ) alone or in the indicated combinations . The values were normalised to vehicle treatment for all cell lines . Mean of 2 independent experiments ± SD . P values calculated by two - tailed paired student t - test * P = 0 . 0235 and * * P = 0 . 002 . Cell lines used in the experiments were : MDA - MB - 468 , HCC70 , HCC1937 , HCC38 , HCC1395 , BT - 549 , HCC70 AZD8186 - resistant and HCC70 MK2206 - resistant .

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(D) p-S6/loading control signal from western blots experiments in which a panel of six TNBC PTEN-null cell lines and HCC70 cells that acquired resistance to AZD8186 or MK2206 were treated with vehicle, vorapaxar (10 μM), GDC0941 (1 μM) or lapatinib (1 μM) alone or in the indicated combinations. The values were normalised to vehicle treatment for all cell lines. Mean of 2 independent experiments ± SD. P values calculated by two-tailed paired student t-test *P=0.0235 and **P=0.002. Cell lines used in the experiments were: MDA-MB-468, HCC70, HCC1937, HCC38, HCC1395, BT-549, HCC70 AZD8186-resistant and HCC70 MK2206-resistant.


( E ) Long - term proliferation assay of MDA - MB - 468 cells treated with vorapaxar ( 5 or 2 . 5 μM ) , GDC0941 ( 1 μM ) or lapatinib ( 1 μM ) , alone or in the indicated combinations . Cells were treated for two weeks and stained by crystal violet . One representative experiment of three is shown .

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(E) Long-term proliferation assay of MDA-MB-468 cells treated with vorapaxar (5 or 2.5 μM), GDC0941 (1 μM) or lapatinib (1 μM), alone or in the indicated combinations. Cells were treated for two weeks and stained by crystal violet. One representative experiment of three is shown.


b , Carboxy - terminal V5immunoblot of full - length ( 68 kDa ) and cleaved ( 36 kDa ) ATG16L1β . Silver stain ( bottom gels ) depicts total protein . Data represent 3 independent experiments . IB , immunoblot .

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b, Carboxy-terminal V5immunoblot of full-length (68 kDa) and cleaved (36 kDa) ATG16L1β. Silver stain (bottom gels) depicts total protein. Data represent 3 independent experiments. IB, immunoblot.


c , TNF - α - mediated processing of humanATG16L1 in monocyte - derived macrophages . Scatter plot of immunoblot densitometry depicts a ratio of cleaved : full - length ATG16L1 ( Extended Data Fig . 2 ) . n = 7 donors .

c, TNF-α-mediated processing of humanATG16L1 in monocyte-derived macrophages. Scatter plot of immunoblot densitometry depicts a ratio of cleaved:full-length ATG16L1 (Extended Data Fig. 2). n = 7 donors.


d , TNF - α - mediated processing of murineATG16L1 in macrophages . Immunoblot represents 5 independent experiments . Scatterplot represents data pooled from 5 independent experiments . WT , wild type .

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d, TNF-α-mediated processing of murineATG16L1 in macrophages. Immunoblot represents 5 independent experiments. Scatterplot represents data pooled from 5 independent experiments. WT, wild type.


a , HeLa cells transfected with control siRNA ( siCTL ) or siCaspase 8 were stimulated with 2 . 0 μM staurosporine or 20 ng ml − 1 TNF + 10 μg ml − 1 CHX .

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a, HeLa cells transfected with control siRNA (siCTL) or siCaspase 8 were stimulated with 2.0 μM staurosporine or 20 ng ml−1 TNF + 10 μg ml−1 CHX.


b , HeLa cells transfected with control siRNA or siCaspase 9 were stimulated as in a .

b, HeLa cells transfected with control siRNA or siCaspase 9 were stimulated as in a.


c , HeLa cells transfected with control , caspase - 3 - or caspase - 7 - specific siRNAs were stimulated with 20 ng ml − 1 TNF + 10 & amp ; amp ; mgr ; g ml − 1 CHX .

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c, HeLa cells transfected with control, caspase-3- or caspase-7-specific siRNAs were stimulated with 20 ng ml−1 TNF + 10 &amp;mgr;g ml−1 CHX.


d , Wild - type or caspase - 3 - knockout macrophages were stimulated with 2 . 0 µM staurosporine or 20 ng ml − 1 TNF + 10 µg ml − 1 CHX . Data in a - c represent 2 independent experiments ; data in d represent 4 independent experiments .

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d, Wild-type or caspase-3-knockout macrophages were stimulated with 2.0 µM staurosporine or 20 ng ml−1 TNF + 10 µg ml−1 CHX. Data in a-c represent 2 independent experiments; data in d represent 4 independent experiments.


a , Intracellular staining for LC3 and ATG16L1 following glucose starvation in the presence of bafilomycin A1 . White mask in LC3 channel illustrates punctate LC3 staining , representing LC3 - II . Histogram depicts mean fluorescence intensity of 10 , 000 cells per sample . b , Quantification of mean ATG16L1 fluorescence intensity in a , n = 4 mice . Glu . , glucose .

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a, Intracellular staining for LC3 and ATG16L1 following glucose starvation in the presence of bafilomycin A1. White mask in LC3 channel illustrates punctate LC3 staining, representing LC3-II. Histogram depicts mean fluorescence intensity of 10,000 cells per sample. b, Quantification of mean ATG16L1 fluorescence intensity in a, n = 4 mice. Glu., glucose.


c , Immunoblot of phosphorylated ( p - ) AMPK & amp ; amp ; agr ; ( Thr 172 ) and lipidated LC3 ( LC3 - II ) .

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c, Immunoblot of phosphorylated (p-) AMPK&amp;agr; (Thr 172) and lipidated LC3 (LC3-II).


d , Quantification of mean LC3 - II area in murine macrophages , n = 4 mice .

d, Quantification of mean LC3-II area in murine macrophages, n = 4 mice.


e , Caspase 3 activity in murine macrophages following glucose starvation , n = 3 mice . RLU , relative light units .

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e, Caspase 3 activity in murine macrophages following glucose starvation, n = 3 mice. RLU, relative light units.


f , Quantification of mean LC3 - II area in wild - type ( Casp3 + / + ) or Casp3 - knockout ( Casp3 − / − ) macrophages , n = 3 mice . Data in a - f represent 2 independent experiments .

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f, Quantification of mean LC3-II area in wild-type (Casp3+/+) or Casp3-knockout (Casp3−/−) macrophages, n = 3 mice. Data in a-f represent 2 independent experiments.


a , Quantification of ATG16L1 mean fluorescence intensity by image - based flow cytometry of 10 , 000 cells , n = 4 ( wild - type , WT ) , 5 ( T300A ) . Data represent 3 independent experiments . b , Quantification of caspase 3 activity upon glucose starvation of 2 × 104 cells , n = 5 . Data represent 2 independent experiments . c , Quantification of mean LC3 - II area of samples in a . Data represent 3 independent experiments .

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a, Quantification of ATG16L1 mean fluorescence intensity by image-based flow cytometry of 10,000 cells, n = 4 (wild-type, WT), 5 (T300A). Data represent 3 independent experiments. b, Quantification of caspase 3 activity upon glucose starvation of 2 × 104 cells, n = 5. Data represent 2 independent experiments. c, Quantification of mean LC3-II area of samples in a. Data represent 3 independent experiments.


d , Visualization of early autophagosomes by transmission electron micrographs ( TEM ) . Red arrows , early autophagosomes , characterized by vesicular structures with a double - membrane . Blue arrows , multivesicular lysosomal structures , identified by degraded intravesicular cargo . Micrographs representative of 10 - 12 images per donor , n = 4 ( WT ) , n = 3 ( T300A ) . A total of 29 ( WT ) and 26 ( T300A ) images containing autophagosomes was analysed . Histogram quantifies autophagic vesicles as a ratio of autophagosome : cytosolic area .

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d, Visualization of early autophagosomes by transmission electron micrographs (TEM). Red arrows, early autophagosomes, characterized by vesicular structures with a double-membrane. Blue arrows, multivesicular lysosomal structures, identified by degraded intravesicular cargo. Micrographs representative of 10-12 images per donor, n = 4 (WT), n = 3 (T300A). A total of 29 (WT) and 26 (T300A) images containing autophagosomes was analysed. Histogram quantifies autophagic vesicles as a ratio of autophagosome:cytosolic area.


a - c , Y . enterocolitica colony forming units ( c . f . u . ) in human and murine macrophages infected for 6 h ( 20 m . o . i . ) . Data in a are pooled from 4 independent cohorts of 3 or 4 donors per genotype , total n = 16 ( non - risk , WT ) , 15 ( T300A , risk ) . Data in b represent 3 independent experiments , n = 4 mice . Data in c represent 3 independent experiments , n = 6 .

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a-c, Y. enterocolitica colony forming units (c.f.u.) in human and murine macrophages infected for 6 h (20 m.o.i.). Data in a are pooled from 4 independent cohorts of 3 or 4 donors per genotype, total n = 16 (non-risk, WT), 15 (T300A, risk). Data in b represent 3 independent experiments, n = 4 mice. Data in c represent 3 independent experiments, n = 6.


d , TNF - α , IL - 1β and IL - 6 levels in culture media of WT and T316A macrophages infected with 20 m . o . i . Y . enterocolitica . Data represent 2 independent experiments , n = 4 mice .

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d, TNF-α, IL-1β and IL-6 levels in culture media of WT and T316A macrophages infected with 20 m.o.i. Y. enterocolitica. Data represent 2 independent experiments, n = 4 mice.


e , Ileum and colon histology ( haematoxylin and eosin staining ) of WT mice . Images are representative of 3 ( PBS ) and 8 ( Y . enterocolitica ) treated male mice .

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e, Ileum and colon histology (haematoxylin and eosin staining) of WT mice. Images are representative of 3 (PBS) and 8 (Y. enterocolitica) treated male mice.