Figure 2 [ A ] Plants grown with 0 . 05 MS and GMVs ( G 0 . 05 ) showed hyper - induction of IPS1 and PS2 gene expression in both shoots and roots , compared to plants grown with 0 . 05 MS alone ( C 0 . 05 ) . Values are means ± SE of three biological replicates . [ B ] Root acid phosphatase activity as detected by the blue color from the 5 - bromo - 4 - chloro - 3 - indolyl phosphate p - toluidine salt ( BCIP ) treatment . Image contrast enhanced for improved visualization of the blue color in roots Supplementation of Pi to the nutrient - deficient plants ( 0 . 05 + Pi ) significantly reduced hyper - accumulation of anthocyanin ( C ) triggered by GMVs . Data information : The boxplots show representative data from three independent experiments ( n = 12 ) . Whiskers represent the min to max data range , the median is represented by the central horizontal line . The upper and lower limits of the box outline represent the first and third quartile . Different letters denote significantly different means at p < 0 . 05 , Tukey ' s multiple comparison test within each group of the same DAT . Supplementation of Pi to the nutrient - deficient plants ( 0 . 05 + Pi ) significantly reduced hyper - induction of IPS1 gene expression ( D ) triggered by GMVs . Data information : The boxplots show representative data from three independent experiments ( n = 12 ) . Whiskers represent the min to max data range , the median is represented by the central horizontal line . The upper and lower limits of the box outline represent the first and third quartile . Different letters denote significantly different means at p < 0 . 05 , Tukey ' s multiple comparison test within each group of the same DAT . [ E ] Pi supplementation to the 0 . 05 MS medium partially restored GMV - induced plant vigor , as indicated by increases in total leaf area per plant . [ F ] A low Pi level ( LP ) , which was equal to 1 / 20 of that in the 0 . 5 MS medium , in plant growth medium blocked GMV - induced plant vigor . Data information : The boxplots show representative data from three independent experiments ( n = 12 ) . Whiskers represent the min to max data range , the median is represented by the central horizontal line . The upper and lower limits of the box outline represent the first and third quartile . Different letters denote significantly different means at p < 0 . 05 , Tukey ' s multiple comparison test within each group of the same DAT . [ G ] The Arabidopsis phr1phl1 mutant showed substantially decreased growth - promotion , compared to the wild type plants . Data information : The boxplots show representative data from three independent experiments ( n = 12 ) . Whiskers represent the min to max data range , the median is represented by the central horizontal line . The upper and lower limits of the box outline represent the first and third quartile . Different letters denote significantly different means at p < 0 . 05 , Tukey ' s multiple comparison test within each group of the same DAT .

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

Figure 2 [A] Plants grown with 0.05 MS and GMVs (G 0.05) showed hyper-induction of IPS1 and PS2 gene expression in both shoots and roots, compared to plants grown with 0.05 MS alone (C 0.05). Values are means ± SE of three biological replicates.  [B] Root acid phosphatase activity as detected by the blue color from the 5-bromo-4-chloro-3-indolyl phosphate p-toluidine salt (BCIP) treatment. Image contrast enhanced for improved visualization of the blue color in roots Supplementation of Pi to the nutrient-deficient plants (0.05 + Pi) significantly reduced hyper-accumulation of anthocyanin (C) triggered by GMVs. Data information: The boxplots show representative data from three independent experiments (n=12). Whiskers represent the min to max data range, the median is represented by the central horizontal line. The upper and lower limits of the box outline represent the first and third quartile. Different letters denote significantly different means at p < 0.05, Tukey's multiple comparison test within each group of the same DAT.Supplementation of Pi to the nutrient-deficient plants (0.05 + Pi) significantly reduced hyper-induction of IPS1 gene expression (D) triggered by GMVs. Data information: The boxplots show representative data from three independent experiments (n=12). Whiskers represent the min to max data range, the median is represented by the central horizontal line. The upper and lower limits of the box outline represent the first and third quartile. Different letters denote significantly different means at p < 0.05, Tukey's multiple comparison test within each group of the same DAT. [E] Pi supplementation to the 0.05 MS medium partially restored GMV-induced plant vigor, as indicated by increases in total leaf area per plant. [F] A low Pi level (LP), which was equal to 1/20 of that in the 0.5 MS medium, in plant growth medium blocked GMV-induced plant vigor. Data information: The boxplots show representative data from three independent experiments (n=12). Whiskers represent the min to max data range, the median is represented by the central horizontal line. The upper and lower limits of the box outline represent the first and third quartile. Different letters denote significantly different means at p < 0.05, Tukey's multiple comparison test within each group of the same DAT. [G] The Arabidopsis phr1phl1 mutant showed substantially decreased growth-promotion, compared to the wild type plants. Data information: The boxplots show representative data from three independent experiments (n=12). Whiskers represent the min to max data range, the median is represented by the central horizontal line. The upper and lower limits of the box outline represent the first and third quartile. Different letters denote significantly different means at p < 0.05, Tukey's multiple comparison test within each group of the same DAT.


Figure 3 [ A ] Images of Arabidopsis plants exposed to four individual GMV components , including diacetyl ( DA ) , 2 , 3 - butanediol ( BTDL ) , 2 - methyl - 1 - propanol ( MP ) and acetoin ( ATN ) . The compounds were applied at dosages that , when the compounds totally evaporate from the agar - containing solid droplets , would yield in volatile concentrations of 9 . 7 µg ( DA ) , 32 . 5 µg ( BTDL ) , 7 . 9 µg ( MP ) and 28 . 5 µg ( ATN ) per mL free space in the petri dish , which resembled the 1 : 3 . 3 : 0 . 8 : 2 . 9 ratio among the four GMV components in natural GMVs22 . anthocyanin hyper accumulation ( B ) were observed in Pi - deficient plants exposed to DA but not to the other three GMV components . Data information : The boxplots show representative data from three independent experiments ( n = 6 ) . Whiskers represent the min to max data range , the median is represented by the central horizontal line . The upper and lower limits of the box outline represent the first and third quartile . Different letters denote significantly different means at p < 0 . 05 , Tukey ' s multiple comparison test . PSR gene hyper induction ( C ) were observed in Pi - deficient plants exposed to DA but not to the other three GMV components . Data information : qPCR results show values of means ± SE of two biological replicates . Different letters denote significantly different means at p < 0 . 05 , Tukey ' s multiple comparison test . Compared to the wild type plants , phr1phl1 showed substantially decreased anthocyanin hyper accumulation ( D ) triggered by DA . Data information : The boxplots show representative data from three independent experiments ( n = 6 ) . Whiskers represent the min to max data range , the median is represented by the central horizontal line . The upper and lower limits of the box outline represent the first and third quartile . Different letters denote significantly different means at p < 0 . 05 , Tukey ' s multiple comparison test . Compared to the wild type plants , phr1phl1 showed substantially decreased PSR gene hyper induction ( E ) triggered by DA . Data information : qPCR results show values of means ± SE of two biological replicates . Different letters denote significantly different means at p < 0 . 05 , Tukey ' s multiple comparison 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 O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 3 [A] Images of Arabidopsis plants exposed to four individual GMV components, including diacetyl (DA), 2,3-butanediol (BTDL), 2-methyl-1-propanol (MP) and acetoin (ATN). The compounds were applied at dosages that, when the compounds totally evaporate from the agar-containing solid droplets, would yield in volatile concentrations of 9.7 µg (DA), 32.5 µg (BTDL), 7.9 µg (MP) and 28.5 µg (ATN) per mL free space in the petri dish, which resembled the 1 : 3.3 : 0.8 : 2.9 ratio among the four GMV components in natural GMVs22. anthocyanin hyper accumulation (B) were observed in Pi-deficient plants exposed to DA but not to the other three GMV components. Data information: The boxplots show representative data from three independent experiments (n=6). Whiskers represent the min to max data range, the median is represented by the central horizontal line. The upper and lower limits of the box outline represent the first and third quartile. Different letters denote significantly different means at p < 0.05, Tukey's multiple comparison test.PSR gene hyper induction (C) were observed in Pi-deficient plants exposed to DA but not to the other three GMV components. Data information: qPCR results show values of means ± SE of two biological replicates. Different letters denote significantly different means at p < 0.05, Tukey's multiple comparison test.Compared to the wild type plants, phr1phl1 showed substantially decreased anthocyanin hyper accumulation (D) triggered by DA. Data information: The boxplots show representative data from three independent experiments (n=6). Whiskers represent the min to max data range, the median is represented by the central horizontal line. The upper and lower limits of the box outline represent the first and third quartile. Different letters denote significantly different means at p < 0.05, Tukey's multiple comparison test.Compared to the wild type plants, phr1phl1 showed substantially decreased PSR gene hyper induction (E) triggered by DA. Data information: qPCR results show values of means ± SE of two biological replicates. Different letters denote significantly different means at p < 0.05, Tukey's multiple comparison test.


Figure 4 [ A ] DA induces genes involved in SA biosynthesis and signaling in Arabidopsis grown with Pi deficiency . Data information : qPCR results show values of means ± SE ( n = 3 ) , two biological replicates were analyzed with similar results . Different letters denote significantly different means at p < 0 . 05 , Tukey ' s multiple comparison test . [ B ] DA elevates SA accumulation levels in Pi - deficient Arabidopsis . Means ± SE of three biological replicates . Data information : The boxplots show representative data from three independent experiments ( n = 6 ) . Whiskers represent the min to max data range , the median is represented by the central horizontal line . The upper and lower limits of the box outline represent the first and third quartile . Different letters denote significantly different means at p < 0 . 05 , Tukey ' s multiple comparison test . [ C ] Images of plants grown with different treatments using DA ( 9 . 7 µg per mL free space ) or SA ( 100 µM in plant growth medium ) . Exogenous application of SA mimics DA - induced anthocyanin accumulation ( D ) patterns in Pi - deficient plants . Data information : The boxplots show representative data from three independent experiments ( n = 6 ) . Whiskers represent the min to max data range , the median is represented by the central horizontal line . The upper and lower limits of the box outline represent the first and third quartile . Different letters denote significantly different means at p < 0 . 05 , Tukey ' s multiple comparison test . Exogenous application of SA mimics DA - induced IPS1 gene induction ( E ) patterns in Pi - deficient plants . Data information : qPCR results show values of means ± SE ( n = 3 ) , two biological replicates were analyzed with similar results . Different letters denote significantly different means at p < 0 . 05 , Tukey ' s multiple comparison test . Compared to the wild type plants , the NahG transgenic plants showed altered responses to DA under Pi - deficiency condition , as shown by plant images ( F ) Compared to the wild type plants , the NahG transgenic plants showed altered responses to DA under Pi - deficiency condition and quantification of anthocyanin accumulation levels ( G ) . Data information : The boxplots show representative data from three independent experiments ( n = 6 ) . Whiskers represent the min to max data range , the median is represented by the central horizontal line . The upper and lower limits of the box outline represent the first and third quartile . Different letters denote significantly different means at p < 0 . 05 , Tukey ' s multiple comparison 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 4 [A] DA induces genes involved in SA biosynthesis and signaling in Arabidopsis grown with Pi deficiency. Data information: qPCR results show values of means ± SE (n=3), two biological replicates were analyzed with similar results. Different letters denote significantly different means at p < 0.05, Tukey's multiple comparison test. [B] DA elevates SA accumulation levels in Pi-deficient Arabidopsis. Means ± SE of three biological replicates. Data information: The boxplots show representative data from three independent experiments (n=6). Whiskers represent the min to max data range, the median is represented by the central horizontal line. The upper and lower limits of the box outline represent the first and third quartile. Different letters denote significantly different means at p < 0.05, Tukey's multiple comparison test. [C] Images of plants grown with different treatments using DA (9.7 µg per mL free space) or SA (100 µM in plant growth medium). Exogenous application of SA mimics DA-induced anthocyanin accumulation (D) patterns in Pi-deficient plants. Data information: The boxplots show representative data from three independent experiments (n=6). Whiskers represent the min to max data range, the median is represented by the central horizontal line. The upper and lower limits of the box outline represent the first and third quartile. Different letters denote significantly different means at p < 0.05, Tukey's multiple comparison test.Exogenous application of SA mimics DA-induced IPS1 gene induction (E) patterns in Pi-deficient plants. Data information: qPCR results show values of means ± SE (n=3), two biological replicates were analyzed with similar results. Different letters denote significantly different means at p < 0.05, Tukey's multiple comparison test.Compared to the wild type plants, the NahG transgenic plants showed altered responses to DA under Pi-deficiency condition, as shown by plant images (F)Compared to the wild type plants, the NahG transgenic plants showed altered responses to DA under Pi-deficiency condition and quantification of anthocyanin accumulation levels (G). Data information: The boxplots show representative data from three independent experiments (n=6). Whiskers represent the min to max data range, the median is represented by the central horizontal line. The upper and lower limits of the box outline represent the first and third quartile. Different letters denote significantly different means at p < 0.05, Tukey's multiple comparison test.


Figure 5 [ A ] Clustering of DA - regulated immunity - related genes in Arabidopsis with sufficient Pi , as revealed by RNAseq analysis . [ B ] DA suppresses plant ROS burst induced by flg22 or elf18 . ROS accumulation was represented in relative luminescence units ( RLU ) . Data bar indicate mean ± SE ( n = 24 ) readings for 60 min . Three independent experiments were performed and similar results were observed . * * p < 0 . 01 , student t - test . [ C ] DA induces gene expression of AZI1 in Arabidopsis . Different letters denote significantly different means at p < 0 . 05 , Tukey ' s multiple comparison test within each group . Bar indicates mean ± SE ( n = 3 ) , two biological replicates were analyzed with similar results . [ D ] flg22 - induced MPK3 / MPK6 phosphorylation in Arabidopsis is not decreased by DA . Kinase assays were performed at 5 min after flg22 treatment . Three independent experiments were performed and similar results were observed . [ E ] Plant resistance to the pathogen Pst DC3000 is not reduced by DA . The severity of pathogen infection was measured as colony forming units ( CFUs ) per mg FW plants at 0 and 3 days after inoculation . Data bar indicate mean ± SE ( n = 8 ) , two independent experiments were performed . Different letters denote significantly different means at p < 0 . 05 , Tukey ' s multiple comparison test within each group . [ F ] Root colonization of GB03 in wild type Arabidopsis ( Col - 0 ) and the mutants / transgenic lines npr1 , NahG , and rbohd . * * * p < 0 . 001compared with Col - 0 without DA treatment , n = 9 biological replicates , student t - test . [ G ] Bacteria chemotaxis with different concentrations of DA . Data point indicates mean ± SE ( n = 6 ) . Two independent experiments were performed and similar results were observed . * * * p < 0 . 001 , student 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 5 [A] Clustering of DA-regulated immunity-related genes in Arabidopsis with sufficient Pi, as revealed by RNAseq analysis.  [B] DA suppresses plant ROS burst induced by flg22 or elf18. ROS accumulation was represented in relative luminescence units (RLU). Data bar indicate mean ± SE (n=24) readings for 60 min. Three independent experiments were performed and similar results were observed. **p < 0.01, student t-test.  [C] DA induces gene expression of AZI1 in Arabidopsis. Different letters denote significantly different means at p < 0.05, Tukey's multiple comparison test within each group. Bar indicates mean ± SE (n=3), two biological replicates were analyzed with similar results.  [D] flg22-induced MPK3/MPK6 phosphorylation in Arabidopsis is not decreased by DA. Kinase assays were performed at 5 min after flg22 treatment. Three independent experiments were performed and similar results were observed.  [E] Plant resistance to the pathogen Pst DC3000 is not reduced by DA. The severity of pathogen infection was measured as colony forming units (CFUs) per mg FW plants at 0 and 3 days after inoculation. Data bar indicate mean ± SE (n=8), two independent experiments were performed. Different letters denote significantly different means at p < 0.05, Tukey's multiple comparison test within each group.  [F] Root colonization of GB03 in wild type Arabidopsis (Col-0) and the mutants/transgenic lines npr1, NahG, and rbohd. *** p < 0.001compared with Col-0 without DA treatment, n= 9 biological replicates, student t-test.  [G] Bacteria chemotaxis with different concentrations of DA. Data point indicates mean± SE (n=6). Two independent experiments were performed and similar results were observed. *** p < 0.001, student t-test.


Figure 1 ( A ) Antibacterial activity of NGJ1 , T6SS - 1 - and T6SS - 2 - strains against target bacteria .

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

Figure 1(A) Antibacterial activity of NGJ1, T6SS-1- and T6SS-2- strains against target bacteria.


Figure 2 ( A ) Growth curve of recombinant BL21 ( DE3 ) cells upon IPTG mediated induction of proteins , represented as colored line graphs . ( B ) Bacterial two - hybrid assay reflecting interaction between proteins of T6SS apparatus and effector operons . Interaction between T25 - zip and T18 - zip was used as positive control while pKNT25 and pUT18C ( empty vectors ) was used as negative control . Appearance of blue color suggests positive interaction while absence of color suggests lack of interaction . ( C ) Yeast two - hybrid assay reflecting interaction of TseTBg1 and TseTBg2 effector proteins with the corresponding PAAR ( PAARTseTBg ) proteins encoded in their operon . Interaction between P53 and SV40 large T - antigen ( T ) proteins was used as positive control . The pGBKT7 and pGADT7 ( empty vectors ) were used as negative control .

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

Figure 2(A) Growth curve of recombinant BL21 (DE3) cells upon IPTG mediated induction of proteins, represented as colored line graphs.(B) Bacterial two-hybrid assay reflecting interaction between proteins of T6SS apparatus and effector operons. Interaction between T25-zip and T18-zip was used as positive control while pKNT25 and pUT18C (empty vectors) was used as negative control. Appearance of blue color suggests positive interaction while absence of color suggests lack of interaction.(C) Yeast two-hybrid assay reflecting interaction of TseTBg1 and TseTBg2 effector proteins with the corresponding PAAR (PAARTseTBg) proteins encoded in their operon. Interaction between P53 and SV40 large T-antigen (T) proteins was used as positive control. The pGBKT7 and pGADT7 (empty vectors) were used as negative control.


Figure 3 ( A , B ) Effect of purified TseTBg ( TseTBg1 / TseTBg2 ) and one of the variants ( TseTBg2D116A , K129A ) proteins on degradation of bacterial genomic DNA . ( C , D ) RNase activity of TseTBg effector proteins , in presence or absence of cognate TsiTBg immunity proteins . ( E ) Effect of TseTBg1 , TseTBg2 , TseTBg2D116A , K129A proteins on degradation of pHM1 , a broad host - range plasmid isolated from E . coli and NGJ1 . ( F ) Effect of TseTBg1 and TseTBg2 proteins on degradation of pHM1 , reintroduced into E . coli from NGJ1 . ( G ) Effect of ectopic expression of DNA adenine methylase ( Dam ) protein on TseTBg proteins mediated degradation of plasmid DNA .

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

Figure 3(A, B) Effect of purified TseTBg (TseTBg1/TseTBg2) and one of the variants (TseTBg2D116A,K129A) proteins on degradation of bacterial genomic DNA.(C, D) RNase activity of TseTBg effector proteins, in presence or absence of cognate TsiTBg immunity proteins.(E) Effect of TseTBg1, TseTBg2, TseTBg2D116A,K129A proteins on degradation of pHM1, a broad host-range plasmid isolated from E. coli and NGJ1. (F) Effect of TseTBg1 and TseTBg2 proteins on degradation of pHM1, reintroduced into E. coli from NGJ1. (G) Effect of ectopic expression of DNA adenine methylase (Dam) protein on TseTBg proteins mediated degradation of plasmid DNA.


Figure 4 ( E , F ) Graph showing the effect of native TsiTBg and HTH or Imm52 domain deleted variant of TsiTBg proteins on Gus reporter gene expression ( quantified using 4 - methylumbelliferyl ß - D - glucuronide , MUG , as substrate ) under the external ( pExtTseTBg1 / pExtTseTBg2 ) and internal ( pIntTseTBg1 / pIntTseTBg2 ) promoters in E . coli .

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

Figure 4(E, F) Graph showing the effect of native TsiTBg and HTH or Imm52 domain deleted variant of TsiTBg proteins on Gus reporter gene expression (quantified using 4-methylumbelliferyl ß-D-glucuronide, MUG, as substrate) under the external (pExtTseTBg1/pExtTseTBg2) and internal (pIntTseTBg1/pIntTseTBg2) promoters in E. coli.


Figure 5 ( A ) Interaction of external ( pExtTseTBg1 ) and internal ( pIntTseTBg1 ) promoter DNA fragments of TseTBg1 operon with the TsiTBg1 protein . ( B ) Interaction of pExtTseTBg1 and pIntTseTBg1 promoter DNA fragments of TseTBg1 operon with the HTH domain deleted variant of TseTBg1 protein ( TsiTBg1ΔHTH ) . ( C ) Interaction of pExtTseTBg2 and pIntTseTBg2 promoter DNA of TseTBg2 operon with the TsiTBg1 protein . ( D ) Interaction of control ( random ) DNA with the TsiTBg1 protein .

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Figure 5(A) Interaction of external (pExtTseTBg1) and internal (pIntTseTBg1) promoter DNA fragments of TseTBg1 operon with the TsiTBg1 protein. (B) Interaction of pExtTseTBg1 and pIntTseTBg1 promoter DNA fragments of TseTBg1 operon with the HTH domain deleted variant of TseTBg1 protein (TsiTBg1ΔHTH). (C) Interaction of pExtTseTBg2 and pIntTseTBg2 promoter DNA of TseTBg2 operon with the TsiTBg1 protein. (D) Interaction of control (random) DNA with the TsiTBg1 protein.


Figure 1 ( A ) MCF10A - eGFP - LC3 cells were left untreated ( Control ) or treated with 5 μM rapamycin ( Rapa ) , 500 ng / ml TRAIL or 10 ng / ml TNF for 24 h . When indicated , 10 mM 3 ‐ MA or 5 μg / ml TRAIL ‐ R2 antagonist antibody was added 1 h before the drugs . Representative confocal images ( 20 μm scale bars ) and the percentages of cells with LC3 translocation are shown . ( B ) MCF10A - eGFP - LC3 cells were transfected with the indicated siRNAs for 48 h and analysed by immunoblotting ( left ) , or stimulated with 500 ng / ml TRAIL for 24 h and analysed for LC3 translocation ( right ) . ( C ) MCF10A cells left untreated ( Control ) or treated with 500 ng / ml TRAIL or 5 μM rapamycin ( Rapa ) for 24 h were analysed for LC3 ‐ I , LC3 ‐ II and tubulin by immunoblotting . Pepstatin A and E64d were added to the cells 4 h before harvesting . Similar results were obtained in two independent experiments . ( D ) MCF10A cells were treated with 5 μM rapamycin ( Rapa ) or indicated concentrations of TRAIL for 24 h , and the increase in the degradation of long ‐ lived proteins as compared with the untreated cultures ( Control ) was measured . ( E ) MCF10A - eGFP - LC3 cells transfected with the indicated siRNAs were left untreated 48 h later ( Control ) or treated with 500 ng / ml TRAIL for 24 h and analysed for the DNA content by flow cytometry . The percentage of apoptotic cells with sub ‐ G1 DNA content is shown . The values represent mean ± s . d . for three ( A , D ) or four ( B , E ) independent experiments . * P ‐ value 0 . 05 , * * P ‐ value 0 . 01 and * * * P ‐ value 0 . 001 as compared with control sample ( A - left panel and D ) , with TRAIL ‐ treated cells without anti ‐ TRAIL ‐ R2 ( A - right panel ) or with TRAIL ‐ treated cells without siRNA ( B , E ) .

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

Figure 1(A) MCF10A-eGFP-LC3 cells were left untreated (Control) or treated with 5 μM rapamycin (Rapa), 500 ng/ml TRAIL or 10 ng/ml TNF for 24 h. When indicated, 10 mM 3‐MA or 5 μg/ml TRAIL‐R2 antagonist antibody was added 1 h before the drugs. Representative confocal images (20 μm scale bars) and the percentages of cells with LC3 translocation are shown.(B) MCF10A-eGFP-LC3 cells were transfected with the indicated siRNAs for 48 h and analysed by immunoblotting (left), or stimulated with 500 ng/ml TRAIL for 24 h and analysed for LC3 translocation (right).(C) MCF10A cells left untreated (Control) or treated with 500 ng/ml TRAIL or 5 μM rapamycin (Rapa) for 24 h were analysed for LC3‐I, LC3‐II and tubulin by immunoblotting. Pepstatin A and E64d were added to the cells 4 h before harvesting. Similar results were obtained in two independent experiments.(D) MCF10A cells were treated with 5 μM rapamycin (Rapa) or indicated concentrations of TRAIL for 24 h, and the increase in the degradation of long‐lived proteins as compared with the untreated cultures (Control) was measured.(E) MCF10A-eGFP-LC3 cells transfected with the indicated siRNAs were left untreated 48 h later (Control) or treated with 500 ng/ml TRAIL for 24 h and analysed for the DNA content by flow cytometry. The percentage of apoptotic cells with sub‐G1 DNA content is shown. The values represent mean±s.d. for three (A, D) or four (B, E) independent experiments. *P‐value 0.05, **P‐value 0.01 and ***P‐value 0.001 as compared with control sample (A-left panel and D), with TRAIL‐treated cells without anti‐TRAIL‐R2 (A-right panel) or with TRAIL‐treated cells without siRNA (B, E).


Figure 2 ( A ) Protein lysates from MCF10A cells left untreated or treated with 500 ng / ml TRAIL or 5 μM rapamycin ( Rapa , positive control ) for 24 h were analysed by immunoblotting for phosphorylated p70S6K ( P ‐ p70S6K ) , total p70S6K and GAPDH ( loading control ) . ( B ) Immunoblot analysis of the levels of P ‐ ACC and tubulin ( loading control ) in lysates from MCF10A cells left untreated or treated with 500 ng / ml TRAIL for 24 h , 1 mM AICAR for 4 h ( positive control ) or 5 μM rapamycin for 24 h ( negative control ) . ( C ) Immunoblot analysis of the level of P ‐ ACC and GAPDH ( loading control ) in lysates from MCF10A cells treated with 500 ng / ml TRAIL for the indicated times . ( D ) MCF10A cells were transfected with indicated siRNAs for 48 h and analysed by immunoblotting for the indicated proteins immediately ( upper panel ) or after additional 24 h ( middle panel ) or 2 h ( lower panel ) incubation with ( + ) or without ( − ) 500 ng / ml TRAIL . ( E ) MCF10A - eGFP - LC3 cells transfected with indicated siRNAs for 48 h and incubated for an additional 24 h with or without 500 ng / ml TRAIL were analysed for LC3 translocation ( top ) and the DNA content ( bottom ) . The values represent mean ± s . d . for four independent experiments . * P ‐ value 0 . 05 and * * * P ‐ value 0 . 001 as compared with TRAIL ‐ treated cells without siRNA . Similar results were obtained in two ( A , B and D ) or three ( C , D ) independent experiments .

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Figure 2(A) Protein lysates from MCF10A cells left untreated or treated with 500 ng/ml TRAIL or 5 μM rapamycin (Rapa, positive control) for 24 h were analysed by immunoblotting for phosphorylated p70S6K (P‐p70S6K), total p70S6K and GAPDH (loading control).(B) Immunoblot analysis of the levels of P‐ACC and tubulin (loading control) in lysates from MCF10A cells left untreated or treated with 500 ng/ml TRAIL for 24 h, 1 mM AICAR for 4 h (positive control) or 5 μM rapamycin for 24 h (negative control).(C) Immunoblot analysis of the level of P‐ACC and GAPDH (loading control) in lysates from MCF10A cells treated with 500 ng/ml TRAIL for the indicated times.(D) MCF10A cells were transfected with indicated siRNAs for 48 h and analysed by immunoblotting for the indicated proteins immediately (upper panel) or after additional 24 h (middle panel) or 2 h (lower panel) incubation with (+) or without (−) 500 ng/ml TRAIL.(E) MCF10A-eGFP-LC3 cells transfected with indicated siRNAs for 48 h and incubated for an additional 24 h with or without 500 ng/ml TRAIL were analysed for LC3 translocation (top) and the DNA content (bottom). The values represent mean±s.d. for four independent experiments. *P‐value 0.05 and ***P‐value 0.001 as compared with TRAIL‐treated cells without siRNA. Similar results were obtained in two (A, B and D) or three (C, D) independent experiments.


Figure 3 ( A ) MCF10A - eGFP - LC3 cells were transfected with indicated siRNAs for 48 h and analysed for the indicated mRNAs ( top ) and proteins ( bottom by RT - PCR and immunoblotting , respectively . Similar results were obtained in three independent experiments . ( B ) MCF10A - eGFP - LC3 cells transfected with indicated siRNAs for 48 h , and left untreated or treated with 500 ng / ml TRAIL for 2 h , 10 μM ionomycin for 24 h or starved for amino acids and glucose for 24 h ( starvation ) were analysed for P ‐ ACC and tubulin ( loading control ) expression by immunoblotting . Similar results were obtained in two independent experiments . ( C ) Cells transfected as in ( B ) and left untreated or treated with 500 ng / ml TRAIL , 10 μM ionomycin or starved for amino acids and glucose for 24 h were analysed for LC3 translocation . The values represent mean ± s . d . of three independent experiments . * * * P ‐ value 0 . 001 as compared with cells treated in a same way , but transfected without siRNA .

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Figure 3(A) MCF10A-eGFP-LC3 cells were transfected with indicated siRNAs for 48 h and analysed for the indicated mRNAs (top) and proteins (bottom by RT-PCR and immunoblotting, respectively. Similar results were obtained in three independent experiments.(B) MCF10A-eGFP-LC3 cells transfected with indicated siRNAs for 48 h, and left untreated or treated with 500 ng/ml TRAIL for 2 h, 10 μM ionomycin for 24 h or starved for amino acids and glucose for 24 h (starvation) were analysed for P‐ACC and tubulin (loading control) expression by immunoblotting. Similar results were obtained in two independent experiments.(C) Cells transfected as in (B) and left untreated or treated with 500 ng/ml TRAIL, 10 μM ionomycin or starved for amino acids and glucose for 24 h were analysed for LC3 translocation. The values represent mean±s.d. of three independent experiments. ***P‐value 0.001 as compared with cells treated in a same way, but transfected without siRNA.


Figure 4 ( A ) The lysates of MCF10A cells treated with 500 ng / ml TRAIL for the indicated times were analysed by immunoblotting . The control lane ( 0 min TRAIL ) originates from the same blot and has been treated and exposed identically . Similar results were obtained in three independent experiments . ( B , C ) The lysates of MCF10A - eGFP - LC3 cells transfected with indicated siRNAs for 48 h and left untreated ( − ) or treated with 500 ng / ml TRAIL for 2 h were analysed by immunoblotting . ( C , lower panel ) MCF10A - eGFP - LC3 cells were transfected with indicated siRNAs for 48 h and analysed for the indicated mRNAs by RT - PCR . Similar results were obtained in two independent experiments . ( D , E ) MCF10A - eGFP - LC3 cells were transfected with indicated siRNAs for 48 h , treated as in Figure 3C and analysed for LC3 translocation ( left ) and sub ‐ G1 DNA content ( right ) . The values represent mean ± s . d . of a minimum of three independent experiments except for the right panel in E ( two experiments ) . * P ‐ value 0 . 05 and * * P ‐ value 0 . 01 as compared with cells treated in a same way , but transfected without siRNA .

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Figure 4(A) The lysates of MCF10A cells treated with 500 ng/ml TRAIL for the indicated times were analysed by immunoblotting. The control lane (0 min TRAIL) originates from the same blot and has been treated and exposed identically. Similar results were obtained in three independent experiments.(B, C) The lysates of MCF10A-eGFP-LC3 cells transfected with indicated siRNAs for 48 h and left untreated (−) or treated with 500 ng/ml TRAIL for 2 h were analysed by immunoblotting. (C, lower panel) MCF10A-eGFP-LC3 cells were transfected with indicated siRNAs for 48 h and analysed for the indicated mRNAs by RT-PCR. Similar results were obtained in two independent experiments.(D, E) MCF10A-eGFP-LC3 cells were transfected with indicated siRNAs for 48 h, treated as in Figure 3C and analysed for LC3 translocation (left) and sub‐G1 DNA content (right). The values represent mean±s.d. of a minimum of three independent experiments except for the right panel in E (two experiments). *P‐value 0.05 and **P‐value 0.01 as compared with cells treated in a same way, but transfected without siRNA.


Figure 5 ( A ) Protein lysates from MCF10A cells left untreated ( 0 ) or treated with 50 ng / ml TNF , 500 ng / ml TRAIL ( positive control ) or 10 ng / ml IL ‐ 1β for indicated times were analysed by immunoblotting for phosphorylated ACC ( P ‐ ACC ) , phosphorylated IκB ( P ‐ IκB ) and GAPDH ( loading control ) . The values show the P ‐ ACC / GAPDH and P ‐ IkB / GAPDH ratios as percentages of the ratios in untreated cells ( left lanes ) and are representative of two independent experiments . ( B , C ) MCF10A - eGFP - LC3 cells left untreated or treated with 500 ng / ml TRAIL , 50 ng / ml TNF or 10 ng / ml IL ‐ 1β for 24 h were analysed for LC3 translocation ( B ) and the DNA content ( C ) . The values represent mean ± s . d . for two independent experiments . * * P ‐ value 0 . 01 and * * * P ‐ value 0 . 001 as compared with untreated cells .

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Figure 5(A) Protein lysates from MCF10A cells left untreated (0) or treated with 50 ng/ml TNF, 500 ng/ml TRAIL (positive control) or 10 ng/ml IL‐1β for indicated times were analysed by immunoblotting for phosphorylated ACC (P‐ACC), phosphorylated IκB (P‐IκB) and GAPDH (loading control). The values show the P‐ACC/GAPDH and P‐IkB/GAPDH ratios as percentages of the ratios in untreated cells (left lanes) and are representative of two independent experiments.(B, C) MCF10A-eGFP-LC3 cells left untreated or treated with 500 ng/ml TRAIL, 50 ng/ml TNF or 10 ng/ml IL‐1β for 24 h were analysed for LC3 translocation (B) and the DNA content (C). The values represent mean±s.d. for two independent experiments. **P‐value 0.01 and ***P‐value 0.001 as compared with untreated cells.


Figure 6 ( A ) The lysates of hTERT - RPE1 cells transfected with indicated siRNAs for 48 h and left untreated or treated with 500 ng / ml TRAIL for 2 h were analysed by immunoblotting . ( B ) hTERT - RPE1 - eGFP - LC3 cells transfected with indicated siRNAs for 48 h were left untreated or treated with 500 ng / ml TRAIL for 24 h and analysed for LC3 translocation ( left ) and sub ‐ G1 DNA content ( middle ) and AMPKα expression ( right ) . ( C ) hTERT - RPE1 - eGFP - LC3 cells were transfected with indicated siRNAs for 48 h and analysed for the indicated mRNAs ( top ) . After 48 h , cells were left untreated or treated with 500 ng / ml TRAIL for 2 h , and analysed for P ‐ ACC and GAPDH ( loading control ) expression by immunoblotting . Similar results were obtained in two independent experiments . ( D ) Cells transfected as described in ( C ) and left untreated or treated with 500 ng / ml TRAIL , 10 μM ionomycin or starved for amino acids and glucose for 24 h were analysed for LC3 translocation . ( E ) Lysates of wild ‐ type ( WT ) MEFs and MEFs with an inactive TAK1 knock ‐ in ( TAK1Δ ) treated with 500 ng / ml TRAIL were analysed by immunoblotting . Similar results were obtained in two independent experiments . ( F ) WT and TAK1Δ MEFs left untreated and treated with 250 or 500 ng / ml TRAIL were analysed for cell death by the LDH release assay . Similar results were obtained in two independent experiments ( A , C and E ) . The values represent mean ± s . d . for two ( D ) or a minimum of three independent experiments ( B , F ) . * P ‐ value 0 . 05 , * * P ‐ value 0 . 01 and * * * P ‐ value 0 . 001 as compared with cells treated in the same way , but transfected without siRNA ( B , D ) or the wild ‐ type cells ( F ) .

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Figure 6(A) The lysates of hTERT-RPE1 cells transfected with indicated siRNAs for 48 h and left untreated or treated with 500 ng/ml TRAIL for 2 h were analysed by immunoblotting.(B) hTERT-RPE1-eGFP-LC3 cells transfected with indicated siRNAs for 48 h were left untreated or treated with 500 ng/ml TRAIL for 24 h and analysed for LC3 translocation (left) and sub‐G1 DNA content (middle) and AMPKα expression (right).(C) hTERT-RPE1-eGFP-LC3 cells were transfected with indicated siRNAs for 48 h and analysed for the indicated mRNAs (top). After 48 h, cells were left untreated or treated with 500 ng/ml TRAIL for 2 h, and analysed for P‐ACC and GAPDH (loading control) expression by immunoblotting. Similar results were obtained in two independent experiments.(D) Cells transfected as described in (C) and left untreated or treated with 500 ng/ml TRAIL, 10 μM ionomycin or starved for amino acids and glucose for 24 h were analysed for LC3 translocation.(E) Lysates of wild‐type (WT) MEFs and MEFs with an inactive TAK1 knock‐in (TAK1Δ) treated with 500 ng/ml TRAIL were analysed by immunoblotting. Similar results were obtained in two independent experiments.(F) WT and TAK1Δ MEFs left untreated and treated with 250 or 500 ng/ml TRAIL were analysed for cell death by the LDH release assay. Similar results were obtained in two independent experiments (A, C and E). The values represent mean±s.d. for two (D) or a minimum of three independent experiments (B, F). *P‐value 0.05, **P‐value 0.01 and ***P‐value 0.001 as compared with cells treated in the same way, but transfected without siRNA (B, D) or the wild‐type cells (F).


Figure 1 ( a ) Instant Blue stained SDS - polyacrylamide gel of Scc1wt cohesin tetramers used in ( b ) . ( b ) Cohesin loading assay . Scc1wt cohesin tetramer was incubated with nicked circular ( CN ) or 1x , 2x or 4x concentration of linearised ( L ) plasmid DNA , immunoprecipitated with anti - Scc1 antibodies , washed with high salt buffer and then eluted with Scc1 peptide . Recovered DNA was separated by agarose gel electrophoresis and stained with GelRedDNA stain . Input DNA = 7 % . ( c ) Silver stainedSDS - polyacrylamide gel of non - cleaved and cleaved Scc1Halo - TEVcohesin tetramers used in ( d ) . ( d ) Non - cleaved and cleaved Scc1Halo - TEVcohesin tetramer was incubated with nicked circular plasmid DNA and processed as in ( b ) . Input DNA = 4 % . ( e ) Kymograph of Scc1GFP - TEVcohesin bound to doubly tethered λ - DNA in cohesin binding buffer and washed with 750 mM NaCl buffer + Sytox Orange . XhoI flow in induced DNA cleavage and cohesin release . Flow in from top and scale bar = 5 µm in this and all subsequent kymographs . ( f ) Kymographs of Scc1GFP - TEVcohesin and Scc1GFPcohesin bound to doubly tethered λ - DNA and washed with 750 mM NaCl buffer . TEV protease flow in released Scc1GFP - TEVbut not Scc1GFP from DNA .

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Figure 1(a) Instant Blue stained SDS-polyacrylamide gel of Scc1wt cohesin tetramers used in (b).(b) Cohesin loading assay. Scc1wt cohesin tetramer was incubated with nicked circular (CN) or 1x, 2x or 4x concentration of linearised (L) plasmid DNA, immunoprecipitated with anti-Scc1 antibodies, washed with high salt buffer and then eluted with Scc1 peptide. Recovered DNA was separated by agarose gel electrophoresis and stained with GelRedDNA stain. Input DNA = 7 %.(c) Silver stainedSDS-polyacrylamide gel of non-cleaved and cleaved Scc1Halo-TEVcohesin tetramers used in (d).(d) Non-cleaved and cleaved Scc1Halo-TEVcohesin tetramer was incubated with nicked circular plasmid DNA and processed as in (b). Input DNA = 4 %.(e) Kymograph of Scc1GFP-TEVcohesin bound to doubly tethered λ-DNA in cohesin binding buffer and washed with 750 mM NaCl buffer + Sytox Orange. XhoI flow in induced DNA cleavage and cohesin release. Flow in from top and scale bar = 5 µm in this and all subsequent kymographs.(f) Kymographs of Scc1GFP-TEVcohesin and Scc1GFPcohesin bound to doubly tethered λ-DNA and washed with 750 mM NaCl buffer. TEV protease flow in released Scc1GFP-TEVbut not Scc1GFP from DNA.


Figure 2 ( a ) Kymograph of a single DNA - bound Scc1Halo - TMR - TEVcohesin complex after 750 mM NaCl buffer wash . ( b ) Distribution of background subtracted EMCCD counts of Scc1Halo - TMR - TEVcohesin complexes immobilized on the coverslip . Peaks at ~ 4 x 103 and ~ 8 x 103 EMCCD counts correspond to single fluorophores and a small fraction of double fluorophores respectively . n = 290 regions with fluorescent molecules , 228 regions with background . ( c ) Photobleaching kinetics of a single DNA - bound Scc1Halo - TMR - TEVcohesin complex . ( d ) Diffusion coefficients of Smc1 / 3 wild type or K38A ATP binding deficient ' KA ' forms of Scc1Halo - TMR - TEV - cohesin in the presence or absence of ATP . Diffusion coefficients were calculated based on the linear fit of the average mean square displacement of ≥ 13 freely diffusing molecules per condition over a 1 second time period .

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Figure 2(a) Kymograph of a single DNA-bound Scc1Halo-TMR-TEVcohesin complex after 750 mM NaCl buffer wash. (b) Distribution of background subtracted EMCCD counts of Scc1Halo-TMR-TEVcohesin complexes immobilized on the coverslip. Peaks at ~ 4 x 103 and ~ 8 x 103 EMCCD counts correspond to single fluorophores and a small fraction of double fluorophores respectively. n = 290 regions with fluorescent molecules, 228 regions with background. (c) Photobleaching kinetics of a single DNA-bound Scc1Halo-TMR-TEVcohesin complex. (d) Diffusion coefficients of Smc1/3 wild type or K38A ATP binding deficient 'KA' forms of Scc1Halo-TMR-TEV-cohesin in the presence or absence of ATP. Diffusion coefficients were calculated based on the linear fit of the average mean square displacement of ≥ 13 freely diffusing molecules per condition over a 1 second time period.


Figure 3 ( a - c ) Kymograph of salt - resistant Scc1GFP - TEV - cohesin diffusing past ( a ) TetO - DNA - bound TetRHalo - TMR . ( a - c ) Kymograph of salt - resistant Scc1GFP - TEV - cohesin diffusing past ( b ) DNA - bound Halo - TMREcoRIE111Q . ( a - c ) Kymograph of salt - resistant Scc1GFP - TEV - cohesin diffusing past ( c ) a DNA - bound TMR - labelled nucleosome . ( d ) Kymograph of salt - resistant Scc1GFP - TEV - cohesin failing to bypass DNA - bound QDotEcoRIE111Q . DNA was stained with Sytox Orange .

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Figure 3(a - c) Kymograph of salt-resistant Scc1GFP-TEV-cohesin diffusing past (a) TetO-DNA-bound TetRHalo-TMR.(a - c) Kymograph of salt-resistant Scc1GFP-TEV-cohesin diffusing past (b) DNA-bound Halo-TMREcoRIE111Q.(a - c) Kymograph of salt-resistant Scc1GFP-TEV-cohesin diffusing past (c) a DNA-bound TMR-labelled nucleosome.(d) Kymograph of salt-resistant Scc1GFP-TEV-cohesin failing to bypass DNA-bound QDotEcoRIE111Q. DNA was stained with Sytox Orange.


Figure 4 ( a ) Kymograph of two Halo - TMRT7 RNAP transcription elongation events . ( b , c ) Kymographs showing ( b ) Halo - TMRT7 RNAP transcription stalling following removal of NTPs and ( c ) subsequent resumption after NTP flow in . ( d ) Kymograph of Halo - TMRT7 RNAP ( diameter ~ 8 nm excluding HaloTag ) displacing Scc1GFP - TEV - cohesin following resumption of transcription in T7 reaction buffer + NTPs . ( e ) Kymograph of Halo - TMRT7 RNAP constraining translocation of salt - resistant Scc1GFP - TEV - cohesin .

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

Figure 4(a) Kymograph of two Halo-TMRT7 RNAP transcription elongation events. (b, c) Kymographs showing (b) Halo-TMRT7 RNAP transcription stalling following removal of NTPs and (c) subsequent resumption after NTP flow in.(d) Kymograph of Halo-TMRT7 RNAP (diameter ~ 8 nm excluding HaloTag) displacing Scc1GFP-TEV-cohesin following resumption of transcription in T7 reaction buffer + NTPs.(e) Kymograph of Halo-TMRT7 RNAP constraining translocation of salt-resistant Scc1GFP-TEV-cohesin.


Figure 5 ( a ) Representative field of view showing Halo - TMRCTCF bound to pPlat - 4xCTCF following 75 mM NaCl , 75 mM KCl buffer wash . Arrows denote CTCF bound at predicted site of 4xCTCF array . ( b ) Kymograph of salt - resistant Scc1GFP - TEV - cohesin failing to bypass DNA - bound Halo - TMRCTCF . DNA was post - stained with Sytox Green .

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

Figure 5(a) Representative field of view showing Halo-TMRCTCF bound to pPlat-4xCTCF following 75 mM NaCl, 75 mM KCl buffer wash. Arrows denote CTCF bound at predicted site of 4xCTCF array.(b) Kymograph of salt-resistant Scc1GFP-TEV-cohesin failing to bypass DNA-bound Halo-TMRCTCF. DNA was post-stained with Sytox Green.


Figure 1C : Western blot analysis of proteins pulled down using the peptides input fraction corresponds to HeLa cell total protein extract . Bound proteins were analyzed using anti - VAP - A , anti VAP - B and anti - MOSPD2 antibodies . Actin was used as a loading control . D : Western blot analysis of proteins pulled down using peptides comprising WT and mutant ( D478S and D478pS ) ORP1 FFAT sequence peptides Bound proteins were analyzed using anti VAP - A , anti - VAP - B and anti MOSPD2 antibodies . Actin was used as a loading control . Data information : Acidic ( D or phosphorylated residues ( pS alcoholic ( S residues are in red , green and blue , respectively ; the other residues are in black .

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Figure 1C: Western blot analysis of proteins pulled down using the peptides input fraction corresponds to HeLa cell total protein extract. Bound proteins were analyzed using anti-VAP-A, anti VAP-B and anti-MOSPD2 antibodies. Actin was used as a loading control.D: Western blot analysis of proteins pulled down using peptides comprising WT and mutant (D478S and D478pS) ORP1 FFAT sequence peptides Bound proteins were analyzed using anti VAP-A, anti-VAP-B and anti MOSPD2 antibodies. Actin was used as a loading control. Data information: Acidic (D or phosphorylated residues (pS alcoholic (S residues are in red, green and blue, respectively; the other residues are in black.


Figure 2B : Coomassie blue staining of the recombinant wild - type MSP domains of VAP - A and VAP - B , and of the KD / MD mutant of VAP - A , after SDS - PAGE . C : Recombinant MSP domains pulled down with peptides corresponding to the Phospho - FFAT motif of STARD3 ( unphosphorylated ; mono - phosphorylated on S209 or S213 ; bi - phosphorylated on S209 and S213 ) , and with the control peptides were revealed by Coomassie blue staining . The recombinant proteins subjected to the assay are showed in the input fraction .

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Figure 2B: Coomassie blue staining of the recombinant wild-type MSP domains of VAP-A and VAP-B, and of the KD/MD mutant of VAP-A, after SDS-PAGE.C: Recombinant MSP domains pulled down with peptides corresponding to the Phospho-FFAT motif of STARD3 (unphosphorylated; mono-phosphorylated on S209 or S213; bi-phosphorylated on S209 and S213), and with the control peptides were revealed by Coomassie blue staining. The recombinant proteins subjected to the assay are showed in the input fraction.


Figure 3A - D : Surface Plasmon Resonance analysis of the MSP domain of VAP - A binding onto immobilized unphosphorylated ( A ) , monophosphorylated pS209 ( B ) and pS213 ( C ) , and bi - phosphorylated pS209 + pS213 ( D ) STARD3 FFAT peptides . Representative sensorgrams resulting from the interaction between the MSP domain of VAP - A injected at different concentrations and the different FFAT peptides are shown in ( A ) , ( B left ) , ( C ) , and ( D left ) . Binding curves display the SPR signal ( RU ) as a function of time . Some samples were measured twice ( concentrations printed in bold ) . Signal obtained for the negative control peptide immobilized on another flowcell is systematically subtracted , as well as the bulk effect recorded with buffer only . Steady - state analysis of the interaction between pS209 ( B right ) and pS209 + pS213 ( D right ) STARD3 FFAT peptide , and the MSP domain of VAP - A . Equilibrium responses ( Req ) extracted from the left panel in association phase were plotted as a function of the dimeric MSP domain of VAP - A concentration , and fitted with a Langmuir binding model . E : Dissociation constants between peptides corresponding to different kind of FFAT motifs and the MSP domains of VAP - A and VAP - B . The kinetic experiments were performed at 25 ° C in Tris - HCl 50 mM , pH 7 . 5 , NaCl 75 mM buffer supplemented with 0 . 005 % ( v / v ) surfactant polysorbate 20 ( P20 , GE Healthcare ) . Mean of n independent experiments : n = 2 for pS209 and pS209 + pS213 ; 3 for pS213 ; and 4 for the non - phosphorylated FFAT . Uncertainties are obtained from the standard deviation considering a t - distribution coefficient for a risk factor of 32 % .

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

Figure 3A-D: Surface Plasmon Resonance analysis of the MSP domain of VAP-A binding onto immobilized unphosphorylated (A), monophosphorylated pS209 (B) and pS213 (C), and bi-phosphorylated pS209+pS213 (D) STARD3 FFAT peptides. Representative sensorgrams resulting from the interaction between the MSP domain of VAP-A injected at different concentrations and the different FFAT peptides are shown in (A), (B left), (C), and (D left). Binding curves display the SPR signal (RU) as a function of time. Some samples were measured twice (concentrations printed in bold). Signal obtained for the negative control peptide immobilized on another flowcell is systematically subtracted, as well as the bulk effect recorded with buffer only. Steady-state analysis of the interaction between pS209 (B right) and pS209+pS213 (D right) STARD3 FFAT peptide, and the MSP domain of VAP-A. Equilibrium responses (Req) extracted from the left panel in association phase were plotted as a function of the dimeric MSP domain of VAP-A concentration, and fitted with a Langmuir binding model.E: Dissociation constants between peptides corresponding to different kind of FFAT motifs and the MSP domains of VAP-A and VAP-B. The kinetic experiments were performed at 25 °C in Tris-HCl 50 mM, pH 7.5, NaCl 75 mM buffer supplemented with 0.005% (v/v) surfactant polysorbate 20 (P20, GE Healthcare). Mean of n independent experiments: n=2 for pS209 and pS209+pS213; 3 for pS213; and 4 for the non-phosphorylated FFAT. Uncertainties are obtained from the standard deviation considering a t-distribution coefficient for a risk factor of 32%.


Figure 4A , B : Binding assays . Immunoprecipitation ( GFP - Trap ) experiments between GFP - tagged VAP - A ( WT and KD / MD mutant ; A ) , VAP - B ( B ) and Flag - tagged STARD3 ( WT and S209A , S209D and S209D / P210A mutants ) . Please note that the mutations modify the FFAT and Phospho - FFAT scores of this region of STARD3 : compared to WT STARD3 ( Phospho - FFAT : 2 . 5 ; conventional FFAT : 6 . 5 ) , the STARD3 S209A , mutant has both low Phospho - FFAT and FFAT scores ( 6 . 5 ) , the S209D mutant has a high conventional FFAT score ( 2 . 5 ) but a low Phospho - FFAT score ( 6 . 5 ) , and the double mutant has a conventional FFAT score of 1 . 5 and a Phospho - FFAT score of 5 . 5 . Approximatively 5 µg of total protein extracts were analyzed by Western blot using anti - Flag , anti - GFP and anti - Actin antibodies . Immunoprecipitated material was analyzed using anti - Flag and anti - GFP antibodies . Recruitment assays . GFP - VAP - A ( C ; green ) were left untransfected ( C ) and labelled using anti - Flag ( magenta ) antibodies . The subpanels on the right are higher magnification ( 3 . 5 × ) images of the area outlined in white . The Overlay panel shows merged green and magenta images . The Coloc panel displays a colocalization mask on which pixels where the green and the magenta channels co - localize are shown in white . Scale bars : 10 µm . Inset scale bars : 2 µm . : Recruitment assays . GFP - VAP - A D , E , G ; green ) and GFP - VAP - A KD / MD - expressing cells ( F ; green ) were transfected with Flag - STARD3 ( D , F ) , Flag - STARD3 S209A ( E ) , and Flag - STARD3 S209D / P210A ( G ) , and labelled using anti - Flag ( magenta ) antibodies . The subpanels on the right are higher magnification ( 3 . 5 × ) images of the area outlined in white . The Overlay panel shows merged green and magenta images . The Coloc panel displays a colocalization mask on which pixels where the green and the magenta channels co - localize are shown in white . Scale bars : 10 µm . Inset scale bars : 2 µm . H : Pearson ' s correlation coefficients between VAP - A ( WT or KD / MD mutant ) and STARD3 ( WT or S209A , S209D , S209D / P210A , FA / YA mutants ) staining are shown . Each dot represents a single cell ( number of cells : VAP - A - STARD3 : 22 ; VAP - A - STARD3 S209A : 25 ; VAP - A - STARD3 S209D : 27 ; VAP - A - STARD3 S209D / P210A : 26 ; VAP - A - STARD3 FA / YA : 20 ; VAP - A KD / MD - STARD3 : 20 , from at least three independent experiments ) . Means and error bars ( SD ) are shown . Kruskal - Wallis with Dunn ' s multiple comparison test ( * * P < 0 . 01 ; * * * P < 0 . 001 ) .

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

Figure 4A,B: Binding assays. Immunoprecipitation (GFP-Trap) experiments between GFP-tagged VAP-A (WT and KD/MD mutant; A), VAP-B (B) and Flag-tagged STARD3 (WT and S209A, S209D and S209D/P210A mutants). Please note that the mutations modify the FFAT and Phospho-FFAT scores of this region of STARD3: compared to WT STARD3 (Phospho-FFAT: 2.5; conventional FFAT: 6.5), the STARD3 S209A, mutant has both low Phospho-FFAT and FFAT scores (6.5), the S209D mutant has a high conventional FFAT score (2.5) but a low Phospho-FFAT score (6.5), and the double mutant has a conventional FFAT score of 1.5 and a Phospho-FFAT score of 5.5. Approximatively 5 µg of total protein extracts were analyzed by Western blot using anti-Flag, anti-GFP and anti-Actin antibodies. Immunoprecipitated material was analyzed using anti-Flag and anti-GFP antibodies.Recruitment assays. GFP-VAP-A (C ; green) were left untransfected (C) and labelled using anti-Flag (magenta) antibodies. The subpanels on the right are higher magnification (3.5×) images of the area outlined in white. The Overlay panel shows merged green and magenta images. The Coloc panel displays a colocalization mask on which pixels where the green and the magenta channels co-localize are shown in white. Scale bars: 10 µm. Inset scale bars: 2 µm.: Recruitment assays. GFP-VAP-A D, E, G; green) and GFP-VAP-A KD/MD-expressing cells (F; green) were transfected with Flag-STARD3 (D, F), Flag-STARD3 S209A (E), and Flag-STARD3 S209D/P210A (G), and labelled using anti-Flag (magenta) antibodies. The subpanels on the right are higher magnification (3.5×) images of the area outlined in white. The Overlay panel shows merged green and magenta images. The Coloc panel displays a colocalization mask on which pixels where the green and the magenta channels co-localize are shown in white. Scale bars: 10 µm. Inset scale bars: 2 µm.H: Pearson's correlation coefficients between VAP-A (WT or KD/MD mutant) and STARD3 (WT or S209A, S209D, S209D/P210A, FA/YA mutants) staining are shown. Each dot represents a single cell (number of cells: VAP-A-STARD3: 22; VAP-A-STARD3 S209A: 25; VAP-A-STARD3 S209D: 27; VAP-A-STARD3 S209D/P210A: 26; VAP-A-STARD3 FA/YA: 20; VAP-A KD/MD-STARD3: 20, from at least three independent experiments). Means and error bars (SD) are shown. Kruskal-Wallis with Dunn's multiple comparison test (**P < 0.01; ***P < 0.001).


Figure 5B : SDS - PAGE gel and Western blot analysis of purified cSTD3 and pS209 cSTD3 proteins . Top : the gel was stained with Sypro Orange to visualize proteins and molecular weight markers . Bottom : two similar gels were blotted onto nitrocellulose and analysed for the presence of total and STARD3 - pS209 using specific antibodies . C : Aggregation assays . LA liposomes ( 50 µM total lipids ) were incubated for 5 minutes with cSTD3 or pS209 cSTD3 ( 380 nM ) . Then , LB liposomes ( 50 µM total lipids ) and VAP - AHIS6 or VAP - A KD / MDHIS6 ( 700 nM ) were successively added . Left panels : mean radius ( black dots ) and polydispersity ( shaded area ) over time . Right panels : size distribution before ( grey bars ) and after the reaction ( black bars ) . D : DHE transport assay . DOPC ( 1 . 2 - dioleoyl - sn - glycero - 3 - phosphocholine ) liposomes ( 62 . 5 µM total lipids , LA ) containing 3 mol % MPB - PE were mixed with cSTD3 or pS209 cSTD3 ( 475 nM ) . After 5 minutes , liposomes ( DOPC / DOGS - NTA - Ni2 + / DNS - PE / DHE liposomes 77 . 5 / 10 / 2 . 5 / 10 mol / mol , 62 . 5 µM total lipids , LB ) , covered or not with 1 µM of VAPHIS6 or VAP - A KD / MDHIS6 , were added . FRET between DHE and DNS - PE in the LB liposomes diminishes as DHE is transported towards LA liposomes . The signal was converted into amount of DHE present in LB liposomes ( in mol % ) . E : Initial DHE transport rate measured with cSTD3 or pS209 cSTD3 in the presence or absence of VAP - A HIS6 or VAP - A KD / MDHIS6 . Data are represented as mean ± S . E . M . ( n = 3 for cSTD3 - VAP and n = 4 for all other data ) . Mann - Whitney test ( * P < 0 . 05 ) .

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

Figure 5B: SDS-PAGE gel and Western blot analysis of purified cSTD3 and pS209 cSTD3 proteins. Top: the gel was stained with Sypro Orange to visualize proteins and molecular weight markers. Bottom: two similar gels were blotted onto nitrocellulose and analysed for the presence of total and STARD3-pS209 using specific antibodies.C: Aggregation assays. LA liposomes (50 µM total lipids) were incubated for 5 minutes with cSTD3 or pS209 cSTD3 (380 nM). Then, LB liposomes (50 µM total lipids) and VAP-AHIS6 or VAP-A KD/MDHIS6 (700 nM) were successively added. Left panels: mean radius (black dots) and polydispersity (shaded area) over time. Right panels: size distribution before (grey bars) and after the reaction (black bars).D: DHE transport assay. DOPC (1.2-dioleoyl-sn-glycero-3-phosphocholine) liposomes (62.5 µM total lipids, LA) containing 3 mol% MPB-PE were mixed with cSTD3 or pS209 cSTD3 (475 nM). After 5 minutes, liposomes (DOPC/DOGS-NTA-Ni2+/DNS-PE/DHE liposomes 77.5/10/2.5/10 mol/mol, 62.5 µM total lipids, LB), covered or not with 1 µM of VAPHIS6 or VAP-A KD/MDHIS6, were added. FRET between DHE and DNS-PE in the LB liposomes diminishes as DHE is transported towards LA liposomes. The signal was converted into amount of DHE present in LB liposomes (in mol%).E: Initial DHE transport rate measured with cSTD3 or pS209 cSTD3 in the presence or absence of VAP-A HIS6 or VAP-A KD/MDHIS6. Data are represented as mean ± S.E.M. (n = 3 for cSTD3-VAP and n=4 for all other data). Mann-Whitney test (*P < 0.05).


Figure 6A - E : HeLa / Ctrl ( A ) , HeLa / STARD3 ( B ) , HeLa / STARD3 S209A ( C ) , HeLa / STARD3 S209D / P210A ( D ) and HeLa / STARD3 Conv - FFAT ( E ) cells were labeled with anti - STARD3 antibodies ( magenta ) , anti - Lamp1 antibodies ( green ) and with the fluorescent cholesterol probe filipin ( Cyan Hot ) . Nuclei are shown in gray ( TO - PRO - 3 ) . Higher magnifications images ( 2x ) of the area outlined in white are shown on the right . The filipin , STARD3 and Lamp1 merged image is labeled Overlay . Scale bars : 10 µm . Inset scale bars : 5 µm . F : Pearson ' s correlation coefficients between STARD3 and Lamp1 staining in HeLa / STARD3 cells . Each dot represents a single cell ( 34 cells from 4 independent experiments ) . Mean and error bars ( SD ) are shown . Note that STARD3 and Lamp1 signals are highly correlated . G : Relative fluorescence intensity of intracellular filipin signals in endosomes of HeLa / Ctrl , HeLa / STARD3 , HeLa / STARD3 S209A , HeLa / STARD3 S209D / P210A and HeLa / STARD3 Conv - FFAT cells . Data are displayed as a Superplot ( Lord et al , 2020 ) showing the relative filipin fluorescence intensity in endosomes of individual cells ( small circles ) from 6 independent experiments ( mean of each experiment shown as a large circle ) . Independent experiments are color - coded . Means and error bars ( SD ) are shown as black bars . Kruskal - Wallis with Dunn ' s multiple comparison test ( * P < 0 . 05 ; * * P < 0 . 01 ; n = 6 independent experiments ) .

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Figure 6A-E: HeLa/Ctrl (A), HeLa/STARD3 (B), HeLa/STARD3 S209A (C), HeLa/STARD3 S209D/P210A (D) and HeLa/STARD3 Conv-FFAT (E) cells were labeled with anti-STARD3 antibodies (magenta), anti-Lamp1 antibodies (green) and with the fluorescent cholesterol probe filipin (Cyan Hot). Nuclei are shown in gray (TO-PRO-3). Higher magnifications images (2x) of the area outlined in white are shown on the right. The filipin, STARD3 and Lamp1 merged image is labeled Overlay. Scale bars: 10 µm. Inset scale bars: 5 µm.F: Pearson's correlation coefficients between STARD3 and Lamp1 staining in HeLa/STARD3 cells. Each dot represents a single cell (34 cells from 4 independent experiments). Mean and error bars (SD) are shown. Note that STARD3 and Lamp1 signals are highly correlated.G: Relative fluorescence intensity of intracellular filipin signals in endosomes of HeLa/Ctrl, HeLa/STARD3, HeLa/STARD3 S209A, HeLa/STARD3 S209D/P210A and HeLa/STARD3 Conv-FFAT cells. Data are displayed as a Superplot (Lord et al, 2020) showing the relative filipin fluorescence intensity in endosomes of individual cells (small circles) from 6 independent experiments (mean of each experiment shown as a large circle). Independent experiments are color-coded. Means and error bars (SD) are shown as black bars. Kruskal-Wallis with Dunn's multiple comparison test (*P < 0.05; **P < 0.01; n=6 independent experiments).


Figure 7A : Coomassie blue staining of the recombinant MSP domain of MOSPD2 after SDS - PAGE . B : Recombinant MSP domains pulled down with STARD3 FFAT peptides ( unphosphorylated , mono - phosphorylated on S209 or S213 , bi - phosphorylated on S209 and S213 , or tri - phosphorylated on S203 , S209 and S213 . and control peptides were revealed by Coomassie blue staining . The recombinant proteins subjected to the assay are shown in the input fraction . SPR analysis of the MSP domain of MOSPD2 binding onto immobilized unphosphorylated ( C ) STARD3 FFAT . Representative sensorgrams resulting from the interaction between the MSP domain of MOSPD2 injected at different concentrations and the different FFAT peptides are shown in ( C ) Binding curves display the SPR signal ( RU ) as a function of time . Concentrations printed in bold indicate samples measured twice . Signal obtained for the negative control peptide immobilized on another flowcell is systematically subtracted , as well as the bulk effect recorded with buffer only . SPR analysis of the MSP domain of MOSPD2 binding onto immobilized pS209 ( D ) STARD3 FFAT . Representative sensorgrams resulting from the interaction between the MSP domain of MOSPD2 injected at different concentrations and the different FFAT peptides are shown in ( D left ) . Binding curves display the SPR signal ( RU ) as a function of time . Concentrations printed in bold indicate samples measured twice . Signal obtained for the negative control peptide immobilized on another flowcell is systematically subtracted , as well as the bulk effect recorded with buffer only . ( D right ) steady - state analysis of the interaction between pS209 STARD3 FFAT peptide and the MSP domain of MOSPD2 . Equilibrium responses ( Req ) extracted from the left panel were plotted as a function of the monomeric concentration of the MSP domain of MOSPD2 , and fitted with a 1 : 1 binding model . The experiments were performed at 25 ° C in Tris - HCl 50 mM , pH 7 . 5 , NaCl 75 mM buffer supplemented with 0 . 005 % ( v / v ) surfactant polysorbate 20 ( P20 , GE Healthcare ) . Mean of 2 independent experiments . Uncertainties are obtained from the standard deviation considering a t - distribution coefficient for a risk factor of 32 % . E : Interaction dissociation constants between the different STARD3 FFAT peptides and the MSP domains of MOSPD2 . F : Immunoprecipitation ( GFP - Trap ) experiments between GFP - tagged MOSPD2 and Flag - tagged STARD3 ( WT and S209A , S209D and S209D / P210A mutants ) . Approximatively 5 µg of total protein extracts were analyzed by Western blot using anti - Flag , anti - GFP and anti - Actin antibodies . Immunoprecipitated material was analyzed using anti - Flag and anti - GFP antibodies . G - I : GFP - MOSPD2 - expressing cells ( green ) were left untransfected ( G ) or transfected with Flag - STARD3 ( H ) and Flag - STARD3 S209A ( I ) , and labelled using anti - Flag ( magenta ) antibodies . The subpanels on the right are higher magnification ( 3 . 5 × ) images of the area outlined in white . The Overlay panel shows merged green and magenta images . The Coloc panel displays a colocalization mask on which pixels where the green and the magenta channels co - localize are shown in white . Scale bars : 10 µm . J : Pearson ' s correlation coefficients between MOSPD2 ( WT or RD / LD ) and STARD3 ( WT , S209A or FA / YA ) staining are shown . Each dot represents a single cell ( number of cells : MOSPD2 - STARD3 : 22 ; MOSPD2 - STARD3 S209A : 21 ; MOSPD2 - STARD3 FA / YA : 20 ; MOSPD2 RD / LD - STARD3 : 17 , from at least three independent experiments ) . Means and error bars ( SD ) are shown . Kruskal - Wallis with Dunn ' s multiple comparison test ( * * * P < 0 . 001 ) .

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

Figure 7A: Coomassie blue staining of the recombinant MSP domain of MOSPD2 after SDS-PAGE.B: Recombinant MSP domains pulled down with STARD3 FFAT peptides (unphosphorylated, mono-phosphorylated on S209 or S213, bi-phosphorylated on S209 and S213, or tri-phosphorylated on S203, S209 and S213.and control peptides were revealed by Coomassie blue staining. The recombinant proteins subjected to the assay are shown in the input fraction.SPR analysis of the MSP domain of MOSPD2 binding onto immobilized unphosphorylated (C) STARD3 FFAT. Representative sensorgrams resulting from the interaction between the MSP domain of MOSPD2 injected at different concentrations and the different FFAT peptides are shown in (C) Binding curves display the SPR signal (RU) as a function of time. Concentrations printed in bold indicate samples measured twice. Signal obtained for the negative control peptide immobilized on another flowcell is systematically subtracted, as well as the bulk effect recorded with buffer only.SPR analysis of the MSP domain of MOSPD2 binding onto immobilized pS209 (D) STARD3 FFAT. Representative sensorgrams resulting from the interaction between the MSP domain of MOSPD2 injected at different concentrations and the different FFAT peptides are shown in (D left). Binding curves display the SPR signal (RU) as a function of time. Concentrations printed in bold indicate samples measured twice. Signal obtained for the negative control peptide immobilized on another flowcell is systematically subtracted, as well as the bulk effect recorded with buffer only. (D right) steady-state analysis of the interaction between pS209 STARD3 FFAT peptide and the MSP domain of MOSPD2. Equilibrium responses (Req) extracted from the left panel were plotted as a function of the monomeric concentration of the MSP domain of MOSPD2, and fitted with a 1:1 binding model. The experiments were performed at 25 °C in Tris-HCl 50 mM, pH 7.5, NaCl 75 mM buffer supplemented with 0.005% (v/v) surfactant polysorbate 20 (P20, GE Healthcare). Mean of 2 independent experiments. Uncertainties are obtained from the standard deviation considering a t-distribution coefficient for a risk factor of 32%.E: Interaction dissociation constants between the different STARD3 FFAT peptides and the MSP domains of MOSPD2.F: Immunoprecipitation (GFP-Trap) experiments between GFP-tagged MOSPD2 and Flag-tagged STARD3 (WT and S209A, S209D and S209D/P210A mutants). Approximatively 5 µg of total protein extracts were analyzed by Western blot using anti-Flag, anti-GFP and anti-Actin antibodies. Immunoprecipitated material was analyzed using anti-Flag and anti-GFP antibodies.G-I: GFP-MOSPD2-expressing cells (green) were left untransfected (G) or transfected with Flag-STARD3 (H) and Flag-STARD3 S209A (I), and labelled using anti-Flag (magenta) antibodies. The subpanels on the right are higher magnification (3.5×) images of the area outlined in white. The Overlay panel shows merged green and magenta images. The Coloc panel displays a colocalization mask on which pixels where the green and the magenta channels co-localize are shown in white. Scale bars: 10 µm.J: Pearson's correlation coefficients between MOSPD2 (WT or RD/LD) and STARD3 (WT, S209A or FA/YA) staining are shown. Each dot represents a single cell (number of cells: MOSPD2-STARD3: 22; MOSPD2- STARD3 S209A: 21; MOSPD2-STARD3 FA/YA: 20; MOSPD2 RD/LD-STARD3: 17, from at least three independent experiments). Means and error bars (SD) are shown. Kruskal-Wallis with Dunn's multiple comparison test (***P < 0.001).


Figure 1 . An S . typhimurium strain devoid of its SPI - 1 TTSS effector proteins is able to induce programmed cell death in macrophages . BMDPM from wild - type ( A ) or caspase - 1 − / − ( B ) mice ( Kuida et al . , 1995 ) were infected with wild - type S . typhimurium or mutant derivative either lacking all TTSS effector proteins ( effectorless ) or defective for TTSS secretion by virtue a having a mutation on an essential component of this system ( invG ) . 6 h after infection , cytotoxicity was evaluated by lactate dehydrogenase release or by terminal deoxynucleatide transferase ( TUNEL ) staining . Values for the lactate dehydrogenase release represent the average of at least three independent measurements . Equivalent results were obtained in several repetitions of these experiments .

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Figure 1. An S. typhimurium strain devoid of its SPI-1 TTSS effector proteins is able to induce programmed cell death in macrophages. BMDPM from wild-type (A) or caspase-1−/− (B) mice (Kuida et al., 1995) were infected with wild-type S. typhimurium or mutant derivative either lacking all TTSS effector proteins (effectorless) or defective for TTSS secretion by virtue a having a mutation on an essential component of this system (invG). 6 h after infection, cytotoxicity was evaluated by lactate dehydrogenase release or by terminal deoxynucleatide transferase (TUNEL) staining. Values for the lactate dehydrogenase release represent the average of at least three independent measurements. Equivalent results were obtained in several repetitions of these experiments.


Figure 2 . Transient expression of SipB in BMDPM from caspase - 1 − / − mice results in cell death . Plasmids expressing either SipB , SipC , or SipD fused to YFP were introduced into BMDPM from caspase - 1 − / − mice as indicated in the Materials and methods section . Macrophages were then fixed , stained with DAPI ( to visualize chromatin ) , and observed under a fluorescence microscope . Cells expressing SipC or SipD do not show any signs of cytotoxicity . In contrast , cells expressing SipB show clear signs of cytotoxicity and chromatin condensation . Arrows indicate the nucleus of transfected cells .

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Figure 2. Transient expression of SipB in BMDPM from caspase-1−/−mice results in cell death. Plasmids expressing either SipB, SipC, or SipD fused to YFP were introduced into BMDPM from caspase-1−/− mice as indicated in the Materials and methods section. Macrophages were then fixed, stained with DAPI (to visualize chromatin), and observed under a fluorescence microscope. Cells expressing SipC or SipD do not show any signs of cytotoxicity. In contrast, cells expressing SipB show clear signs of cytotoxicity and chromatin condensation. Arrows indicate the nucleus of transfected cells.


Figure 3 . Transient expression of SipB in COS - 2 cells . ( A ) A plasmid expressing YFP - tagged SipB was transfected into COS - 2 cells , and its localization was examined under a confocal microscope . ( B ) SipB colocalized with mitochondrial markers in transfected cells . COS - 2 cells were transfected with a plasmid encoding either SipB - CFP ( top ) or SipB - YFP ( bottom ) . Transfected cells were stained with MitoTracker ® Red ( Molecular Probes , Inc . ) to visualize mitochondria and were examined by confocal microscopy . The SipB - induced structures were readily stained with mitochondrial markers ( top ) . Mitochondria were also seen contained within the SipB - induced structures ( bottom ) . ( C ) SipB - induced structures were labeled with the ER marker Sec - 61 . COS - 2 cells were cotransfected with plasmids encoding SipB - YFP and Sec61 - CFP , and transfected cells were examined by confocal microscopy .

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

Figure 3.Transient expression of SipB in COS-2 cells. (A) A plasmid expressing YFP-tagged SipB was transfected into COS-2 cells, and its localization was examined under a confocal microscope. (B) SipB colocalized with mitochondrial markers in transfected cells. COS-2 cells were transfected with a plasmid encoding either SipB-CFP (top) or SipB-YFP (bottom). Transfected cells were stained with MitoTracker® Red (Molecular Probes, Inc.) to visualize mitochondria and were examined by confocal microscopy. The SipB-induced structures were readily stained with mitochondrial markers (top). Mitochondria were also seen contained within the SipB-induced structures (bottom).  (C) SipB-induced structures were labeled with the ER marker Sec-61. COS-2 cells were cotransfected with plasmids encoding SipB-YFP and Sec61-CFP, and transfected cells were examined by confocal microscopy.


Figure 4 . Immuno - EM of SipB - transfected COS - 2 cells . COS - 2 cells were transfected with a plasmid expressing SipB - YFP , and 24 h after transfection , cells were fixed and prepared for immuno - EM using an antibody specific for GFP and a gold - labeled anti - rabbit antibody . SipB was seen localized to structures made up of closely apposed membranes that were often in close proximity to mitochondria ( A ) . Mitochondria were often seen apparently fusing to the SipB - induced membranous structure ( B , see arrows in inset on top ; C , inset to the right ) . Mitochondria were often seen contained with the SipB - induced membrane structures ( D ) . Bars , 250 nm .

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Figure 4. Immuno-EM of SipB-transfected COS-2 cells. COS-2 cells were transfected with a plasmid expressing SipB-YFP, and 24 h after transfection, cells were fixed and prepared for immuno-EM using an antibody specific for GFP and a gold-labeled anti-rabbit antibody. SipB was seen localized to structures made up of closely apposed membranes that were often in close proximity to mitochondria (A). Mitochondria were often seen apparently fusing to the SipB-induced membranous structure (B, see arrows in inset on top; C, inset to the right). Mitochondria were often seen contained with the SipB-induced membrane structures (D). Bars, 250 nm.


Figure 5 . The nonfusogenic SipB428 - 593 mutant localizes to the mitochondria , but does not form multivesicular structures and interferes with the activity of the wild - type protein . ( A ) COS - 2 cells were transfected with a plasmid expressing YFP - tagged SipB428 - 593 , stained with an antibody to the mitochondrial protein CoxIV , subunit VIc ( Molecular Probes , Inc . ) , and examined by confocal microscopy . SipB428 - 593 - YFP localized to mitochondria and did not induce the formation of multivesicular structures seen after expression of the wild - type protein . ( B ) COS - 2 cells were cotransfected with plasmids expressing either SipB428 - 593 - YFP or SipB - CFP , and transfected cells were examined under a confocal microscope . Expression of SipB428 - 593 - YFP prevented wild - type SipB - CFP from forming multivesicular structures ( notice that vesicular structures are only apparent in the absence , but not in the presence , of SipB428 - 593 - YFP ; right ) .

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Figure 5. The nonfusogenic SipB428-593 mutant localizes to the mitochondria, but does not form multivesicular structures and interferes with the activity of the wild-type protein. (A) COS-2 cells were transfected with a plasmid expressing YFP-tagged SipB428-593, stained with an antibody to the mitochondrial protein CoxIV, subunit VIc (Molecular Probes, Inc.), and examined by confocal microscopy. SipB428-593-YFP localized to mitochondria and did not induce the formation of multivesicular structures seen after expression of the wild-type protein.  (B) COS-2 cells were cotransfected with plasmids expressing either SipB428-593-YFP or SipB-CFP, and transfected cells were examined under a confocal microscope. Expression of SipB428-593-YFP prevented wild-type SipB-CFP from forming multivesicular structures (notice that vesicular structures are only apparent in the absence, but not in the presence, of SipB428-593-YFP; right).


Figure 6 . The S . typhimurium SPI - 1 TTSS - secreted protein SipB localizes to the mitochondria of infected macrophages . ( A ) Bone marrow - derived macrophages were infected with either wild - type S . typhimurium or the TTSS translocation - defective sipD mutant strain with a multiplicity of infection of 50 . 3 h after infection , macrophages were collected and processed for immuno - EM using gold - labeled antibodies . SipB could be readily detected in macrophages infected with wild - type ( A and B ) , but not in macrophages infected with the sipD mutant strain ( C ) . Bars , 100 nm . ( B ) Alternatively , mitochondria were isolated from the infected macrophages and the presence of SipB or the resident mitochondrial protein CoxIV , subunit IV , and were examined by Western immunoblot analysis using specific antibodies .

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Figure 6. The S. typhimurium SPI-1 TTSS-secreted protein SipB localizes to the mitochondria of infected macrophages. (A) Bone marrow-derived macrophages were infected with either wild-type S. typhimurium or the TTSS translocation-defective sipD mutant strain with a multiplicity of infection of 50. 3 h after infection, macrophages were collected and processed for immuno-EM using gold-labeled antibodies. SipB could be readily detected in macrophages infected with wild-type (A and B), but not in macrophages infected with the sipD mutant strain (C). Bars, 100 nm.  (B) Alternatively, mitochondria were isolated from the infected macrophages and the presence of SipB or the resident mitochondrial protein CoxIV, subunit IV, and were examined by Western immunoblot analysis using specific antibodies.