Figure 7A Malondialdehyde ( MDA ) content in leaves of 14 - d - old DJ and osprr73 plants treated by 180 mM NaCl for 3 d and normal condition . Data represent means ± SD . n = 6 , biological replicates . The asterisks indicate the significant difference ( * * * ) P ≤ 0 . 001 by student ' s t - test . B DAB staining showing the greater ROS accumulation in the leaves of osprr73 mutant treated by 180 mM NaCl treatment for 3 days . Scale bar , 1 cm . C H2O2 concentration was detected by using H2DCFDA in the root tip of DJ and osprr73 mutant plants . Scale bar , 5 mm . - NaCl and + NaCl represented the untreated or treated plants by NaCl respectively . D Quantitative analysis of H2O2 concentration in the root of DJ and osprr73 mutant . Data represent means ± SD . n ≥ 6 . The asterisks indicate the significant difference ( * * * ) P ≤ 0 . 001 by student ' s t - test . E , F Measurement of SOD ( Superoxide dismutase ) ( E ) and CAT ( Catalase ) ( F ) enzymes activity in shoots of DJ and osprr73 seedlings with or without NaCl stress . Data represent means ± SD . n = 3 technical replicates . Data are representative from two independent biological replicates with similar result . The asterisks indicate the significant difference ( * ) P ≤ 0 . 05 by student ' s t - test .

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

Figure 7A Malondialdehyde (MDA) content in leaves of 14-d-old DJ and osprr73 plants treated by 180 mM NaCl for 3 d and normal condition. Data represent means ± SD. n = 6, biological replicates. The asterisks indicate the significant difference (***) P ≤ 0.001 by student's t-test.B DAB staining showing the greater ROS accumulation in the leaves of osprr73 mutant treated by 180 mM NaCl treatment for 3 days. Scale bar, 1 cm.C H2O2 concentration was detected by using H2DCFDA in the root tip of DJ and osprr73 mutant plants. Scale bar, 5 mm. -NaCl and +NaCl represented the untreated or treated plants by NaCl respectively.D Quantitative analysis of H2O2 concentration in the root of DJ and osprr73 mutant. Data represent means ± SD. n ≥ 6. The asterisks indicate the significant difference (***) P ≤ 0.001 by student's t-test.E, F Measurement of SOD (Superoxide dismutase) (E) and CAT (Catalase) (F) enzymes activity in shoots of DJ and osprr73 seedlings with or without NaCl stress. Data represent means ± SD. n = 3 technical replicates. Data are representative from two independent biological replicates with similar result. The asterisks indicate the significant difference (*) P ≤ 0.05 by student's t-test.


Figure 1scRNA - Seq of the expression of the entire glycolytic pathway or of glucose phosphate isomerase only ( GPI ) in primary CD4 + T - cells infected in vitro ( D , E ) or CD4 + T - cells of PLWH ( F ) . In panels ( D , E ) , cells were infected with VSVG - HIV - 1 - GFP and sorted for viral expression as detailed in ( Golumbeanu et al , 2018 ) . Following latency establishment , cells were left untreated or HIV - 1 expression was reactivated through suberoyl anilide hydroxamic acid ( SAHA ) or α - CD3 - CD28 engagement . Clusters 1 and 2 were identified by principal component analysis as described in ( Golumbeanu et al , 2018 ) . In panel ( F ) , CD4 + T - cells were isolated from total blood of PLWH under ART as described in ( Cohn et al , 2018 ) . Viral expression was reactivated by treatment with phytohemagglutinin ( PHA ) and cells were sorted using antibodies against Env and Gag . Sorted cells were then subjected to scRNA - Seq analysis . The expression level of the HUMAN - GLYCOLYSIS pathway in ( D ) was calculated as the average expression of genes comprising the gene list ; expression levels in cluster 1 and 2 were compared using Wilcoxon rank sum test . For panels ( E , F ) , significance of GPI differential expression level between clusters ( E ) or between control and Env + Gag + conditions ( F ) was assessed by the Wilcoxon rank sum test encoded in FindMarkers Seurat R function . * * p < 0 . 01 , * * * p < 0 . 001 ; * * * p < 0 . 0001 . For panels ( D , E ) n = 1 donor , 43 cells ( untreated ) , 90 cells ( SAHA ) , 91 cells ( TCR ) , for panel ( F ) n = 3 donors , 109 cells ( control ) and 85 cells ( Gag + Env + ) .

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 O O O

Figure 1scRNA-Seq of the expression of the entire glycolytic pathway or of glucose phosphate isomerase only (GPI) in primary CD4+ T-cells infected in vitro (D,E) or CD4+ T-cells of PLWH (F). In panels (D, E), cells were infected with VSVG-HIV-1-GFP and sorted for viral expression as detailed in (Golumbeanu et al, 2018). Following latency establishment, cells were left untreated or HIV-1 expression was reactivated through suberoyl anilide hydroxamic acid (SAHA) or α-CD3-CD28 engagement. Clusters 1 and 2 were identified by principal component analysis as described in (Golumbeanu et al, 2018). In panel (F), CD4+ T-cells were isolated from total blood of PLWH under ART as described in (Cohn et al, 2018). Viral expression was reactivated by treatment with phytohemagglutinin (PHA) and cells were sorted using antibodies against Env and Gag. Sorted cells were then subjected to scRNA-Seq analysis. The expression level of the HUMAN-GLYCOLYSIS pathway in (D) was calculated as the average expression of genes comprising the gene list; expression levels in cluster 1 and 2 were compared using Wilcoxon rank sum test. For panels (E, F), significance of GPI differential expression level between clusters (E) or between control and Env+ Gag+ conditions (F) was assessed by the Wilcoxon rank sum test encoded in FindMarkers Seurat R function. ** p< 0.01, *** p< 0.001; *** p< 0.0001. For panels (D,E) n = 1 donor, 43 cells (untreated), 90 cells (SAHA ), 91 cells (TCR), for panel (F) n = 3 donors, 109 cells (control) and 85 cells (Gag+ Env+).


Figure 2Relative ratios of NADH / NAD + ( D ) and ATP / ADP ( E ) in latently infected and reactivated cells . Data were normalized using the matching uninfected control .

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Figure 2Relative ratios of NADH/NAD+ (D) and ATP/ADP (E) in latently infected and reactivated cells. Data were normalized using the matching uninfected control.


Figure 4Relative expression of HIV - 1 gag in cells infected with Tat - deficient HIV - 1pNL4 - 3 as compared to cells infected with wild type HIV - 1 pNL4 - 3 . relative expression of the limiting rate enzyme of the pentose phosphate pathway , i . e . G6PD ( B ) in HIV - 1 infected as compared to mock infected cells . relative expression of genes regulating the thioredoxin and glutathione antioxidant pathways , i . e . TrxR1 ( C ) and GCLC ( D ) , in HIV - 1 infected as compared to mock infected cells .

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Figure 4Relative expression of HIV-1 gag in cells infected with Tat-deficient HIV-1pNL4-3 as compared to cells infected with wild type HIV-1 pNL4-3.relative expression of the limiting rate enzyme of the pentose phosphate pathway, i.e. G6PD (B) in HIV-1 infected as compared to mock infected cells.relative expression of genes regulating the thioredoxin and glutathione antioxidant pathways, i.e. TrxR1 (C) and GCLC (D), in HIV-1 infected as compared to mock infected cells.


Figure 5Reactivation from HIV - 1 latency ( A ) and relative cell viability ( B ) in primary Th17 cells following 24 h treatment with the Trx inhibitor auranofin ( AF ; 500 nM ) , the GSH inhibitor buthionine sulfoximine ( BSO ; 250 μM ) , or a combination of the two . GFP - HIV - 1 expression was determined by FACS . Data are expressed as mean ± SD of three replicates and were analyzed by One - Way ANOVA followed by Tukey ' s post - test ( A ) or Two - Way ANOVA followed by Sidak ´ s post - test ( B ) . Solid lines represent the means . Levels of integrated HIV - 1 DNA following treatment for 48 h with AF and / or BSO in CD4 + T - cells derived from PLWH under suppressive antiretroviral therapy . Live cells were sorted after treatment , and integrated DNA was measured by Alu - PCR . The latency reactivating agent SAHA was used as a reference compound ( Archin et al , 2012 ) . Data were analyzed by non - parametric Friedman ´ s test followed by Dunn ' s post - test .

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

Figure 5Reactivation from HIV-1 latency (A) and relative cell viability (B) in primary Th17 cells following 24 h treatment with the Trx inhibitor auranofin (AF; 500 nM), the GSH inhibitor buthionine sulfoximine (BSO; 250 μM), or a combination of the two. GFP-HIV-1 expression was determined by FACS. Data are expressed as mean ± SD of three replicates and were analyzed by One-Way ANOVA followed by Tukey's post-test (A) or Two-Way ANOVA followed by Sidak´s post-test (B). Solid lines represent the means.Levels of integrated HIV-1 DNA following treatment for 48 h with AF and/or BSO in CD4+ T-cells derived from PLWH under suppressive antiretroviral therapy. Live cells were sorted after treatment, and integrated DNA was measured by Alu-PCR. The latency reactivating agent SAHA was used as a reference compound (Archin et al, 2012). Data were analyzed by non-parametric Friedman´s test followed by Dunn's post-test.


Figure 1EVs were isolated from CCM of uninfected or HIV - 1 - infected Jurkat cells by differential ultracentrifugation ( A - C ) ( A , C ) The successive pellets ( 2K , 10K , 100K ) recovered from the CCM of 20 x 106 cells were analyzed by Western blot for the indicated proteins side - by - side with the lysate of 0 . 4 x 106 producing cells : C . Images from two different representative experiments are shown . Amount of proteins recovered in 2K , 10K and 100K pellets analyzed in stain - free gel images was similar between pellets : AU ( protein amount in a given pellet / total protein amount in 2K + 10K + 100K ) for 2K , 10K and 100K pellets were 1 . 13 ± 0 . 28 , 0 , 96 ± 0 . 10 and 0 . 91 ± 0 . 33 , respectively in control cells ( A ) , and 1 . 22 ± 0 . 29 , 0 , 84 ± 0 . 35 and 0 . 94 ± 0 . 30 , respectively in infected cells ( C ) ( mean ± SD ) . EVs were isolated from CCM of uninfected Jurkat cells by differential ultracentrifugation SD ) . ( B ) Whole - mount EM analysis showing representative images and size distribution of particles in 2K , 10K and 100K pellets ( Scale bar , 200 nm ) . The diameter ( nm ) of particles was determined with ImageJ for five different experiments . 387 , 363 and 385 particles were counted for 2K , 10K and 100K pellets , respectivelly . ( A - D ) EVs were isolated from CCM of uninfected or HIV - 1 - infected Jurkat cells by differential ultracentrifugation and sEVs contained in the 100K pellet were further separated by velocity iodixanol gradient ( D ) . ( D ) 12 fractions were recovered from iodixanol gradients of the 100K pellets obtained from CCM of non - infected and infected Jurkat cells and analysed by Western blot for the indicated proteins .

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Figure 1EVs were isolated from CCM of uninfected or HIV-1-infected Jurkat cells by differential ultracentrifugation (A-C) (A, C) The successive pellets (2K, 10K, 100K) recovered from the CCM of 20 x 106 cells were analyzed by Western blot for the indicated proteins side-by-side with the lysate of 0.4 x 106 producing cells: C. Images from two different representative experiments are shown. Amount of proteins recovered in 2K, 10K and 100K pellets analyzed in stain-free gel images was similar between pellets: AU (protein amount in a given pellet/total protein amount in 2K+10K+100K) for 2K, 10K and 100K pellets were 1.13±0.28, 0,96±0.10 and 0.91±0.33, respectively in control cells (A), and 1.22±0.29, 0,84±0.35 and 0.94±0.30, respectively in infected cells (C) (mean±SD).EVs were isolated from CCM of uninfected Jurkat cells by differential ultracentrifugation SD). (B) Whole-mount EM analysis showing representative images and size distribution of particles in 2K, 10K and 100K pellets (Scale bar, 200 nm). The diameter (nm) of particles was determined with ImageJ for five different experiments. 387, 363 and 385 particles were counted for 2K, 10K and 100K pellets, respectivelly.(A-D) EVs were isolated from CCM of uninfected or HIV-1-infected Jurkat cells by differential ultracentrifugation and sEVs contained in the 100K pellet were further separated by velocity iodixanol gradient (D). (D) 12 fractions were recovered from iodixanol gradients of the 100K pellets obtained from CCM of non-infected and infected Jurkat cells and analysed by Western blot for the indicated proteins.


Figure 2 ( A ) Proteomic profiles of different proteins , showing the relative abundance distribution across the 3x3 subfractions obtained from Jurkat cells . Proteins with very similar profiles ( represented by the same colours ) are likely part of the same EV subtypes . Each profile consists of three independent data triplets ( F1 - F2 - F3A , F1 - F2 - F3B and F1 - F2 - F3C ) . ( C ) Violin plots showing enrichment of the protein markers of intracellular organelles across the F1 - F2 - F3 subfractions as compared to expression in the total cell proteome . Solid horizontal lines indicate medians and dashed line indicate quartiles ( n = 3 ) . Mitochondria and ER markers are de - enriched , and plasma membrane markers are enriched in EV fractions , with progressively pronounced effects from F1 to F3 .

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

Figure 2(A) Proteomic profiles of different proteins, showing the relative abundance distribution across the 3x3 subfractions obtained from Jurkat cells. Proteins with very similar profiles (represented by the same colours) are likely part of the same EV subtypes. Each profile consists of three independent data triplets (F1- F2- F3A, F1- F2- F3B and F1-F2-F3C).(C) Violin plots showing enrichment of the protein markers of intracellular organelles across the F1-F2-F3 subfractions as compared to expression in the total cell proteome. Solid horizontal lines indicate medians and dashed line indicate quartiles (n=3). Mitochondria and ER markers are de-enriched, and plasma membrane markers are enriched in EV fractions, with progressively pronounced effects from F1 to F3.


Figure 3EVs were purified by SEC from supernatants of Jurkat cells . ( A ) EVs were subjected to immuno - isolation with beads coupled to antibodies against CD81 or CD63 . Bead - associated ( Pull - down : PD ) vesicles and those left behind ( Flow - Through : FT ) were loaded on a gel for Western blot analysis with antibodies specific for CD63 , Syntenin - 1 , CD81 , ADAM10 and CD3G . A representative blot ( top panel ) and quantification ( mean ± SD ) of the proportion of signal in FT as compared with total ( PD + FT ) from four independent experiments ( bottom panel ) are shown . ( B ) Representative images of double - immunogold labelling of EVs purified by SEC from Jurkat cells . Left panel , CD81 / CD63 ; right panel , CD81 / CD3E . Arrows show CD63 staining in CD81 + CD63 + EVs . EVs were purified by SEC from supernatants of Jurkat cells . ( C , D ) Multiplex bead - based flow cytometry assay for detection of EV surface markers . Antibody - coated capture beads were incubated with 2 x 109 particles . Captured EVs were detected with either APC - labeled anti - CD81 , anti - CD63 or anti - CD3E . ( C ) Median APC fluorescence values for the different bead populations is shown as the ratio to the median APC fluorescence of control beads ( log10 scale ) . shown . ( D ) Heat - map representation of the median APC fluorescence values for the different bead populations detected with anti - CD63 or anti - CD3E antibodies relative to the values detected with anti - CD81 .

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 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 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

Figure 3EVs were purified by SEC from supernatants of Jurkat cells. (A) EVs were subjected to immuno-isolation with beads coupled to antibodies against CD81 or CD63. Bead-associated (Pull-down: PD) vesicles and those left behind (Flow-Through: FT) were loaded on a gel for Western blot analysis with antibodies specific for CD63, Syntenin-1, CD81, ADAM10 and CD3G. A representative blot (top panel) and quantification (mean ± SD) of the proportion of signal in FT as compared with total (PD+FT) from four independent experiments (bottom panel) are shown.(B) Representative images of double-immunogold labelling of EVs purified by SEC from Jurkat cells. Left panel, CD81/CD63; right panel, CD81/CD3E. Arrows show CD63 staining in CD81+CD63+ EVs.EVs were purified by SEC from supernatants of Jurkat cells. (C,D) Multiplex bead-based flow cytometry assay for detection of EV surface markers. Antibody-coated capture beads were incubated with 2 x 109 particles. Captured EVs were detected with either APC-labeled anti-CD81, anti-CD63 or anti-CD3E. (C) Median APC fluorescence values for the different bead populations is shown as the ratio to the median APC fluorescence of control beads (log10 scale). shown. (D) Heat-map representation of the median APC fluorescence values for the different bead populations detected with anti-CD63 or anti-CD3E antibodies relative to the values detected with anti-CD81.


Figure 4 ( B ) Proteomic profiles of different proteins showing the relative abundance distribution across the six subfractions . Each profile consists of two independent data triplets ( F1 - F2 - F3A and F1 - F2 - F3B ) . Abundance profiles of the same groups of proteins showed the same clustering behaviour , although in this second dataset , the proportion of proteins recovered in the F2 was more variable . These differences , however , did not affect the results of the NNP or movement analysis . ( C ) Unbiased identification of significant translocation events triggered by HIV - 1 infection . Each protein is scored for magnitude of translocation ( M score , x - axis ) and reproducibility of translocation direction ( R score , y - axis ) across the two replicates . MR plot analysis reveals significant translocations in the top right quadrant . Proteins with M > 1 and R > 0 . 9 are candidate hits for changing localization ( estimated FDR = 8 % ) .

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 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

Figure 4(B) Proteomic profiles of different proteins showing the relative abundance distribution across the six subfractions. Each profile consists of two independent data triplets (F1- F2- F3A and F1- F2- F3B). Abundance profiles of the same groups of proteins showed the same clustering behaviour, although in this second dataset, the proportion of proteins recovered in the F2 was more variable. These differences, however, did not affect the results of the NNP or movement analysis.(C) Unbiased identification of significant translocation events triggered by HIV-1 infection. Each protein is scored for magnitude of translocation (M score, x-axis) and reproducibility of translocation direction (R score, y-axis) across the two replicates. MR plot analysis reveals significant translocations in the top right quadrant. Proteins with M>1 and R>0.9 are candidate hits for changing localization (estimated FDR = 8%).


Figure 5 ( A , B ) ( A ) Analysis of the three candidate markers in EV pellets isolated from CCM of control and infected ( red boxes ) Jurkat cells . The successive pellets ( 2K , 10K , 100K ) were analyzed by Western blot , using antibodies against SPN , MOV10 , SERINC3 and p24 ( HIV - 1 protein ) . The 2K , 10K , and 100K pellets obtained from 20 x 106 cells were loaded on the gel . Representative images ( A ) and quantifications in three to five independent experiments ( B ) are shown . For each pellet , arbitrary units ( AU ) represent the ratio of signal intensity for the analysed protein in the given pellet to the total amount of proteins in the same pellet . In cell lysates from the same experiments , AU ( given protein / total amount of protein ) for SPN and MOV10 were 1 . 29 ± 0 . 15 and 0 . 84 ± 0 . 33 times the levels in control cells , respectively ( Mean ± SD ) . ( C ) WB showing the distribution of the same proteins in six fractions recovered after velocity gradient separation of vesicles contained in the 100K pellets of control or HIV - 1 infected ( red boxes ) cells . Viral p24 protein was recovered in the bottom fractions of infected samples together with MOV10 and SPN . SERINC3 was recovered in the middle fractions . ( T = top ; B = bottom ) . ( D , E ) Quantification of MOV10 , SPN , SERINC3 and p24 signals in 4 to 7 independent Western blots from HIV - 1 infected samples . AU = band intensity in a given pellet / sum ( band intensity in all six pellets ) . ( F ) Quantification of SERINC3 signals in the different fractions from control and infected samples . AU = band intensity in a given pellet / sum ( band intensity in all 12 pellets ) . Lines represent the medians of three independent experiments . ( G , H ) EVs from supernatant of HIV - infected Jurkat cells were subjected to immuno - isolation with beads coupled to antibody against SPN . Bead - associated ( Pull - down : PD ) vesicles and those left behind ( Flow - Through : FT ) were loaded on a gel for Western blot analysis with antibodies specific for SPN , MOV10 , CD63 , SERINC3 and viral p24 . A representative image ( G ) and quantification ( H ) Mean ± SD of the proportion of signal in PD as compared with total ( PD + FT ) in three independent experiments are shown . ( I ) EVs remaining in the FT , or EVs secreted by the same number of cells but not subjected to immuno - isolation ( input ) were used to infect a reporter cell line . Mean ± SD of six independent experiments is shown .

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 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 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

Figure 5(A, B) (A) Analysis of the three candidate markers in EV pellets isolated from CCM of control and infected (red boxes) Jurkat cells. The successive pellets (2K, 10K, 100K) were analyzed by Western blot, using antibodies against SPN, MOV10, SERINC3 and p24 (HIV-1 protein). The 2K, 10K, and 100K pellets obtained from 20 x 106 cells were loaded on the gel. Representative images (A) and quantifications in three to five independent experiments (B) are shown. For each pellet, arbitrary units (AU) represent the ratio of signal intensity for the analysed protein in the given pellet to the total amount of proteins in the same pellet. In cell lysates from the same experiments, AU (given protein/total amount of protein) for SPN and MOV10 were 1.29±0.15 and 0.84±0.33 times the levels in control cells, respectively (Mean±SD).(C) WB showing the distribution of the same proteins in six fractions recovered after velocity gradient separation of vesicles contained in the 100K pellets of control or HIV-1 infected (red boxes) cells. Viral p24 protein was recovered in the bottom fractions of infected samples together with MOV10 and SPN. SERINC3 was recovered in the middle fractions. (T = top; B = bottom). (D, E) Quantification of MOV10, SPN, SERINC3 and p24 signals in 4 to 7 independent Western blots from HIV-1 infected samples. AU = band intensity in a given pellet / sum (band intensity in all six pellets). (F) Quantification of SERINC3 signals in the different fractions from control and infected samples. AU = band intensity in a given pellet / sum (band intensity in all 12 pellets). Lines represent the medians of three independent experiments. (G, H) EVs from supernatant of HIV-infected Jurkat cells were subjected to immuno-isolation with beads coupled to antibody against SPN. Bead-associated (Pull-down: PD) vesicles and those left behind (Flow-Through: FT) were loaded on a gel for Western blot analysis with antibodies specific for SPN, MOV10, CD63, SERINC3 and viral p24. A representative image (G) and quantification (H) Mean ± SD of the proportion of signal in PD as compared with total (PD+FT) in three independent experiments are shown.(I) EVs remaining in the FT, or EVs secreted by the same number of cells but not subjected to immuno-isolation (input) were used to infect a reporter cell line. Mean± SD of six independent experiments is shown.


Figure 6Jurkat cells were infected with lentivirus expressing control luciferase shRNA or shRNAs targeting SERINC3 ( A ) GFP + Ghost X4R5 cells obtained after treatment with supernatant from infected SERINC3 KD cells . Results are expressed as a ratio to the number of GFP + cells after treatment with supernatant from infected control cells . . Mean ± SD of three independent experiments is shown . Jurkat cells were infected with lentivirus expressing control luciferase shRNA or shRNAs targeting SERINC3 , followed or not by infection with NL4 - 3 EGFP - Nef + HIV - 1 ( red boxes ) . ( B ) Number of particles secreted were quantified by NTA . Results are expressed as a ratio to the number of particles secreted by uninfected luciferase shRNA cells . Jurkat cells were infected with lentivirus expressing control luciferase shRNA or shRNAs targeting SERINC3 , followed or not by infection with NL4 - 3 EGFP - Nef + HIV - 1 ( red boxes ) . ( C ) 100K pellets from infected cells were subjected to iodixanol velocity gradient separation . Six fractions were recovered and analyzed by Western blot for the presence of SPN , SERINC3 and p24 . ( T = top ; B = bottom ) . ( D ) Quantification of SPN and p24 signals in the different fractions of two independent iodixanol fractionations ( exp 1 : open symbols , exp 2 : closed symbols ) . AU = band intensity in a given pellet / sum ( band intensity in all 12 pellets ) . Jurkat cells were infected with lentivirus expressing control luciferase shRNA or shRNAs targeting SERINC3 , followed or not by infection with NL4 - 3 EGFP - Nef + HIV - 1 ( red boxes ) . 2K , 10K , 100K obtained from CCM of control cells and SERINC3 KD cells either uninfected of infected with NL4 - 3 EGFP - Nef + were analyzed by Western blot with antibodies against SPN , and p24 . The 2K , 10K , and 100K pellets obtained from 20 x 106 cells were loaded on the gel . Representative images ( E ) Jurkat cells were infected with lentivirus expressing control luciferase shRNA or shRNAs targeting SERINC3 , followed or not by infection with NL4 - 3 EGFP - Nef + HIV - 1 ( red boxes ) . 2K , 10K , 100K from CCM of control cells and SERINC3 KD cells either uninfected of infected with NL4 - 3 EGFP - Nef + were analyzed and quantification of SPN and p24 signals ( F ) are shown . For each pellet , arbitrary units ( AU ) represent the ratio of signal intensity in the given pellet to the total amount of protein in the same given pellet and relative to the total amount in 2K + 10K + 100K from control cells . Jurkat cells were infected with lentivirus expressing control luciferase shRNA or shRNAs targeting SERINC3 , followed or not by infection with NL4 - 3 EGFP - Nef + HIV - 1 ( red boxes ) . ( G ) Multiplex bead - based flow cytometry assay for detection of EV surface markers . Antibody - coated capture beads were incubated with EV samples . Captured EVs were detected with a mixture of APC - labeled anti - CD9 , anti - CD63 , and anti - CD81 . Quantification of the median APC fluorescence values for all bead populations after background and isotype antibody correction . Results were relativized to the values obtained with control sample for each experiment . Dotted line represents values obtained for EVs isolated from control cell CM .

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

Figure 6Jurkat cells were infected with lentivirus expressing control luciferase shRNA or shRNAs targeting SERINC3 (A) GFP+ Ghost X4R5 cells obtained after treatment with supernatant from infected SERINC3 KD cells. Results are expressed as a ratio to the number of GFP+ cells after treatment with supernatant from infected control cells. . Mean ± SD of three independent experiments is shown.Jurkat cells were infected with lentivirus expressing control luciferase shRNA or shRNAs targeting SERINC3, followed or not by infection with NL4-3 EGFP-Nef+ HIV-1 (red boxes). (B) Number of particles secreted were quantified by NTA. Results are expressed as a ratio to the number of particles secreted by uninfected luciferase shRNA cells.Jurkat cells were infected with lentivirus expressing control luciferase shRNA or shRNAs targeting SERINC3, followed or not by infection with NL4-3 EGFP-Nef+ HIV-1 (red boxes). (C) 100K pellets from infected cells were subjected to iodixanol velocity gradient separation. Six fractions were recovered and analyzed by Western blot for the presence of SPN, SERINC3 and p24. (T = top; B = bottom). (D) Quantification of SPN and p24 signals in the different fractions of two independent iodixanol fractionations (exp 1: open symbols, exp 2: closed symbols). AU = band intensity in a given pellet / sum (band intensity in all 12 pellets). Jurkat cells were infected with lentivirus expressing control luciferase shRNA or shRNAs targeting SERINC3, followed or not by infection with NL4-3 EGFP-Nef+ HIV-1 (red boxes). 2K, 10K, 100K obtained from CCM of control cells and SERINC3 KD cells either uninfected of infected with NL4-3 EGFP-Nef+ were analyzed by Western blot with antibodies against SPN, and p24. The 2K, 10K, and 100K pellets obtained from 20 x 106 cells were loaded on the gel. Representative images (E)Jurkat cells were infected with lentivirus expressing control luciferase shRNA or shRNAs targeting SERINC3, followed or not by infection with NL4-3 EGFP-Nef+ HIV-1 (red boxes). 2K, 10K, 100K from CCM of control cells and SERINC3 KD cells either uninfected of infected with NL4-3 EGFP-Nef+ were analyzed and quantification of SPN and p24 signals (F) are shown. For each pellet, arbitrary units (AU) represent the ratio of signal intensity in the given pellet to the total amount of protein in the same given pellet and relative to the total amount in 2K+10K+100K from control cells.Jurkat cells were infected with lentivirus expressing control luciferase shRNA or shRNAs targeting SERINC3, followed or not by infection with NL4-3 EGFP-Nef+ HIV-1 (red boxes). (G) Multiplex bead-based flow cytometry assay for detection of EV surface markers. Antibody-coated capture beads were incubated with EV samples. Captured EVs were detected with a mixture of APC-labeled anti-CD9, anti-CD63, and anti-CD81. Quantification of the median APC fluorescence values for all bead populations after background and isotype antibody correction. Results were relativized to the values obtained with control sample for each experiment. Dotted line represents values obtained for EVs isolated from control cell CM.


Figure 1Immunostaining of H3K27ac in mouse germinal vesicle ( GV ) oocytes ( n = 8 ) , metaphase II ( MII ) oocytes ( n = 7 ) , zygotes ( n = 7 ) , early 2 - cell ( 2C ) ( n = 7 ) and late 2C ( n = 9 ) embryos . Right panel is the quantification of H3K27ac immunostaining relative signal intensities at different stages . Scale bar : 20 μm . Data information : For boxplots in A the central band represents the median . The lower and upper edges of the box represent the first and third quartiles , respectively . The whiskers of the boxplot extend to 1 . 5 times inter - quartile range ( IQR ) .

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

Figure 1Immunostaining of H3K27ac in mouse germinal vesicle (GV) oocytes (n = 8), metaphase II (MII) oocytes (n = 7), zygotes (n = 7), early 2-cell (2C) (n = 7) and late 2C (n = 9) embryos. Right panel is the quantification of H3K27ac immunostaining relative signal intensities at different stages. Scale bar: 20 μm. Data information: For boxplots in A the central band represents the median. The lower and upper edges of the box represent the first and third quartiles, respectively. The whiskers of the boxplot extend to 1.5 times inter-quartile range (IQR).


Figure 4Immunostaining of H3K27ac in A - 485 treated embryos versus control ( DMSO treated ) at different time points . Scale bar : 20 μm . Developmental rate of embryos treated with A - 485 versus control .

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Figure 4Immunostaining of H3K27ac in A-485 treated embryos versus control (DMSO treated) at different time points. Scale bar: 20 μm.Developmental rate of embryos treated with A-485 versus control.


Figure 6Immunostaining of H3K27ac in TSA treated embryos under different concentrations versus control ( DMSO treated ) at 28 hpi . Scale bar : 20 μm . Developmental rate of embryos treated with TSA at different time points ( 0 - 20 hpi and 8 - 28 hpi ) versus control .

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Figure 6Immunostaining of H3K27ac in TSA treated embryos under different concentrations versus control (DMSO treated) at 28 hpi. Scale bar: 20 μm.Developmental rate of embryos treated with TSA at different time points (0-20 hpi and 8-28 hpi) versus control.


Figure 1 ( A ) HT29 cells were infected with Shigella WT , S325 , or ospD deletion mutants , and incubated for 8 h . Aliquots of cellular supernatants were subjected to cytotoxicity assays . * P < 0 . 05 ( one - way ANOVA ) . ( B ) HT29 cells were infected with Shigella WT or ∆ ospD3 and incubated for 8 h . Infected cells were fixed and subjected to TUNEL and PI staining . Percentages of positive cells ( TUNEL , green ; PI , red ) are shown in graph at right . The nuclei were stained with DAPI ( blue ) . Scale bar : 100 μm . n . s . , not significant ; * P < 0 . 05 ( unpaired two - tailed Student ' s t - test ) . ( C ) HT29 cells were infected with Shigella WT , S325 , or ∆ ospD3 and incubated for 8 h . Infected cells were subjected to Giemsa staining . Arrows indicate cells in which the cytoplasm disappeared . Scale bar : 20 μm .

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 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

Figure 1(A) HT29 cells were infected with Shigella WT, S325, or ospD deletion mutants, and incubated for 8 h. Aliquots of cellular supernatants were subjected to cytotoxicity assays. *P<0.05 (one-way ANOVA).(B) HT29 cells were infected with Shigella WT or ∆ospD3 and incubated for 8 h. Infected cells were fixed and subjected to TUNEL and PI staining. Percentages of positive cells (TUNEL, green; PI, red) are shown in graph at right. The nuclei were stained with DAPI (blue). Scale bar: 100 μm. n.s., not significant; *P<0.05 (unpaired two-tailed Student's t-test).(C) HT29 cells were infected with Shigella WT, S325, or ∆ospD3 and incubated for 8 h. Infected cells were subjected to Giemsa staining. Arrows indicate cells in which the cytoplasm disappeared. Scale bar: 20 μm.


Figure 2 ( A ) HT29 cells were infected with Shigella WT , S325 , or ospD deletion mutants , and incubated for 8 h . Cell lysates were subjected to immunoblotting . ( B HT29 cells were infected with the indicated Shigella strains in the presence or absence of RIPK1 inhibitor , RIPK3 inhibitor , or caspase inhibitor ( z - VAD ) , and then incubated for 8 h . Cell lysates and aliquots of cellular supernatants were subjected to immunoblottingC ) HT29 cells were infected with the indicated Shigella strains in the presence or absence of RIPK1 inhibitor , RIPK3 inhibitor , or caspase inhibitor ( z - VAD ) , and then incubated for 8 h . Cell lysates and aliquots of cellular supernatants were subjected to cytotoxicity assay ( D HT29 cells treated with the indicated siRNAs were infected with Shigella WT or ∆ ospD3 . Cell lysates and aliquots of cellular supernatants were subjected to immunoblotting ( top ) and cytotoxicity assays ( bottom ) , respectively . The knockdown efficiency of the indicated siRNAs was assessed by immunoblotting ( inset ) . n . s . , not significant ; * P < 0 . 05 ( unpaired two - tailed Student ' s t - test ) . E ) HT29 cells treated with the indicated siRNAs were infected with Shigella WT or ∆ ospD3 . Cell lysates and aliquots of cellular supernatants were subjected to immunoblotting ( top ) and cytotoxicity assays ( bottom ) , respectively . The knockdown efficiency of the indicated siRNAs was assessed by immunoblotting ( inset ) . n . s . , not significant ; * P < 0 . 05 ( unpaired two - tailed Student ' s t - test ) .

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

Figure 2(A) HT29 cells were infected with Shigella WT, S325, or ospD deletion mutants, and incubated for 8 h. Cell lysates were subjected to immunoblotting.(B HT29 cells were infected with the indicated Shigella strains in the presence or absence of RIPK1 inhibitor, RIPK3 inhibitor, or caspase inhibitor (z-VAD), and then incubated for 8 h. Cell lysates and aliquots of cellular supernatants were subjected to immunoblottingC) HT29 cells were infected with the indicated Shigella strains in the presence or absence of RIPK1 inhibitor, RIPK3 inhibitor, or caspase inhibitor (z-VAD), and then incubated for 8 h. Cell lysates and aliquots of cellular supernatants were subjected to cytotoxicity assay(D HT29 cells treated with the indicated siRNAs were infected with Shigella WT or ∆ospD3. Cell lysates and aliquots of cellular supernatants were subjected to immunoblotting (top) and cytotoxicity assays (bottom), respectively. The knockdown efficiency of the indicated siRNAs was assessed by immunoblotting (inset). n.s., not significant; *P<0.05 (unpaired two-tailed Student's t-test).E) HT29 cells treated with the indicated siRNAs were infected with Shigella WT or ∆ospD3. Cell lysates and aliquots of cellular supernatants were subjected to immunoblotting (top) and cytotoxicity assays (bottom), respectively. The knockdown efficiency of the indicated siRNAs was assessed by immunoblotting (inset). n.s., not significant; *P<0.05 (unpaired two-tailed Student's t-test).


Figure 3 ( A ) HT29 cells were infected with the indicated Shigella strains and incubated for 8 h , and then cell lysates were subjected to immunoblotting . ( B ) 293T cells were transfected with Myc - tagged ospD expression plasmids . After 24 h , cells were harvested and subjected to immunoblotting . Arrows indicate cleaved RIPK1 . D ) 293T cells were transfected with a series of plasmids expressing ospD3 point mutants . After 24 h , cells were harvested and subjected to immunoblotting . Arrows indicate cleaved RIPK1 . ( E HT29 cells were infected with Shigella WT , S325 , ∆ ospD3 , ∆ ospD3 / D3 ( ∆ ospD3 complemented with wild - type ospD3 ) , or ∆ ospD3 / D3CS ( ∆ ospD3 complemented with a protease activity - deficient mutant , in which the cysteine residue at position 64 was replaced by serine ) strains and incubated for 8 h . Cell lysates and aliquots of cellular supernatants were subjected to immunoblottingF ) HT29 cells were infected with Shigella WT , S325 , ∆ ospD3 , ∆ ospD3 / D3 ( ∆ ospD3 complemented with wild - type ospD3 ) , or ∆ ospD3 / D3CS ( ∆ ospD3 complemented with a protease activity - deficient mutant , in which the cysteine residue at position 64 was replaced by serine ) strains and incubated for 8 h . Cell lysates and aliquots of cellular supernatants were subjected to cytotoxicity

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

Figure 3(A) HT29 cells were infected with the indicated Shigella strains and incubated for 8 h, and then cell lysates were subjected to immunoblotting.(B) 293T cells were transfected with Myc-tagged ospD expression plasmids. After 24 h, cells were harvested and subjected to immunoblotting. Arrows indicate cleaved RIPK1.D) 293T cells were transfected with a series of plasmids expressing ospD3 point mutants. After 24 h, cells were harvested and subjected to immunoblotting. Arrows indicate cleaved RIPK1.(E HT29 cells were infected with Shigella WT, S325, ∆ospD3, ∆ospD3/D3 (∆ospD3 complemented with wild-type ospD3), or ∆ospD3/D3CS (∆ospD3 complemented with a protease activity-deficient mutant, in which the cysteine residue at position 64 was replaced by serine) strains and incubated for 8 h. Cell lysates and aliquots of cellular supernatants were subjected to immunoblottingF) HT29 cells were infected with Shigella WT, S325, ∆ospD3, ∆ospD3/D3 (∆ospD3 complemented with wild-type ospD3), or ∆ospD3/D3CS (∆ospD3 complemented with a protease activity-deficient mutant, in which the cysteine residue at position 64 was replaced by serine) strains and incubated for 8 h. Cell lysates and aliquots of cellular supernatants were subjected to cytotoxicity assays


Figure 4 ( A ) HT29 cells were infected with the indicated Shigella strains and incubated for 8 h . Cells were harvested and subjected to measurement of caspase - 8 activation . Caspase - 8 activity is reported as relative light units ( RLU ) of infected samples , minus the value in uninfected samples . * P < 0 . 05 ( one - way ANOVA ) . ( B ) HT29 cells were infected with the indicated Shigella strains and incubated for 8 h . Cell lysates were subjected to immunoblotting . Arrows indicate cleaved forms of caspase - 8 , PARP , GSDMD , and IL - 18 , respectively . ( C ) HT29 cells were infected with the indicated Shigella strains and incubated for 8 h . Aliquots of cellular supernatants were subjected to cytotoxicity assay . * P < 0 . 05 ; n . s . , not significant ( one - way ANOVA ) . ( D ) HT29 cells were infected with Shigella WT or ∆ ospC1 mutant and incubated for 8 h . Infected cells were then fixed and stained with cleaved caspase - 8 ( green ) , rhodamine - phalloidin ( red ) , and DAPI ( blue ) . Percentages of positive cells are shown in the graph at right ( * P < 0 . 05 ; unpaired two - tailed Student ' s t - test ) . Scale bar : 100 μm . ( E ) HT29 cells were infected with the indicated Shigella strains and incubated for 8 h . Cells were harvested and subjected to measurement of caspase - 3 / 7 activation . Caspase - 3 / 7 activity is reported as relative light units ( RLU ) of infected samples minus uninfected samples . * P < 0 . 05 ( one - way ANOVA ) . ( F ) HT29 cells were infected with Shigella WT , S325 , or ∆ ospC1 strains , and incubated for up to 8 h . After incubation for the indicated times , cells were subjected to real - time Annexin V apoptosis assay . Annexin V binding is reported as relative light units ( RLU ) in infected samples minus the value in uninfected samples . * P < 0 . 05 ( one - way ANOVA ) .

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 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 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 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 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

Figure 4(A) HT29 cells were infected with the indicated Shigella strains and incubated for 8 h. Cells were harvested and subjected to measurement of caspase-8 activation. Caspase-8 activity is reported as relative light units (RLU) of infected samples, minus the value in uninfected samples. *P<0.05 (one-way ANOVA).(B) HT29 cells were infected with the indicated Shigella strains and incubated for 8 h. Cell lysates were subjected to immunoblotting. Arrows indicate cleaved forms of caspase-8, PARP, GSDMD, and IL-18, respectively.(C) HT29 cells were infected with the indicated Shigella strains and incubated for 8 h. Aliquots of cellular supernatants were subjected to cytotoxicity assay. *P<0.05; n.s., not significant (one-way ANOVA).(D) HT29 cells were infected with Shigella WT or ∆ospC1 mutant and incubated for 8 h. Infected cells were then fixed and stained with cleaved caspase-8 (green), rhodamine-phalloidin (red), and DAPI (blue). Percentages of positive cells are shown in the graph at right (*P<0.05; unpaired two-tailed Student's t-test). Scale bar: 100 μm.(E) HT29 cells were infected with the indicated Shigella strains and incubated for 8 h. Cells were harvested and subjected to measurement of caspase-3/7 activation. Caspase-3/7 activity is reported as relative light units (RLU) of infected samples minus uninfected samples. *P<0.05 (one-way ANOVA).(F) HT29 cells were infected with Shigella WT, S325, or ∆ospC1 strains, and incubated for up to 8 h. After incubation for the indicated times, cells were subjected to real-time Annexin V apoptosis assay. Annexin V binding is reported as relative light units (RLU) in infected samples minus the value in uninfected samples. *P<0.05 (one-way ANOVA).


Figure 5 ( A HT29 cells were infected with Shigella WT , ∆ ospC1 , ∆ ospD3 , ∆ ospC1 ∆ ospD3 , or ∆ ospC1 ∆ ospD3 / ospC1 ( ∆ ospC1 ∆ ospD3 complemented with ospC1 ) strains and incubated for 8 h . Cell lysates and aliquots of cellular supernatants were subjected to immunoblottingB ) HT29 cells were infected with Shigella WT , ∆ ospC1 , ∆ ospD3 , ∆ ospC1 ∆ ospD3 , or ∆ ospC1 ∆ ospD3 / ospC1 ( ∆ ospC1 ∆ ospD3 complemented with ospC1 ) strains and incubated for 8 h . Cell lysates and aliquots of cellular supernatants were subjected to cytotoxicity assay ( C ) HT29 cells were infected with Shigella WT , ∆ ospD3 , or ∆ ospC1 . Cell lysates obtained at the indicated time points were subjected to immunoblotting . ( D HT29 cells treated with DMSO or caspase - 8 inhibitor were infected with Shigella WT , ∆ ospC1 , ∆ ospD3 , or ∆ ospC1 ∆ ospD3 strains and incubated for 8 h . Cell lysates and aliquots of cellular supernatants were subjected to immunoblottingE ) HT29 cells treated with DMSO or caspase - 8 inhibitor were infected with Shigella WT , ∆ ospC1 , ∆ ospD3 , or ∆ ospC1 ∆ ospD3 strains and incubated for 8 h . Cell lysates and aliquots of cellular supernatants were subjected to cytotoxicity assay ( F ) HT29 cells treated with the control or caspase - 8 siRNAs were infected with the indicated Shigella strains and incubated for 8 h . Cell lysates were subjected to immunoblotting . The knockdown efficiency of the indicated siRNAs was assessed by immunoblotting .

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 O O O O O O O O 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 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 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

Figure 5(A HT29 cells were infected with Shigella WT, ∆ospC1, ∆ospD3, ∆ospC1∆ospD3, or ∆ospC1∆ospD3/ospC1 (∆ospC1∆ospD3 complemented with ospC1) strains and incubated for 8 h. Cell lysates and aliquots of cellular supernatants were subjected to immunoblottingB) HT29 cells were infected with Shigella WT, ∆ospC1, ∆ospD3, ∆ospC1∆ospD3, or ∆ospC1∆ospD3/ospC1 (∆ospC1∆ospD3 complemented with ospC1) strains and incubated for 8 h. Cell lysates and aliquots of cellular supernatants were subjected to cytotoxicity assay(C) HT29 cells were infected with Shigella WT, ∆ospD3, or ∆ospC1. Cell lysates obtained at the indicated time points were subjected to immunoblotting.(D HT29 cells treated with DMSO or caspase-8 inhibitor were infected with Shigella WT, ∆ospC1, ∆ospD3, or ∆ospC1∆ospD3 strains and incubated for 8 h. Cell lysates and aliquots of cellular supernatants were subjected to immunoblottingE) HT29 cells treated with DMSO or caspase-8 inhibitor were infected with Shigella WT, ∆ospC1, ∆ospD3, or ∆ospC1∆ospD3 strains and incubated for 8 h. Cell lysates and aliquots of cellular supernatants were subjected to cytotoxicity assay(F) HT29 cells treated with the control or caspase-8 siRNAs were infected with the indicated Shigella strains and incubated for 8 h. Cell lysates were subjected to immunoblotting. The knockdown efficiency of the indicated siRNAs was assessed by immunoblotting.


Figure 1A Peroxisomes were induced by growing the WT strain expressing Pot1 ‐ GFP in oleate medium to mid ‐ log ‐ phase , then transferred to SD ‐ N starvation medium with or without GABA to trigger pexophagy for 6 h . GFP cleavage was analyzed at the indicated time points by immunoblotting . B Mitochondria were induced by growing the WT strain expressing OM45 ‐ GFP in YPL medium to mid ‐ log ‐ phase and subsequently transferring cells to either SD ‐ N with or without GABA to trigger mitophagy for 12 h . GFP cleavage was analyzed at the indicated time points by immunoblotting . C Mitophagy was monitored by fluorescence microscopy using a WT strain expressing OM45 ‐ GFP grown in YPL medium for 12 h to mid ‐ log ‐ phase in the presence of FM4 ‐ 64 , and transferred to either SD ‐ N medium with or without GABA for 24 h . Bar , 5 μm . D The Cvt pathway was monitored using the WT strain in SD medium with or without GABA , grown to mid ‐ log ‐ phase , after which samples were analyzed for Ape1 maturation . E Ribophagy was monitored by growing the WT strain expressing Rpl25 ‐ GFP in SD medium to mid ‐ log ‐ phase and transferring cells to SD ‐ N either with or without GABA for 24 h . F Autophagy was monitored by growing the WT strain expressing GFP ‐ Atg8 in SD medium to mid ‐ log ‐ phase and transferring cells to SD ‐ N either with or without GABA for 6 h .

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Figure 1A Peroxisomes were induced by growing the WT strain expressing Pot1‐GFP in oleate medium to mid‐log‐phase, then transferred to SD‐N starvation medium with or without GABA to trigger pexophagy for 6 h. GFP cleavage was analyzed at the indicated time points by immunoblotting.B Mitochondria were induced by growing the WT strain expressing OM45‐GFP in YPL medium to mid‐log‐phase and subsequently transferring cells to either SD‐N with or without GABA to trigger mitophagy for 12 h. GFP cleavage was analyzed at the indicated time points by immunoblotting.C Mitophagy was monitored by fluorescence microscopy using a WT strain expressing OM45‐GFP grown in YPL medium for 12 h to mid‐log‐phase in the presence of FM4‐64, and transferred to either SD‐N medium with or without GABA for 24 h. Bar, 5 μm.D The Cvt pathway was monitored using the WT strain in SD medium with or without GABA, grown to mid‐log‐phase, after which samples were analyzed for Ape1 maturation.E Ribophagy was monitored by growing the WT strain expressing Rpl25‐GFP in SD medium to mid‐log‐phase and transferring cells to SD‐N either with or without GABA for 24 h.F Autophagy was monitored by growing the WT strain expressing GFP‐Atg8 in SD medium to mid‐log‐phase and transferring cells to SD‐N either with or without GABA for 6 h.


Figure 2APeroxisomes were induced in oleate medium and pexophagy was monitored as described for Fig 1A . B Pexophagy was monitored by fluorescence microscopy using a WT strain expressing Pot1 ‐ GFP grown in oleate medium to mid ‐ log ‐ phase in the presence of FM4 ‐ 64 , and transferred to either SD ‐ N medium with or without GABA or to SD ‐ N with GABA and rapamycin for 6 h . Bar , 5 μm . C Mitochondria were induced in YPL medium and mitophagy was assessed as described for Fig .

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Figure 2APeroxisomes were induced in oleate medium and pexophagy was monitored as described for Fig 1A.B Pexophagy was monitored by fluorescence microscopy using a WT strain expressing Pot1‐GFP grown in oleate medium to mid‐log‐phase in the presence of FM4‐64, and transferred to either SD‐N medium with or without GABA or to SD‐N with GABA and rapamycin for 6 h. Bar, 5 μm.C Mitochondria were induced in YPL medium and mitophagy was assessed as described for Fig .


Figure 3A WT cells expressing OM45 ‐ GFP , along with the uga2 ∆ strain over ‐ expressing the GAD1 gene and expressing OM45 ‐ GFP were grown in YPL medium to mid ‐ log ‐ phase . To monitor mitophagy , strains were transferred to SD ‐ N starvation medium ( with or without rapamycin ) . B WT strain along with the uga2 ∆ strain over ‐ expressing the GAD1 gene was grown in oleate medium and pexophagy was monitored as described in Fig , with or without rapamycin . Samples were taken at the indicated time points , and Pot1 degradation was analyzed by immunoblotting ( 45 kD ) . C To monitor autophagy , WT cells expressing GFP ‐ Atg8 along with the uga2 ∆ strain over ‐ expressing the GAD1 gene and expressing GFP ‐ Atg8 were grown in SD medium and transferred to SD ‐ N .

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Figure 3A WT cells expressing OM45‐GFP, along with the uga2∆ strain over‐expressing the GAD1 gene and expressing OM45‐GFP were grown in YPL medium to mid‐log‐phase. To monitor mitophagy, strains were transferred to SD‐N starvation medium (with or without rapamycin).B WT strain along with the uga2∆ strain over‐expressing the GAD1 gene was grown in oleate medium and pexophagy was monitored as described in Fig , with or without rapamycin. Samples were taken at the indicated time points, and Pot1 degradation was analyzed by immunoblotting (45 kD).C To monitor autophagy, WT cells expressing GFP‐Atg8 along with the uga2∆ strain over‐expressing the GAD1 gene and expressing GFP‐Atg8 were grown in SD medium and transferred to SD‐N.


Figure 4A , B WT cells were cultured under pexophagy ( A ) or mitophagy ( B ) conditions with or without GABA and rapamycin . S6 phosphorylation at the indicated time points was analyzed by immunoblotting with a loading control . C Samples were analyzed for Pot1 degradation by immunoblotting ( 45 kD ) . D GFP production during mitophagy was analyzed by immunoblotting .

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Figure 4A,B WT cells were cultured under pexophagy (A) or mitophagy (B) conditions with or without GABA and rapamycin. S6 phosphorylation at the indicated time points was analyzed by immunoblotting with a loading control.C Samples were analyzed for Pot1 degradation by immunoblotting (45 kD).D GFP production during mitophagy was analyzed by immunoblotting.


Figure 5A S6 phosphorylation after 6 h in SD ‐ N was analyzed by immunoblotting with a loading control . B GFP production monitoring autophagy at the indicated time points was analyzed by immunoblotting .

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Figure 5A S6 phosphorylation after 6 h in SD‐N was analyzed by immunoblotting with a loading control.B GFP production monitoring autophagy at the indicated time points was analyzed by immunoblotting.


Figure 6A , B WT , WT with GABA , WT with GABA and 10 mM GSH and WT with GABA and 200 nM rapamycin were tested for intracellular ROS levels under ( A ) pexophagy and ( B ) mitophagy conditions . After 24 h incubation , cells were stained with DHR − 123 and propidium iodide for 1 h . Living cells were analyzed for DHR − 123 fluorescence by flow cytometry . Data represent mean + s . d . ( n = 3 ) . * P < 0 . 005 , * * P < 0 . 01C , D Yeast cells stained with 5 μM propidium iodide were used to differentiate between living and dead cells under ( C ) pexophagy or ( D ) mitophagy conditions . Significant differences between the treatments and strains were determined using an unpaired two ‐ tailed t ‐ test . * * P < 0 . 01 . E Pexophagy assay was monitored by the degradation of Pot1 ‐ GFP and analyzed for GFP cleavage by immunoblotting . F Mitophagy assay was monitored by the degradation of Om45 ‐ GFP and analyzed for GFP cleavage by immunoblotting .

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

Figure 6A,B WT, WT with GABA, WT with GABA and 10 mM GSH and WT with GABA and 200 nM rapamycin were tested for intracellular ROS levels under (A) pexophagy and (B) mitophagy conditions. After 24 h incubation, cells were stained with DHR−123 and propidium iodide for 1 h. Living cells were analyzed for DHR−123 fluorescence by flow cytometry. Data represent mean + s.d. (n = 3). *P < 0.005, **P < 0.01C,D Yeast cells stained with 5 μM propidium iodide were used to differentiate between living and dead cells under (C) pexophagy or (D) mitophagy conditions. Significant differences between the treatments and strains were determined using an unpaired two‐tailed t‐test. **P < 0.01.E Pexophagy assay was monitored by the degradation of Pot1‐GFP and analyzed for GFP cleavage by immunoblotting.F Mitophagy assay was monitored by the degradation of Om45‐GFP and analyzed for GFP cleavage by immunoblotting.


Figure 7A , B Example images of Parkin ‐ expressing HeLa cells analyzed using a tandem fluorochrome protein ( mito ‐ RFP ‐ GFP ) mitophagy assay under ( A ) control conditions or ( B ) displaying mitophagy depicted by the red mitochondrial structures localized to lysosomes . Bar , 10 μm . C Percentage of cells displaying mitophagy + s . d . , * * P < 0 . 01 using an unpaired two ‐ tailed t ‐ test , n > 80 .

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Figure 7A,B Example images of Parkin‐expressing HeLa cells analyzed using a tandem fluorochrome protein (mito‐RFP‐GFP) mitophagy assay under (A) control conditions or (B) displaying mitophagy depicted by the red mitochondrial structures localized to lysosomes. Bar, 10 μm.C Percentage of cells displaying mitophagy + s.d., **P < 0.01 using an unpaired two‐tailed t‐test, n > 80.


Figure 8A Electron microscopy images of mitochondria from WT ( n = 44 ) and SSADH ‐ deficient mice ( Aldh5a1 − / − ) ( n = 80 ) were calculated for area size . B Electron microscopy images showing typical sizes of WT and Aldh5a1 − / − mice liver mitochondria . Bar , 0 . 5 μm . C Quantification of mitochondrial numbers from electron microscopy images of liver from WT ( n = 31 ) and Aldh5a1 − / − mice treated with vehicle ( n = 39 ) or rapamycin ( n = 34 ) ( 5 mg / kg body weight per day ) via intraperitoneal injections for 3 successive days starting at day 7 of life . D Quantification of mitochondrial numbers from electron microscopy images of brain from WT ( n = 23 ) and Aldh5a1 − / − mice treated with vehicle ( n = 30 ) or rapamycin ( n = 41 ) ( 5 mg / kg body weight per day ) via intraperitoneal injections for 3 successive days starting at day 7 of life . E Aldh5a1 − / − mice were treated with vehicle or rapamycin ( 10 mg / kg body weight per day ) via intraperitoneal injections for 10 successive days starting at day 10 of life . WT mice served as non ‐ disease controls ( set to 1 ) . After sacrifice , liver homogenates were used to measure SOD enzyme activity using a colorimetric SOD activity assay . F Mitochondrial SOD2 protein levels were quantified from liver microsections using immunofluorescence microscopy and automated image analysis ( WT set to 1 ) . G Immunofluorescence images showing typical nuclear staining ( DAPI , blue ) and SOD2 staining ( red ) from WT , Aldh5a1 − / − mice treated with vehicle and Aldh5a1 − / − mice treated with rapamycin . Bar , 10 μm .

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Figure 8A Electron microscopy images of mitochondria from WT (n = 44) and SSADH‐deficient mice (Aldh5a1−/−) (n = 80) were calculated for area size.B Electron microscopy images showing typical sizes of WT and Aldh5a1−/− mice liver mitochondria. Bar, 0.5 μm.C Quantification of mitochondrial numbers from electron microscopy images of liver from WT (n = 31) and Aldh5a1−/− mice treated with vehicle (n = 39) or rapamycin (n = 34) (5 mg/kg body weight per day) via intraperitoneal injections for 3 successive days starting at day 7 of life.D Quantification of mitochondrial numbers from electron microscopy images of brain from WT (n = 23) and Aldh5a1−/− mice treated with vehicle (n = 30) or rapamycin (n = 41) (5 mg/kg body weight per day) via intraperitoneal injections for 3 successive days starting at day 7 of life.E Aldh5a1−/− mice were treated with vehicle or rapamycin (10 mg/kg body weight per day) via intraperitoneal injections for 10 successive days starting at day 10 of life. WT mice served as non‐disease controls (set to 1). After sacrifice, liver homogenates were used to measure SOD enzyme activity using a colorimetric SOD activity assay.F Mitochondrial SOD2 protein levels were quantified from liver microsections using immunofluorescence microscopy and automated image analysis (WT set to 1).G Immunofluorescence images showing typical nuclear staining (DAPI, blue) and SOD2 staining (red) from WT, Aldh5a1−/− mice treated with vehicle and Aldh5a1−/− mice treated with rapamycin. Bar, 10 μm.


Figure 9A Quantification of S6 phosphorylation of liver lysates from WT ( n = 5 ) and Aldh5a1 − / − mice treated with vehicle ( n = 4 ) or rapamycin ( n = 5 ) after normalization ( WT set to 1 ) . B S6 phosphorylation of liver lysates analyzed by immunoblotting . C Quantification of S6 phosphorylation of brain lysates from WT ( n = 2 ) and Aldh5a1 − / − mice treated with vehicle ( n = 3 ) or rapamycin ( n = 3 ) after normalization ( WT set to 1 ) . D S6 phosphorylation of brain lysates analyzed by immunoblotting .

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Figure 9A Quantification of S6 phosphorylation of liver lysates from WT (n = 5) and Aldh5a1−/− mice treated with vehicle (n = 4) or rapamycin (n = 5) after normalization (WT set to 1).B S6 phosphorylation of liver lysates analyzed by immunoblotting.C Quantification of S6 phosphorylation of brain lysates from WT (n = 2) and Aldh5a1−/− mice treated with vehicle (n = 3) or rapamycin (n = 3) after normalization (WT set to 1).D S6 phosphorylation of brain lysates analyzed by immunoblotting.


Figure 1 ( A ) Serum levels of miR - 483 - 3p / - 5p in IPAH patients ( n = 139 ) and HC ( n = 95 ) measured by qPCR . The data are fold change normalized to the averaged level of HC . Data information : Values are expressed as median ± interquartile range . Statistical test : Mann Whitney U test ( A ) ( B ) ROC curve with sensitivity and specificity of serum levels of miR - 483 - 3p / - 5p for differentiating IPAH patients from HCs at diagnosis ( IPAH , n = 139 ; HC , n = 95 ) . Data information : Values are expressed as median ± interquartile range . ( C ) Levels of miR - 483 - 3p / - 5p associated with PAH risk in 3 groups . IPAH patients were divided into a low risk group ( Low ) and an intermediate plus high risk group ( Inter & high ) according to the World Symposium on Pulmonary Hypertension 2018 [ 14 ] . Data information : Values are expressed as median ± interquartile range . Statistical test : Kruskal - Wallis U test ( C ) . ( D ) Levels of miR - 483 - 3p were inversely correlated with serum levels of ET - 1 ( IPAH , n = 118 ; HC , n = 95 ) and IL - 6 ( IPAH , n = 112 ; HC , n = 93 ) . Data information : Values are expressed as median ± interquartile range .

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

Figure 1(A) Serum levels of miR-483-3p/-5p in IPAH patients (n = 139) and HC (n = 95) measured by qPCR. The data are fold change normalized to the averaged level of HC. Data information: Values are expressed as median ± interquartile range. Statistical test: Mann Whitney U test (A)(B) ROC curve with sensitivity and specificity of serum levels of miR-483-3p/-5p for differentiating IPAH patients from HCs at diagnosis (IPAH, n = 139; HC, n = 95). Data information: Values are expressed as median ± interquartile range.(C) Levels of miR-483-3p/-5p associated with PAH risk in 3 groups. IPAH patients were divided into a low risk group (Low) and an intermediate plus high risk group (Inter&high) according to the World Symposium on Pulmonary Hypertension 2018 [14]. Data information: Values are expressed as median ± interquartile range. Statistical test: Kruskal-Wallis U test (C).(D) Levels of miR-483-3p were inversely correlated with serum levels of ET-1 (IPAH, n = 118; HC, n = 95) and IL-6 (IPAH, n = 112; HC, n = 93). Data information: Values are expressed as median ± interquartile range.


Figure 2 ( A ) CD144 - enriched EVs were isolated from serum of IPAH patients ( n = 37 ) and HCs ( n = 34 ) . The levels of miR - 483 - 3p / - 5p were measured by qPCR . Data information : Values are expressed as median ± interquartile range . Statistical test : Mann Whitney U test . PAECs were transfected with miR - 483 - 3p / - 5p mimic or scramble RNA for 36 hr before RNA isolation and then analyzed by RNA - seq . Data are results from two biological repeats . ( C ) Heat map comparison of log2 fold changes of the indicated genes . Data information : Values are expressed as median ± interquartile range . Statistical test : Mann Whitney U test .

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 O O O O O O O O O O O

Figure 2(A) CD144-enriched EVs were isolated from serum of IPAH patients (n = 37) and HCs (n = 34). The levels of miR-483-3p/-5p were measured by qPCR. Data information: Values are expressed as median ± interquartile range. Statistical test: Mann Whitney U test.PAECs were transfected with miR-483-3p/-5p mimic or scramble RNA for 36 hr before RNA isolation and then analyzed by RNA-seq. Data are results from two biological repeats. (C) Heat map comparison of log2 fold changes of the indicated genes. Data information: Values are expressed as median ± interquartile range. Statistical test: Mann Whitney U test.


Figure 3PAECs were infected with Lenti - pre - miR - 483 or Lenti - null for 24 hr . Expression levels of miR - 483 - 3p / - 5p measured by qPCR Data information : Values are expressed as mean ± SEM from 3 independent experiments . Statistical test : t - test ( * P < 0 . 05 between the indicated groupsPAECs were infected with Lenti - pre - miR - 483 or Lenti - null for 24 hr . Expression levels of TGF - β , TGFBR2 , β - catenin , CTGF , IL - 1β , and ET - 1 mRNA were measured by qPCR Data information : Values are expressed as mean ± SEM from 3 independent experiments . Statistical test : t - test ( * P < 0 . 05 between the indicated groupsPAECs were infected with Lenti - pre - miR - 483 or Lenti - null for 24 hr . Expression levels of TGF - β , TGFBR2 , β - catenin , CTGF , IL - 1β , and ET - 1 protein were measured by Western blot Data information : Values are expressed as mean ± SEM from 3 independent experiments . Statistical test : t - test ( * P < 0 . 05 between the indicated groups ( E ) Bovine aortic ECs were transfected with a luciferase reporter fused with the 3 ' UTR of TGF - β ( Luc - TGF - β WT ) , TGFBR2 ( Luc - TGFBR2 WT ) , IL - 1β ( Luc - IL - 1β WT ) or ET - 1 ( Luc - ET - 1 WT ) or a binding site mutation ( Luc - TGF - β Mut , Luc - TGFBR2 Mut , Luc - IL - 1β Mut , ET - 1 - Mut ) , then infected with Lenti - pre - miR - 483 for additional 36 hr . Luciferase activity was measured . Data information : Values are expressed as mean ± SEM from 3 independent experiments . Statistical test : t - test ( * P < 0 . 05 between the indicated groupsPAECs were infected with Lenti - pre - miR - 483 or Lenti - null for 36 hr . The Ago1 - or Ago2 - associated miRNAs and mRNAs were enriched by immunoprecipitation with anti - Ago1 or anti - Ago2 . Levels of miR - 483 - 3p / - 5p mRNA were detected by qPCR and normalized to those of Ago1 or Ago2 protein . Data information : Values are expressed as mean ± SEM from 3 independent experiments . Statistical test : t - test ( * P < 0 . 05 between the indicated groupsPAECs were infected with Lenti - pre - miR - 483 or Lenti - null for 36 hr . The Ago1 - or Ago2 - associated miRNAs and mRNAs were enriched by immunoprecipitation with anti - Ago1 or anti - Ago2 . Levels of TGF - β , TGFBR2 , β - catenin , CTGF , IL - 1β , and ET - 1 mRNA were detected by qPCR and normalized to those of Ago1 or Ago2 protein . Data information : Values are expressed as mean ± SEM from 3 independent experiments . Statistical test : t - test ( * P < 0 . 05 between the indicated

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

Figure 3PAECs were infected with Lenti-pre-miR-483 or Lenti-null for 24 hr. Expression levels of miR-483-3p/-5p measured by qPCR Data information: Values are expressed as mean ± SEM from 3 independent experiments. Statistical test: t-test (*P < 0.05 between the indicated groupsPAECs were infected with Lenti-pre-miR-483 or Lenti-null for 24 hr. Expression levels of TGF-β, TGFBR2, β-catenin, CTGF, IL-1β, and ET-1 mRNA were measured by qPCR Data information: Values are expressed as mean ± SEM from 3 independent experiments. Statistical test: t-test (*P < 0.05 between the indicated groupsPAECs were infected with Lenti-pre-miR-483 or Lenti-null for 24 hr. Expression levels of TGF-β, TGFBR2, β-catenin, CTGF, IL-1β, and ET-1 protein were measured by Western blot Data information: Values are expressed as mean ± SEM from 3 independent experiments. Statistical test: t-test (*P < 0.05 between the indicated groups(E) Bovine aortic ECs were transfected with a luciferase reporter fused with the 3'UTR of TGF-β (Luc-TGF-β WT), TGFBR2 (Luc-TGFBR2 WT), IL-1β (Luc-IL-1β WT) or ET-1 (Luc-ET-1 WT) or a binding site mutation (Luc-TGF-β Mut, Luc-TGFBR2 Mut, Luc-IL-1β Mut, ET-1-Mut), then infected with Lenti-pre-miR-483 for additional 36 hr. Luciferase activity was measured. Data information: Values are expressed as mean ± SEM from 3 independent experiments. Statistical test: t-test (*P < 0.05 between the indicated groupsPAECs were infected with Lenti-pre-miR-483 or Lenti-null for 36 hr. The Ago1- or Ago2-associated miRNAs and mRNAs were enriched by immunoprecipitation with anti-Ago1 or anti-Ago2. Levels of miR-483-3p/-5p mRNA were detected by qPCR and normalized to those of Ago1 or Ago2 protein. Data information: Values are expressed as mean ± SEM from 3 independent experiments. Statistical test: t-test (*P < 0.05 between the indicated groupsPAECs were infected with Lenti-pre-miR-483 or Lenti-null for 36 hr. The Ago1- or Ago2-associated miRNAs and mRNAs were enriched by immunoprecipitation with anti-Ago1 or anti-Ago2. Levels of TGF-β, TGFBR2, β-catenin, CTGF, IL-1β, and ET-1 mRNA were detected by qPCR and normalized to those of Ago1 or Ago2 protein. Data information: Values are expressed as mean ± SEM from 3 independent experiments. Statistical test: t-test (*P < 0.05 between the indicated groups


Figure 4 ( A ) Lung ECs were isolated from EC - miR - 483 transgenic rats ( Tg ) and their wild - type littermates ( WT ) , n = 3 in each group . MiR - 483 - 3p / - 5p levels were measured by qPCR . Data information : Values are expressed as mean ± SEM . ( B ) Serum from WT and Tg rats ( n = 6 ) were collected . MiR - 483 - 3p and - 5p associated with CD144 were enriched by immunoprecipitation with anti - CD144 . Levels of miR - 483 - 3p / - 5p were measured by qPCR . Data information : Values are expressed as mean ± SEM . Lung ECs isolated from miR - 483 - Tg rat and WT littermates were exposed to normoxia or hypoxia ( 0 . 2 % O2 ) for 24 hr . Proliferation of ECs were measured by flow cytometry Data information : Values are expressed as mean ± SEM . Data are representative of three independent experiments Statistical test : t - test ( * P < 0 . 05 vs . respective controls or between the indicated groupsLung ECs isolated from miR - 483 - Tg rat and WT littermates were exposed to normoxia or hypoxia ( 0 . 2 % O2 ) for 24 hr . migration of ECs were measured by wound healing assay Data information : Values are expressed as mean ± SEM . Data are representative of three independent experiments Statistical test : t - test ( * P < 0 . 05 vs . respective controls or between the indicated groupsTg and WT rats were injected with saline or MCT . Expression levels of TGF - β , TGFBR2 , β - catenin , CTGF , IL - 1β , and ET - 1 mRNA and protein in lung tissues were measured by qPCR and Western blot , respectively ( E ) . Data information : Values are expressed as mean ± SEM . Tg and WT rats were injected with saline or MCT . Levels of miR - 483 - 3p / - 5p in serum and lung tissues were measured by qPCR ( F ) . Data information : Values are expressed as mean ± SEM . Tg and WT rats were injected with saline or MCT . Ago1 - or Ago2 - associated miRNAs ( G ) were enriched from the isolated lung tissues by immunoprecipitation with anti - Ago1 or anti - Ago2 and quantified by qPCR ( n = 3 × 3 , samples were pooled from three animals for each assay , and 3 independent experiments [ a total of 9 animals ] were performed ) . Data information : Values are expressed as mean ± SEM . Tg and WT rats were injected with saline or MCT . Ago1 - or Ago2 - associated mRNAs ( H ) were enriched from the isolated lung tissues by immunoprecipitation with anti - Ago1 or anti - Ago2 and quantified by qPCR ( n = 3 × 3 , samples were pooled from three animals for each assay , and 3 independent experiments [ a total of 9 animals ] were performed ) . Data information : Values are expressed as mean ± SEM .

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

Figure 4(A) Lung ECs were isolated from EC- miR-483 transgenic rats (Tg) and their wild-type littermates (WT), n = 3 in each group. MiR-483-3p/-5p levels were measured by qPCR. Data information: Values are expressed as mean ± SEM.(B) Serum from WT and Tg rats (n = 6) were collected. MiR-483-3p and -5p associated with CD144 were enriched by immunoprecipitation with anti-CD144. Levels of miR-483-3p/-5p were measured by qPCR. Data information: Values are expressed as mean ± SEM. Lung ECs isolated from miR-483-Tg rat and WT littermates were exposed to normoxia or hypoxia (0.2% O2) for 24 hr. Proliferation of ECs were measured by flow cytometry Data information: Values are expressed as mean ± SEM. Data are representative of three independent experiments Statistical test: t-test (*P < 0.05 vs. respective controls or between the indicated groupsLung ECs isolated from miR-483-Tg rat and WT littermates were exposed to normoxia or hypoxia (0.2% O2) for 24 hr. migration of ECs were measured by wound healing assay Data information: Values are expressed as mean ± SEM. Data are representative of three independent experiments Statistical test: t-test (*P < 0.05 vs. respective controls or between the indicated groupsTg and WT rats were injected with saline or MCT. Expression levels of TGF-β, TGFBR2, β-catenin, CTGF, IL-1β, and ET-1 mRNA and protein in lung tissues were measured by qPCR and Western blot, respectively (E). Data information: Values are expressed as mean ± SEM.Tg and WT rats were injected with saline or MCT. Levels of miR-483-3p/-5p in serum and lung tissues were measured by qPCR (F). Data information: Values are expressed as mean ± SEM.Tg and WT rats were injected with saline or MCT. Ago1- or Ago2-associated miRNAs (G) were enriched from the isolated lung tissues by immunoprecipitation with anti-Ago1 or anti-Ago2 and quantified by qPCR (n = 3×3, samples were pooled from three animals for each assay, and 3 independent experiments [a total of 9 animals] were performed). Data information: Values are expressed as mean ± SEM. Tg and WT rats were injected with saline or MCT. Ago1- or Ago2-associated mRNAs (H) were enriched from the isolated lung tissues by immunoprecipitation with anti-Ago1 or anti-Ago2 and quantified by qPCR (n = 3×3, samples were pooled from three animals for each assay, and 3 independent experiments [a total of 9 animals] were performed). Data information: Values are expressed as mean ± SEM.


Figure 5EC - miR - 483 - Tg and WT rats were injected with saline or MCT . Analysis of mPAP for the indicated groups ( 7 rats per group ) Data information : Values are expressed as mean ± SEM . Statistical test : t - test ( * P < 0 . 05 vs . respective controls or between the indicated groups ) . EC - miR - 483 - Tg and WT rats were injected with saline or MCT . Analysis of RV hypertrophy [ RV / ( LV + S ) ] for the indicated groups ( 7 rats per group ) Data information : Values are expressed as mean ± SEM . Statistical test : t - test ( * P < 0 . 05 vs . respective controls or between the indicated groups ) . Vascularization evaluated by H & E staining of pulmonary arteries ( C ) . Data information : Values are expressed as mean ± SEM . Statistical test : t - test ( * P < 0 . 05 vs . respective controls or between the indicated groups ) . Scale bar : 20 µm ( C ) Vascular integrity evaluated by intravenous injection of Evans blue dye ( D ) . Data information : Values are expressed as mean ± SEM . Statistical test : t - test ( * P < 0 . 05 vs . respective controls or between the indicated groups ) . Scale bar : 0 . 5 cm ( D ) . EC - miR - 483 - Tg rats and WT littermates were injected with SU5416 and then exposed to hypoxia for 3 weeks and had reoxygenation for 2 weeks or injected with DMSO and exposed to normoxia for 5 weeks . Analysis of mPAP ( E ) for the indicated groups . Data information : Values are expressed as mean ± SEM . Statistical test : t - test ( * P < 0 . 05 vs . respective controls or between the indicated groups ) . EC - miR - 483 - Tg rats and WT littermates were injected with SU5416 and then exposed to hypoxia for 3 weeks and had reoxygenation for 2 weeks or injected with DMSO and exposed to normoxia for 5 weeks . Analysis of RV hypertrophy [ RV / ( LV + S ) ] ( F ) for the indicated groups . Data information : Values are expressed as mean ± SEM . Statistical test : t - test ( * P < 0 . 05 vs . respective controls or between the indicated groups ) .

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 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 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 B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 5EC-miR-483-Tg and WT rats were injected with saline or MCT. Analysis of mPAP for the indicated groups (7 rats per group) Data information: Values are expressed as mean ± SEM. Statistical test: t-test (*P < 0.05 vs. respective controls or between the indicated groups).EC-miR-483-Tg and WT rats were injected with saline or MCT. Analysis of RV hypertrophy [RV/(LV+S)] for the indicated groups (7 rats per group) Data information: Values are expressed as mean ± SEM. Statistical test: t-test (*P < 0.05 vs. respective controls or between the indicated groups).Vascularization evaluated by H&E staining of pulmonary arteries (C). Data information: Values are expressed as mean ± SEM. Statistical test: t-test (*P < 0.05 vs. respective controls or between the indicated groups). Scale bar: 20 µm (C)Vascular integrity evaluated by intravenous injection of Evans blue dye (D). Data information: Values are expressed as mean ± SEM. Statistical test: t-test (*P < 0.05 vs. respective controls or between the indicated groups). Scale bar: 0.5 cm (D).EC-miR-483-Tg rats and WT littermates were injected with SU5416 and then exposed to hypoxia for 3 weeks and had reoxygenation for 2 weeks or injected with DMSO and exposed to normoxia for 5 weeks. Analysis of mPAP (E) for the indicated groups. Data information: Values are expressed as mean ± SEM. Statistical test: t-test (*P < 0.05 vs. respective controls or between the indicated groups).EC-miR-483-Tg rats and WT littermates were injected with SU5416 and then exposed to hypoxia for 3 weeks and had reoxygenation for 2 weeks or injected with DMSO and exposed to normoxia for 5 weeks. Analysis of RV hypertrophy [RV/(LV+S)] (F) for the indicated groups. Data information: Values are expressed as mean ± SEM. Statistical test: t-test (*P < 0.05 vs. respective controls or between the indicated groups).


Figure 6qPCR analysis of miR - 483 - 3p / - 5p in serum from PH ( MCT - treated ) rats . Data information : Values are expressed as mean ± SEM . Statistical test : t - test ( * P < 0 . 05 vs . respective controls or between the indicated groupsqPCR analysis of miR - 483 - 3p / - 5p in lung tissue from PH ( MCT - treated ) rats . Data information : Values are expressed as mean ± SEM . Statistical test : t - test ( * P < 0 . 05 vs . respective controls or between the indicated groups ( D ) Protein and mRNA levels of TGF - β , TGFBR2 , β - catenin , CTGF , IL - 1β , and ET - 1 measured by Western blot and qPCR , respectively ( n = 3 × 3 , samples were pooled from three animals for each assay , and 3 independent experiments [ a total of 9 animals ] were performed ) . Data information : Values are expressed as mean ± SEM . Statistical test : t - test ( * P < 0 . 05 vs . respective controls or between the indicated groupsAnalysis of mPAP ( E ) for the indicated groups ( 7 rats in each group ) . Data information : Values are expressed as mean ± SEM . Statistical test : t - test ( * P < 0 . 05 vs . respective controls or between the indicated groupsAnalysis of RV hypertrophy [ RV / ( LV + S ) ] for the indicated groups ( 7 rats in each group ) . Data information : Values are expressed as mean ± SEM . Statistical test : t - test ( * P < 0 . 05 vs . respective controls or between the indicated groupsAnalysis of systemic blood pressure ( SBP ) ( G ) for the indicated groups ( 7 rats in each group ) . Data information : Values are expressed as mean ± SEM . Statistical test : t - test ( * P < 0 . 05 vs . respective controls or between the indicated groups ( H ) RV hypertrophy shown by the H & E staining and RV / ( LV + S ) quantification . Data information : Values are expressed as mean ± SEM . Statistical test : t - test ( * P < 0 . 05 vs . respective controls or between the indicated groups Scale bar : 0 . 5 cm ( H ) ( I ) Vascularization revealed by H & E staining ( n = 7 rats in each group ) . Data information : Values are expressed as mean ± SEM . Statistical test : t - test ( * P < 0 . 05 vs . respective controls or between the indicated groups Scale bar : 20 µm ( I ) . ( J ) Survival rate of 3 groups ( n = 15 rats in each group ) . Data information : Values are expressed as mean ± SEM . Statistical test : t - test ( * P < 0 . 05 vs . respective controls or between the indicated

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 O O O O O O O O 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 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 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 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 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

Figure 6qPCR analysis of miR-483-3p/-5p in serum from PH (MCT-treated) rats. Data information: Values are expressed as mean ± SEM. Statistical test: t-test (*P < 0.05 vs. respective controls or between the indicated groupsqPCR analysis of miR-483-3p/-5p in lung tissue from PH (MCT-treated) rats. Data information: Values are expressed as mean ± SEM. Statistical test: t-test (*P < 0.05 vs. respective controls or between the indicated groups(D) Protein and mRNA levels of TGF-β, TGFBR2, β-catenin, CTGF, IL-1β, and ET-1 measured by Western blot and qPCR, respectively (n = 3×3, samples were pooled from three animals for each assay, and 3 independent experiments [a total of 9 animals] were performed). Data information: Values are expressed as mean ± SEM. Statistical test: t-test (*P < 0.05 vs. respective controls or between the indicated groupsAnalysis of mPAP (E) for the indicated groups (7 rats in each group). Data information: Values are expressed as mean ± SEM. Statistical test: t-test (*P < 0.05 vs. respective controls or between the indicated groupsAnalysis of RV hypertrophy [RV/(LV+S)] for the indicated groups (7 rats in each group). Data information: Values are expressed as mean ± SEM. Statistical test: t-test (*P < 0.05 vs. respective controls or between the indicated groupsAnalysis of systemic blood pressure (SBP) (G) for the indicated groups (7 rats in each group). Data information: Values are expressed as mean ± SEM. Statistical test: t-test (*P < 0.05 vs. respective controls or between the indicated groups(H) RV hypertrophy shown by the H&E staining and RV/(LV+S) quantification. Data information: Values are expressed as mean ± SEM. Statistical test: t-test (*P < 0.05 vs. respective controls or between the indicated groups Scale bar: 0.5 cm (H)(I) Vascularization revealed by H&E staining (n = 7 rats in each group). Data information: Values are expressed as mean ± SEM. Statistical test: t-test (*P < 0.05 vs. respective controls or between the indicated groups Scale bar: 20 µm (I).(J) Survival rate of 3 groups (n = 15 rats in each group). Data information: Values are expressed as mean ± SEM. Statistical test: t-test (*P < 0.05 vs. respective controls or between the indicated groups