( A , B ) Wild - type ( WT ) ( BY4741 ) expressing GFP − Atg8were grown to mid - log - phase in YPD were then preincubated with 50 µM cerulenin or 50 µM cerulenin + 0 . 1 mM palmitic / stearic / myristic or with DMSO ( - ) in rich medium for 30 min . were washed shifted to nitrogen - starvation medium ( SD - N ) for 4 h in presence of 50 µM cerulenin or 50 µM cerulenin + 0 . 1 mM palmitic / stearic / myristic or DMSO . were then lysed subjected to SDS − PAGE , followed by western blot using anti - GFP anti - Pgk1 antibodies ( A ) , or were visualized by microscopy ( B )

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

(A, B) Wild-type (WT) (BY4741) cells expressing GFP−Atg8were grown to mid-log-phase in YPD and were then preincubated with 50 µM cerulenin or 50 µM cerulenin + 0.1 mM palmitic/stearic/myristic acids or with DMSO (-) in the rich medium for 30 min. Cells were washed and shifted to nitrogen-starvation medium (SD-N) for 4 h in the presence of 50 µM cerulenin or 50 µM cerulenin + 0.1 mM palmitic/stearic/myristic acids or DMSO. Cells were then lysed and subjected to SDS−PAGE, followed by western blot analysis using anti-GFP and anti-Pgk1 antibodies (A), or were visualized by fluorescence microscopy (B)


( C ) ( TOS038 ) expressing Atg1 - GFP were grown to mid - log phase were then preincubated in YPD with 50 µM cerulenin or with DMSO for 30 min . were washed , shifted to SD - N for 4 h , then visualized by 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 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

(C) Cells (TOS038) expressing Atg1-GFP were grown to mid-log phase and were then preincubated in YPD with 50 µM cerulenin or with DMSO for 30 min. Cells were washed, shifted to SD-N for 4 h, and then visualized by fluorescence microscopy. Scale bar, 5 μm.


( D ) WT ( BY4741 ) pep4 ∆ ( TOS015 ) strains were grown to mid - log phase preincubated in YPD with cerulenin or DMSO for 30 min . were - labeled for 10 min with [ 35S ] methionine cysteine chased for indicated time periods . lysates were subjected to immunoprecipitation with anti - Ape1 antibodies followed by SDS ‒ PAGE X - ray film to detect radioactive signals . prApe1 , premature Ape1 ; mApe1 , mature Ape1 . (

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

(D) WT (BY4741) and pep4∆ (TOS015) strains were grown to mid-log phase and preincubated in YPD with cerulenin or DMSO for 30 min. Cells were pulse-labeled for 10 min with [35S] methionine and cysteine and chased for the indicated time periods. Cell lysates were subjected to immunoprecipitation with anti-Ape1 antibodies followed by SDS‒PAGE and X-ray film to detect radioactive signals. prApe1, premature Ape1; mApe1, mature Ape1. (


( E ) of fas1 ∆ ( TOS029 ) expressing GFP ‒ Atg8 were grown to mid - log phase in YPD + 0 . 1 mM palmitic / stearic / myristic shifted either to same medium or to YPD without fatty for 30 min . were then shifted to SD - N for indicated times . lysates were subjected to SDS ‒ PAGE , followed by western blot using anti - GFP 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 B-CONTROLLED_VAR O B-CONTROLLED_VAR O B-CONTROLLED_VAR O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O

(E) Cells of the fas1∆ (TOS029) strain expressing GFP‒Atg8 were grown to mid-log phase in YPD + 0.1 mM palmitic/stearic/myristic acids and shifted either to the same medium or to YPD without fatty acids for 30 min. Cells were then shifted to SD-N for the indicated times. Cell lysates were subjected to SDS‒PAGE, followed by western blot analysis using anti-GFP antibodies.


( F ) WT ( BY4741 ) were grown to mid - log phase then preincubated in YPD with 25 µM or 50 µM cerulenin ( 25 , 50 ) or with DMSO ( - ) in rich medium for 30 min . were washed shifted to SD - N in presence of cerulenin ( 25 , 50 ) or DMSO ( - ) . were lysed subjected to SDS ‒ PAGE , followed by western blot using anti - GFP anti - Pgk1 antibodies . cer , cerulenin ; DIC , Differential interference contrast ; FA , fatty ; 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 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 B-CONTROLLED_VAR O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O B-CONTROLLED_VAR O B-CONTROLLED_VAR O O O O O O O O O O O O O O

(F) WT (BY4741) cells were grown to mid-log phase and then preincubated in YPD with 25 µM or 50 µM cerulenin (25, 50) or with DMSO (-) in rich medium for 30 min. Cells were washed and shifted to SD-N in the presence of cerulenin (25, 50) or DMSO (-). Cells were lysed and subjected to SDS‒PAGE, followed by western blot analysis using anti-GFP and anti-Pgk1 antibodies. cer, cerulenin; DIC, Differential interference contrast; FA, fatty acids; SD-N, nitrogen-starvation medium; WT, wild-type YPD, complete medium.


( A ) ( TN124 ) were grown to mid - log phase preincubated in YPD with 50 µM cerulenin for indicated time periods ( Cer preincubation ) or incubated with DMSO without preincubation ] ( - ) . were then shifted to SD - N for 3 h with addition of DMSO or 50 µM cerulenin ( cer ) . Autophagic activity was measured by alkaline phosphatase . Error bars represent s . e . m . of three independent experiments . * P < 0 . 05 ( Student ' s t - ) .

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

(A) Cells (TN124 strain) were grown to mid-log phase and preincubated in YPD with 50 µM cerulenin for the indicated time periods (Cer preincubation) or incubated with DMSO without preincubation] (-). Cells were then shifted to SD-N for 3 h with the addition of DMSO or 50 µM cerulenin (cer). Autophagic activity was measured by alkaline phosphatase assay. Error bars represent the s.e.m. of three independent experiments. *P < 0.05 (Student's t-test).


( B ) were grown as in ( A ) , stained with BODIPY , visualized by 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

(B) Cells were grown as in (A), stained with BODIPY, and visualized by fluorescence microscopy. Scale bar, 5 μm.


( C ) WT ( SCY62 ) tagΔsteΔ ( H1246 ) were grown to mid - log phase in YPD shifted to SD - N for 3 h . were stained with Nile red visualized by microscopy . Scale bar , 5 μm .

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

(C) WT (SCY62) and tagΔsteΔ (H1246) cells were grown to mid-log phase in YPD and shifted to SD-N for 3 h. Cells were stained with Nile red and visualized by fluorescence microscopy. Scale bar, 5 μm.


( D ) WT ( SCY62 ) tag ∆ ste ∆ ( H1246 ) expressing GFP − Atg8 were grown to mid - log phase in YPD shifted to SD - N for indicated time periods . lysates were subjected to SDS − PAGE , followed by western blot using anti - GFP , anti - Ape1 ( prApe1 , premature Ape1 ; mApe1 , mature Ape1 ) , anti - Fas1 anti - Pgk1 antibodies .

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

(D) WT (SCY62) and tag∆ste∆ (H1246) cells expressing GFP−Atg8 were grown to mid-log phase in YPD and shifted to SD-N for the indicated time periods. Cell lysates were subjected to SDS−PAGE, followed by western blot analysis using anti-GFP, anti-Ape1 (prApe1, premature Ape1; mApe1, mature Ape1), and anti-Fas1 and anti-Pgk1 antibodies.


( E ) WT ( SCY62 ) tag ∆ ste ∆ ( H1246 ) were grown to mid - log phase in YPD shifted to SD - N for 2 h . GFP − Atg8 was visualized by microscopy . Scale bar , 5 μm . cer , cerulenin ; DIC , Differential interference contrast ; FA , fatty ; SD - N , nitrogen - starvation medium ; WT , wild - type , YPD , complete medium .

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

(E) WT (SCY62) and tag∆ste∆ (H1246) cells 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. cer, cerulenin; DIC, Differential interference contrast; FA, fatty acids; SD-N, nitrogen-starvation medium; WT, wild-type, YPD, complete medium.


( A ) GAL - DGA1GAL - ARE2lro1Δ are1Δ ( FYS118 ) was grown on SC ( synthetic minimal medium without dextrose ) + raffinoseovernight , diluted to OD 0 . 4 , grown to mid - log phase in either SC + glucose or SC + galactose medium . were shifted to SD - N for 2 h , stained with BODIPY , visualized by fluorescencemicroscopy . 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 B-CONTROLLED_VAR O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O

(A) GAL-DGA1GAL-ARE2lro1Δ are1Δ (FYS118 strain) was grown on SC (synthetic minimal medium without dextrose) + raffinoseovernight, diluted to OD 0.4, and grown to mid-log phase in either SC + glucose or SC + galactose medium. Cells were shifted to SD-N for 2 h, stained with BODIPY, and visualized by fluorescencemicroscopy. Scale bar, 5 μm.


( B ) WT ( BY4741 ) GAL - DGA1 GAL - ARE2 lro1 ∆ are1 ∆ ( FYS118 ) expressing GFP − Atg8 were grown as in A . were lysed at indicated times subjected to SDS ‒ PAGE , followed by western blot using anti - GFP antibodies . (

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

(B) WT (BY4741) and GAL-DGA1 GAL-ARE2 lro1∆ are1∆ (FYS118) cells expressing GFP−Atg8 were grown as in A. Cells were lysed at the indicated times and subjected to SDS‒PAGE, followed by western blot analysis using anti-GFP antibodies. (


( C , D ) WT ( BY4741 ) GAL - DGA1 GAL - ARE2 lro1 ∆ are1 ∆ ( FYS118 ) expressing GFP − Atg8 ( C ) or GFP − Atg1 ( D ) were grown as in A visualized by microscopy after 2 h in SD - N . 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 B-CONTROLLED_VAR O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(C, D) WT (BY4741) and GAL-DGA1 GAL-ARE2 lro1∆ are1∆ (FYS118) cells expressing GFP−Atg8 (C) or GFP−Atg1 (D) were grown as in A and visualized by fluorescence microscopy after 2 h in SD-N. Scale bar, 5 μm. DIC, Differential interference contrast; SD-N, nitrogen-starvation medium; WT, wild-type.


( A ) WT ( SCY62 ) , tag ∆ ste ∆ ( H1246 ) , tag ∆ ( H1226 ) ste ∆ ( H1112 ) expressing GFP − Atg8 were grown to mid - log phase in YPD shifted to SD - N for 6 h . were stained with BODIPY visualized by microscopy . Scale bar , 5 μm .

O O O O O O O O B-CONTROLLED_VAR O B-CONTROLLED_VAR O O O O O B-CONTROLLED_VAR O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR 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 6 h. Cells were stained with BODIPY and visualized by fluorescence microscopy. Scale bar, 5 μm.


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

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

(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 ) ste ∆ ( H1112 ) expressing GFP - Atg8 were grown to mid - log phase in YPD shifted to SD - N for 2 h . GFP − Atg8 was visualized by microscopy . Scale bar , 5 μm . (

O O O O O O O O O B-CONTROLLED_VAR O B-CONTROLLED_VAR O O O O O B-CONTROLLED_VAR O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR 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 ) ste ∆ ( H1112 ) were grown as in ( A ) . Lysates were subjected to SDS − PAGE in urea gel , followed by western blot using anti - Atg8 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 B-CONTROLLED_VAR O B-CONTROLLED_VAR O O O O O B-CONTROLLED_VAR O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O B-MEASURED_VAR O O O O O B-MEASURED_VAR O B-MEASURED_VAR O O O B-MEASURED_VAR 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 ) tag ∆ pep4 ∆ ( tag ∆ pep4 ∆ ) ( G , H ) were grown to an exponential phase in YPD before being starved in SD - N for 2 h , were then processed for electron microscopy . H is a magnification of box in panel G . Magnification of proliferating ER in a tag ∆ ste ∆ ( H1246 ) is shown in ( I ) Asterisks indicate autophagic bodies . N , nucleus ; PM , plasma ; CW , wall ; ER , endoplasmic reticulum ; M , mitochondrion ; V , vacuole ; # , trehalose ; F , ER proliferation . Scale bars : 1 µm ( A - H ) 200 nm ( I ) . ( J ) Average number of autophagic bodies per section was determined by counting 100 randomly selected profiles . Error bars represent standard deviations fromcounting of three grids . * * * P < 0 . 001 ( Student ' s t - ) . 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 B-CONTROLLED_VAR O O B-CONTROLLED_VAR O O O O O O O O B-CONTROLLED_VAR O B-CONTROLLED_VAR O B-CONTROLLED_VAR O O B-CONTROLLED_VAR O B-CONTROLLED_VAR O B-CONTROLLED_VAR O O O O O O O O B-CONTROLLED_VAR O B-CONTROLLED_VAR O O B-CONTROLLED_VAR O B-CONTROLLED_VAR O O O O O O O B-CONTROLLED_VAR O B-CONTROLLED_VAR O O B-CONTROLLED_VAR O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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

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

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

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

O O O O O O O B-CONTROLLED_VAR 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 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

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

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 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

(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 ∆ expressing GFP − Atg8 were grown , treated , lysed , western blotted as in ( A ) . of GFP / GFP − Atg8 is presented on right . Error bars represent s . e . m . of three independent experiments . * * P < 0 . 01 ( Student ' s - t ) .

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 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

(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 ∆ ldb16 ∆ expressing GFP − Atg8 were grown , treated , lysed , western blotted as in ( A ) . of GFP / GFP − Atg8 is presented on right . Error bars represent s . e . m . of three independent experiments . * P < 0 . 05 , * * P < 0 . 01 * * * P < 0 . 001 ( Student ' s t - ) .

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 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

(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 ∆ yeh1 ∆ expressing GFP − Atg8 were grown to mid - log phase in YPD shifted to SD - N for 4 h . GFP − Atg8 was visualized by 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 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 B-CONTROLLED_VAR 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 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 using anti - Pdia4 anti - actin antibodies . Data information : Data from 3 experiments are presented as mean ± SD .

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

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

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 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

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

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 B-MEASURED_VAR 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

(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 of Pdia4 markers in ( Cyto ) , ( Nuc ) , ( Mem ) , mitochondrial ( Mito ) , ER compartments of Ming 6 using indicated antibodies . Data information : Data from 3 experiments are presented as mean ± SD .

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

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

O O O 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 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

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

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

(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 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 mean ± SD . one - way ANOVA were used for statistical of differences between groups , P ( * ) < 0 . 05 ; P ( * * ) < 0 . 01 P ( * * * ) < 0 . 001 are considered statistically significant .

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

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

(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 ) islets of 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 mean ± SD . One - way ANOVA was used for statistical of differences between groups , P ( * ) < 0 . 05 ; P ( * * ) < 0 . 01 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

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

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


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

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.


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

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

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

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

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

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

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

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

(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 infected with a lentivirus expressing a scramble RNAi ( GK ) an RNAi of Ndufs3 ( Ndufs3 KD ) or p22phox ( p22phox KD ) were selected tested for of Ndufs3 ( 1st column ) , mitochondrial ROS ( 2nd column , MitoSOX ) , p22phox ( 3rd column ) , ROS ( 4th column , CellROX ) . were grown in presence of 0 . 5 mM ( LG ) 25 mM ( HG ) glucose . ( C ) same experiments as ( B ) were conducted except that Min6 were infected with a lentivirus expressing a cDNA of truncated Ndufs3 ( tNdufs3 ) p22phox ( tp22phox ) . ( Data information : Data from 3 experiments are presented as mean ± SD . One - way ANOVA was used for statistical of differences between groups , P ( * ) < 0 . 05 ; P ( * * ) < 0 . 01 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 B-CONTROLLED_VAR O B-CONTROLLED_VAR O O O B-CONTROLLED_VAR O B-CONTROLLED_VAR O O O O O O O B-MEASURED_VAR O O O O O O B-MEASURED_VAR O O O O O O O B-MEASURED_VAR 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 B-CONTROLLED_VAR O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 , which were transfected with construct encoding Flag - Pdia4 that expressing Myc / Flag - tagged p22phox or Ndufs3 , were treated with GHTT ( 28 μM ) for 30 min . were lysed incubated with anti - Pdia4 antibodies plus G beads . Their total lysates ( TL ) immunoprecipitates ( IP ) underwent immunoblotting 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 B-CONTROLLED_VAR O B-CONTROLLED_VAR O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O 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

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

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 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 MST experiments . Data information : measurements of inhibition potency were result of two independent experiments performed in triplicate ( n = 6 ) points were used for calculating values where 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

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 . MST profiles shown are one of two independent measurements . Data information : measurements of inhibition potency were result of two independent experiments performed in triplicate ( n = 6 ) points were used for calculating values where 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

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 GAT1WT used in study displaying a KM value of 11 . 4 μM .

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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 substrate analogue nipecotic acid displaying a Ki value of 14 . 4 μM . Data information : measurements of inhibition potency were result of two independent experiments performed in triplicate ( n = 6 ) points were used for calculating values where error bars represent s . e . m .

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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 ) SKF89976a ( 7 . 3 μM ) . Data information : measurements of inhibition potency were result of two independent experiments performed in triplicate ( n = 6 ) points were used for calculating values where error bars represent s . e . m .

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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 consequences on transport activity . L300W has maximal effect on transport activity . histogram represents mean of a total 6 measurements ( n = 6 ) done as two independent measurements . datapoints are represented as black dots around error bar that represents s . e . m .

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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 . inhibition potencies of inhibitors display lowered value suggesting an improved ability to compete for GABA uptake . Data information : data plots were performed as two independent experiments each time done in triplicate ( n = 6 ) . Error bars in data display s . e . m . Statistical significance of inhibition potencies were measured between mutant GAT1WT transporter measured with an unpaired t - 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 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 B-CONTROLLED_VAR 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 . inhibition potencies of inhibitors display lowered value suggesting an improved ability to compete for GABA uptake . Data information : data plots were performed as two independent experiments each time done in triplicate ( n = 6 ) . Error bars in data display s . e . m . Statistical significance of inhibition potencies were measured between mutant GAT1WT transporter measured with an unpaired t - display p values as follows panel E - 0 . 0001 ; tiagabine , panel F - 0 . 0017 ; NO711 , panel G - 0 . 018 ;

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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 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 inhibitors were decided on Ki values calculated for inhibitors . Each data point was plotted using 6 measurements carried out in two independent measurements in triplicates . Error bars represent s . e . m .

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

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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 substrate analogue nipecotic acid for GAT1 S359E displaying a Ki value of 30 . 1 μM Data information : kinetic plots were plotted as a mean of 6 measurements carried out in two independent measurements in triplicate . error bars represent s . e . m .

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

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

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

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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 were incubated with 20 µg / ml of whole extract of Y . pseudotuberculosis expressing full - CNFY or N - or C - terminally deleted toxin variants for 24 h . nuclei were strained with DAPI ( blue ) actin cytoskeleton was stained using FITC - phalloidin ( green ) . formation of large , multinuclear was ob served by micro scopy formation of thick actin stress actin folding were only observed with CNFY - treated . scale bar is 40 µm . incubated with extracts of YP147 ( ΔcnfY ) harboring 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 were incubated with 20 µg / ml of whole extract of Y . pseudotuberculosis expressing full - CNFY , N - or C - terminal deletion variants fused to GFP ( green ) for 90 or 180 min . were fixed processed for microscopy . red signal represents late endosomes ( CellLight Late Endosomes - RFP ( Rab7a ) ) . Nuclei were stained with DAPI ( blue ) . A merged image of different channels is shown , smaller are magnified views of boxed areas . 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 supernatant from 25 ° C overnight Yersinia cultures expressing indicated CNFY derivatives to determine β - lactamase activity by measuring changes in absorbance at 390 nm ( yellow ) 486 nm ( red ) . data represent 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.


viability of Y . pseudotuberculosis YPIII expressing indicated CNFY derivatives was assessed in equalized cultures using BacTiter - Glo Microbial Viability kit ( Promega ) . data represent 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 of RhoA activation in HEp - 2 lysate by CNFY recombinant D4 - 5 . Purified CNFY or D4 - 5 fragment ( 1 µM ) was added to extracts of HEp - 2 incubated for 10 , 20 , 30 or 60 min . De amidation of RhoA was analyzed by shift of modified Rho GTPase band in SDS PAGE gels after detection with anti - RhoA antibodies .

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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 of recombinant RhoA deamidation by CNFY D4 - 5 fragment . Recombinant RhoA was incubated with purified CNFY or D4 - 5 fragment samples were separated by SDS - PAGE after indicated times before subjecting to trypsin digestion 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 fold change ( log2 ) in number of reads between vehicle AZD8186 - treated conditions vs p - value of difference between two treatment conditions for each shRNA . 9 out of 18 shRNAs targeting EGFR showed a p - value < 0 . 2 are highlighted in plot . plot was generated considering results from biological triplicate of 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 indicated shRNAs targeting EGFR selected by puromycin . EGFR mRNA was then measured by RT - qPCR ( C ) Average ± SD of triplicates 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 indicated shRNAs targeting EGFR selected by puromycin . viability was measured after 4 days of treatment with serial dilutions of AZD8186 ( D ) . Average ± SD of triplicates 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 presence of vehicle , AZD8186 ( 0 . 25 μM ) , GDC0941 ( 0 . 25 μM ) or MK2206 ( 0 . 45 μM ) , as indicated . viability was measured after 4 days normalised within each of PI3K pathway inhibitor - treated condition to viability in absence of gefitinib Average ± SD of triplicates 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 presence of vehicle , AZD8186 ( 0 . 25 μM ) , GDC0941 ( 0 . 25 μM ) or MK2206 ( 0 . 45 μM ) , as indicated . viability was measured after 4 days normalised within each of PI3K pathway inhibitor - treated condition to viability in absence of lapatinib . Average ± SD of triplicates 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 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 - unpaired student t - 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 lines used in experiments were : MDA - MB - 468 , HCC70 , HCC1937 , HCC38 , HCC1395 BT - 549 ; PTEN - WT lines were : MDA - MB - 157 , MDA - MB - 231 , HCC1187 , HCC1428 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 five PTEN - WT TNBC lines for 6 days in three independent experiments . score was obtained analysing viability data through software Chalice Analyser . Mean of synergy scores ± SD . Statistical significance of Mann Whitney two - * 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 ) samples from METABRIC dataset classified as TNBCs were assigned to groups " PTEN - low " when falling in lower quartile for PTEN expression , " PTEN - mut " when harbouring a non - synonymous mutation on PTEN gene , " PTEN - WT " in other cases . Comparison of expression of EGFR between PTEN low or mut PTEN - WT groups . Data presented in a box whisker plot with central band indicating median , upper lower extremes of box or hinge being third first quartiles respectively whiskers extending to most extreme data values Mean ± SD . P value calculated by unpaired t - .

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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 ) 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 ± ) . Statistical significance of two - way ANOVA statistical * * P = 0 . 0028 * * * P < 0 . 0001 . ( B ) 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 ± ) . Growth curves were compared using two - way ANOVA statistical . Statistical significance of two - way ANOVA statistical * * * P = 0 . 0006 * * * * 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 volume of HCC - 70 tumors during treatment . Statistical comparison between different treatment groups by Mann - Whitney * * P = 0 . 0023 * * * 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 of mice harbouring HCC70 xenograft tumors ( 6 - 7 mice per group , mean ± ) 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 derived from a mammary spontaneously developed in a Wap - cre : Ptenfl / fl : Tp53fl / fl mouse was cloned injected in mammary fat pad of syngeneic C57BL6 / J recipient mice . Tumours grew in 12 out of 35 transplanted mice only these tumours were selected for treatments described in figure . When tumours reached an average volume of 100mm3 , they were treated with vehicle or a combination of AZD8186 ( 150mg / kg , og once / day ) erlotinib ( 50mg / kg IP once / day ) . Tumours were then measured during treatment ( 6 mice per group , mean ± ) . Statistical significance of two - way ANOVA statistical * * * * 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 were treated for 24h with vehicle , PI3Kβi ( AZD8186 250 nM ) , EGFRi ( gefitinib 3 μM ) or cetuximab ( 100 μg / ml ) , alone or in combination . lysates were probed with 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 were treated for 24h with vehicle , AKTi ( MK2206 450 nM ) , gefitinib ( 3 μM ) or cetuximab ( 100 μg / ml ) , alone or in indicated combinations . lysates were probed with 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 ) ZR - 75 - 1 ( D ) parental or PI3Kβi - Res ( derivative 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 . whole lysates were then probed with indicated antibodies . Spliced of parental resistant paired samples were taken from same original blot blots showing P - S6 S6 tot in ( D ) have been cut 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 were treated with vehicle , AKTi ( MK2206 250 nM ) or gefitinib ( 3 μM ) , alone or in indicated combinations . lysates were probed with 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 , viability measured after 4 days of treatment . Average ± SD of triplicates 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 , viability measured after 4 days . Average ± SD of triplicates 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 ) in HCC70 ( I ) . 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 PI3Kβi . lysates were incubated with IgG control or anti - EGFR antibody immuno - complexes or total lysates were immune - blotted with 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 pre - treated with vehicle or PI3Kβi ( AZD8186 250 nM ) for 1h . lysates were probed with indicated antibodies . Phospho - AKT pan - AKT bands were quantified by use of ImageLite software : of phospho - AKT to pan - AKT normalised to control ( left 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 ) dot - plots show for each gene knocked - out by sgRNAs fold change ( log2 ) between treated conditions ( AZD8186 vehicle in signal ( anti - phosphoS6 immunofluorescence ) versus p - value of difference calculated by two sided t - . plots represent means of biological triplicates . Genes for which it was calculated a p < 0 . 0001 Log2 ( fold change ) > 0 . 25 are reported highlighted in green ; genes having a 0 . 0001 0 . 25 are reported shown in red . Gefitinib combined with AZD8186 represents positive control of 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 ) . dot - plots show for each gene knocked - out by sgRNAs fold change ( log2 ) between treated conditions GDC0941 , vehicle in signal ( anti - phosphoS6 immunofluorescence ) versus p - value of difference calculated by two sided t - . plots represent means of biological triplicates . Genes for which it was calculated a p < 0 . 0001 Log2 ( fold change ) > 0 . 25 are reported highlighted in green ; genes having a 0 . 0001 0 . 25 are reported 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 fold change in signal between vehicle GDC0941 - treated conditions for MDA - MB - 468 transduced with non - target Control , GNB2 or GNG5 sgRNAs ( N = 2 or 3 ) . Data presented in a box whisker plot with central band indicating median , upper lower extremes of box or hinge being third first quartiles respectively whiskers extending to most extreme data values within 1 . 5 times inter - quartile range . Statistical significance of unpaired t - * * * * 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 of GNB2 KO . lysates of MDA - MB - 468 parental three MDA - MB - 468 GNB2 KO clones were probed with indicated antibodies . of 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 three MDA - MB - 468 GNB2 KO clones were treated with vehicle , GDC0941 1 μM , gefitinib 3 μM or lapatinib 1 μM for 24 hours . lysates were probed with 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 three MDA - MB - 468 GNB2 KO clones treated with serial dilutions of indicated drugs for six days . Mean ± SD of triplicates 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 ) samples from METABRIC dataset classified as TNBCs ( N = 299 ) were assigned to groups " PTEN - low " when falling in lower quartile for PTEN expression , " PTEN - mut " when harbouring a non - synonymous mutation on PTEN gene , " PTEN - WT " in other cases . Comparison of expression of GNB2 between PTEN low or mut PTEN - WT groups . Box & Whisker plot ( Median / IQR / 1 . 5 * IQR Whiskers ) . P value calculated by unpaired t - .

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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 results from drug screening with compounds targeting GPCR signaling . MDA - MB - 468 were treated with compounds of GPCR - targeted library at three different concentrations ( 0 . 1 , 1 or 10 μM ) in combination with vehicle , GDC0941 450 nM or lapatinib 1 μM . pS6 signal was measured by IF after 24 hours of treatment normalized to DAPI . Z scores of normalized values for each drug measured in presence of vehicle , GDC0941 or lapatinib are reported on y axis or on left or right x axis of dot plot , respectively . Readings acquired following treatment with three different concentrations of GPCR - targeted drugs are reported in 3 different colours . Dots corresponding to vorapaxar treatments at three different concentrations are highlighted in plot . values reported are mean of a biological triplicate of 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 indicated combinations . lysates were probed with 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 or GNB2 KO clones were starved treated with scramble or PAR1 activating peptide for 5 minutes , alone or in combination with vorapaxar . lysates were probed with indicated antibodies . Phospho - AKT , pan - AKT , phospho - ERK1 / 2 pan - ERK1 / 2 bands were quantified by use of ImageLite software : of phospho - AKT to pan - AKT phospho - ERK1 / 2 to pan - ERK1 / 2 normalised to control peptide - treated conditions for WT GNB2 KO ( first 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.