Journal: The EMBO Journal

Article Title: TFEB controls vascular development by regulating the proliferation of endothelial cells

doi: 10.15252/embj.201798250

Figure Lengend Snippet: A–C Increase in Vegfr2 expression in the vasculature of Tfeb EC−/− and Tfeb iEC−/− mice. (A) Representative immunostaining images of Tfeb EC−/− embryonic vessels (E10.5) stained with anti‐endomucin and anti‐Vegfr2 Abs (scale bars: 50 μm). Bar graph indicates the Vegfr2 mean intensity only in endomucin + vessel areas (embryos n = 6, mean ± SEM; *** P < 0.0001 versus control embryos by Student's t‐ test). (B) Representative immunostaining images (i) and detail (ii) of the vascular front and vascular plexus of the retina (p5) of control and Tfeb iEC−/− mice with anti‐iB4 and anti‐Vegfr2 Abs (scale bars: 50 μm). Bar graph indicates the Vegfr2 mean intensity only in iB4 + vessel areas (mice n = 6, mean ± SEM; ** P < 0.001 and *** P < 0.0001 versus control mice by Student's t‐ test). (C) Representative immunostaining images of the glomerulus (p17) of control and Tfeb iEC−/− mice with anti‐podocin, anti‐CD31, and anti‐Vegfr2 Abs (scale bars: 50 μm). Bar graphs indicate the Vegfr2 mean intensity in CD31 + vessel areas (mice n = 6, mean ± SEM; ** P < 0.001 versus control mice by Student's t‐ test). D–F VEGFR2 expression is regulated by TFEB. (D, E) qPCR of VEGFR2 in lung ECs obtained from control and Tfeb iEC−/− mice (D) and in human sh‐TFEB (E). Data are expressed as relative fold‐change compared with the expression in control cells after normalization to the housekeeping gene TBP ( n = 3, mean ± SEM; ** P < 0.001 and *** P < 0.0001, by Student's t‐ test). (F) Immunoblots of total lysates from scr‐shRNA and sh‐TFEB ECs probed with anti‐VEGFR2 and α‐tubulin Abs. The bar graph shows the densitometric analysis expressed as the ratio between VEGFR2 and α‐tubulin ( n = 3, mean ± SEM; *** P < 0.0001 versus scr‐shRNA by Student's t‐ test). G Analysis of TFEB binding to the VEGFR2 promoter in human ECs. ChIP was performed using digested chromatin from human control ECs and TFEBS142A ECs incubated with IgG (indicated in the bar graph as “+IgG”) or with Ab anti‐TFEB (indicated in the bar graph as “+Ab anti‐TFEB”), followed by qPCR for VEGFR2 . Bar graph shows the percent enrichment ( n = 3, mean ± SD). Source data are available online for this figure.

Article Snippet: Anti‐GFP and anti‐Ki67 (SP6) from Thermo Fisher Scientific; isolectin‐B4, anti‐MYO1C, and anti‐α‐tubulin (B‐5‐1‐2), anti‐LAMP1, anti‐PCNA (PC10) from Sigma‐Aldrich; anti‐CD31, anti‐FLK1‐APC‐conjugated (Avas12alpha1), anti‐CD71‐FITC‐conjugated (C2), anti‐CD117 (2B8)‐PE‐cy7‐conjugated, anti‐CD31‐FITC‐conjugated (Mec 13.3), anti‐CD102, anti‐IgG 2a k isotype ‐ APC‐conjugated (R3595), anti‐IgG2bk isotype‐PE‐Cy7 conjugated (2B8), anti‐CD326, anti‐Rab4, and anti‐Rab5 from BD Biosciences; anti‐VEGFR2 (55B11), anti‐p‐Tyr‐1175‐VEGFR2 (D5B11), anti‐PLCγ‐1, anti‐p‐Tyr‐783‐PLCγ‐1, anti‐ERK1/2, anti‐p‐ERK‐1/2 (T202/Y204; E10), anti‐Src (36D10), anti‐p‐Src (Tyr416; D49G4), anti‐CDK4 (D9G3E), anti‐Rb (4H1), anti‐p‐Rb (Ser780; C84F6), and anti‐p‐Rb (Ser807/811; D20B12), anti‐PCNA (PC10), anti‐ULK1 (D8H5), anti‐ATG9A (D409D) from Cell Signaling Technology; anti‐endomucin (V.7C7), anti‐Flk‐1 (A‐3), anti‐podocin (G‐20), anti‐caveolin‐1 (N‐20) and anti‐E2F2 (TFE‐25), anti‐E2F1 from Santa Cruz Biotechnology; anti‐human VEGFR2 (89109) from R&D System; anti‐TFEB from MyBiosource; hypoxyprobe‐1‐FITC‐conjugated antibody (Chemicon); anti‐LC3 from Novus Biologicals; anti‐cyclin D1 (SP4), anti‐E2F1, and anti‐TGN46 (2F7.1) from Abcam.

Techniques: Expressing, Immunostaining, Staining, Control, Western Blot, shRNA, Binding Assay, Incubation

Journal: The EMBO Journal

Article Title: TFEB controls vascular development by regulating the proliferation of endothelial cells

doi: 10.15252/embj.201798250

Figure Lengend Snippet: A, B Silenced TFEB alters the localization of VEGFR2. (A) FACS analysis of surface VEGFR2 expression on human scr‐shRNA and sh‐TFEB ECs. Bar graph shows the ratio between total and PM VEGFR2 ( n = 6, mean ± SEM; ** P < 0.001 versus scr‐shRNA by Student's t‐ test). (B) Representative TIRF and epifluorescence images of human scr‐shRNA and sh‐TFEB ECs after staining with anti‐VEGFR2 Ab (scale bars: 10 μm). Bar graphs show the ratio of VEGFR2 analyzed in epifluorescence and TIRF mode analyzed by TIRF ( n = 40, mean ± SEM; *** P < 0.0001 versus scr‐shRNA by Student's t‐ test). C Silenced TFEB alters the localization and the phosphorylation state of VEGFR2 and its signal transduction. Representative immunoblot of PM biotinylated portion and total cell lysates of scr‐shRNA and sh‐TFEB ECs after VEGF‐A stimulation (30 ng/ml). Blots of total or PM cell lysates were probed with anti‐VEGFR2. Blots of total cell lysates were probed with anti‐p‐Y1175‐VEGFR2, anti‐PLCγ, p‐PLCγ, anti‐ERK‐1/2, anti‐pERK1/2, anti‐p‐Src, anti‐Src, anti‐CD31, and α‐tubulin Abs. The bar graphs (i,ii) show densitometric analysis of stimulated versus unstimulated scr‐shRNA and sh‐TFEB ECs expressed as: (i) % of VEGFR2 on PM fraction ( n = 3, mean ± SEM; ANOVA P < 0.02; ** P < 0.001 versus scr‐shRNA by Bonferroni post‐test), (ii) % of VEGFR2 total ( n = 3, mean ± SEM; ANOVA P > 0.05; * P < 0.05 and ** P < 0.001 versus scr‐shRNA by Bonferroni post‐test). D, E Regulation of autophagy and lysosome pathway by TFEB silencing. (D) qPCR and (E) immunoblots showing the differentially expressed autophagy‐ and lysosome‐related genes between human scr‐shRNA and sh‐TFEB ECs. (D) Data are expressed as relative fold‐change compared with the expression in control cells after normalization to the housekeeping gene TBP ( n = 3, mean ± SEM; *** P < 0.0001 by Student's t‐ test). (E) Immunoblots of total lysates from human scr‐shRNA and sh‐TFEB ECs probed with anti‐ULK‐1, anti‐ATG9, anti‐LC3‐I/II, anti‐LAMP‐1, and α‐tubulin Abs. The bar graph shows the densitometric analysis expressed as the ratio between scr‐shRNA and sh‐TFEB ECs ( n = 3, mean ± SEM; ** P < 0.001, *** P < 0.0001 versus scr‐shRNA by Student's t‐ test). F TFEB silencing inhibits VEGFR2 internalization. Bar graphs of VEGFR2 internalization expressed as the percent of internalized VEGFR2 versus PM VEGFR2 after VEGF‐A stimulation ( n = 6, mean ± SEM, ANOVA P < 0.0001; *** P < 0.0001 versus scr‐shRNA by Bonferroni post‐test). Source data are available online for this figure.

Article Snippet: Anti‐GFP and anti‐Ki67 (SP6) from Thermo Fisher Scientific; isolectin‐B4, anti‐MYO1C, and anti‐α‐tubulin (B‐5‐1‐2), anti‐LAMP1, anti‐PCNA (PC10) from Sigma‐Aldrich; anti‐CD31, anti‐FLK1‐APC‐conjugated (Avas12alpha1), anti‐CD71‐FITC‐conjugated (C2), anti‐CD117 (2B8)‐PE‐cy7‐conjugated, anti‐CD31‐FITC‐conjugated (Mec 13.3), anti‐CD102, anti‐IgG 2a k isotype ‐ APC‐conjugated (R3595), anti‐IgG2bk isotype‐PE‐Cy7 conjugated (2B8), anti‐CD326, anti‐Rab4, and anti‐Rab5 from BD Biosciences; anti‐VEGFR2 (55B11), anti‐p‐Tyr‐1175‐VEGFR2 (D5B11), anti‐PLCγ‐1, anti‐p‐Tyr‐783‐PLCγ‐1, anti‐ERK1/2, anti‐p‐ERK‐1/2 (T202/Y204; E10), anti‐Src (36D10), anti‐p‐Src (Tyr416; D49G4), anti‐CDK4 (D9G3E), anti‐Rb (4H1), anti‐p‐Rb (Ser780; C84F6), and anti‐p‐Rb (Ser807/811; D20B12), anti‐PCNA (PC10), anti‐ULK1 (D8H5), anti‐ATG9A (D409D) from Cell Signaling Technology; anti‐endomucin (V.7C7), anti‐Flk‐1 (A‐3), anti‐podocin (G‐20), anti‐caveolin‐1 (N‐20) and anti‐E2F2 (TFE‐25), anti‐E2F1 from Santa Cruz Biotechnology; anti‐human VEGFR2 (89109) from R&D System; anti‐TFEB from MyBiosource; hypoxyprobe‐1‐FITC‐conjugated antibody (Chemicon); anti‐LC3 from Novus Biologicals; anti‐cyclin D1 (SP4), anti‐E2F1, and anti‐TGN46 (2F7.1) from Abcam.

Techniques: Expressing, shRNA, Staining, Phospho-proteomics, Transduction, Western Blot, Control

Journal: The EMBO Journal

Article Title: TFEB controls vascular development by regulating the proliferation of endothelial cells

doi: 10.15252/embj.201798250

Figure Lengend Snippet: A–C Analysis of TFEB binding and modulation of the MYO1C promoter in human ECs. (A) Representative snapshot of TFEB binding on MYO1C in human ECs. (B) ChIP was performed using digested chromatin from control ECs and TFEBS142A ECs incubated with IgG (indicated in the bar graph as “+IgG”) or with Ab anti‐TFEB (indicated in the bar graph as “+Ab anti‐TFEB”), followed by qPCR for MYO1C . Bar graph shows the percent enrichment ( n = 3, mean ± SD). (C) qPCR of MYO1C expression in scr‐shRNA, sh‐TFEB, sh‐MYO1C, and sh‐TFEB+sh‐MYO1C ECs. Data are expressed as relative fold‐change compared with the expression in scr‐shRNA ECs after normalization to the housekeeping gene TBP ( n = 3, mean ± SEM; ** P < 0.001 and *** P < 0.0001 by Student's t‐ test). D MYO1C silencing reverses the effect of TFEB silencing on the up‐regulation of PM VEGFR2. Analyses were performed on human ECs carrying appropriate scr‐shRNA or sh‐TFEB in the presence or absence of sh‐MYO1C. Representative Western blot of MYOC1, total and PM biotinylated VEGFR2 (representative experiment out of 4 with similar results). E, F Representative immunostaining images of the vascular plexus of the retina (p5) (E) and glomerulus (p17) (F) of control and Tfeb iEC−/− mice with anti‐CD31 and anti‐MYO1C Abs (scale bars: 50 μm). Bar graphs indicate the Myo1C mean intensity only in vessel areas CD31 + ( n = 6, mean ± SEM; *** P < 0.0001 versus control mice by Student's t‐ test). G Representative TIRF and epifluorescence images of VEGFR2 in scr‐shRNA, sh‐TFEB, sh‐MYO1C, and sh‐TFEB+sh‐MYO1C human ECs (scale bars: 10 μm). Bar graph shows the ratio between PM and total VEGFR2 ( n = 40, mean ± SEM; ** P < 0.001 and *** P < 0.0001 versus scr‐shRNA by Student's t‐ test). Source data are available online for this figure.

Article Snippet: Anti‐GFP and anti‐Ki67 (SP6) from Thermo Fisher Scientific; isolectin‐B4, anti‐MYO1C, and anti‐α‐tubulin (B‐5‐1‐2), anti‐LAMP1, anti‐PCNA (PC10) from Sigma‐Aldrich; anti‐CD31, anti‐FLK1‐APC‐conjugated (Avas12alpha1), anti‐CD71‐FITC‐conjugated (C2), anti‐CD117 (2B8)‐PE‐cy7‐conjugated, anti‐CD31‐FITC‐conjugated (Mec 13.3), anti‐CD102, anti‐IgG 2a k isotype ‐ APC‐conjugated (R3595), anti‐IgG2bk isotype‐PE‐Cy7 conjugated (2B8), anti‐CD326, anti‐Rab4, and anti‐Rab5 from BD Biosciences; anti‐VEGFR2 (55B11), anti‐p‐Tyr‐1175‐VEGFR2 (D5B11), anti‐PLCγ‐1, anti‐p‐Tyr‐783‐PLCγ‐1, anti‐ERK1/2, anti‐p‐ERK‐1/2 (T202/Y204; E10), anti‐Src (36D10), anti‐p‐Src (Tyr416; D49G4), anti‐CDK4 (D9G3E), anti‐Rb (4H1), anti‐p‐Rb (Ser780; C84F6), and anti‐p‐Rb (Ser807/811; D20B12), anti‐PCNA (PC10), anti‐ULK1 (D8H5), anti‐ATG9A (D409D) from Cell Signaling Technology; anti‐endomucin (V.7C7), anti‐Flk‐1 (A‐3), anti‐podocin (G‐20), anti‐caveolin‐1 (N‐20) and anti‐E2F2 (TFE‐25), anti‐E2F1 from Santa Cruz Biotechnology; anti‐human VEGFR2 (89109) from R&D System; anti‐TFEB from MyBiosource; hypoxyprobe‐1‐FITC‐conjugated antibody (Chemicon); anti‐LC3 from Novus Biologicals; anti‐cyclin D1 (SP4), anti‐E2F1, and anti‐TGN46 (2F7.1) from Abcam.

Techniques: Binding Assay, Control, Incubation, Expressing, shRNA, Western Blot, Immunostaining

Journal: The EMBO Journal

Article Title: TFEB controls vascular development by regulating the proliferation of endothelial cells

doi: 10.15252/embj.201798250

Figure Lengend Snippet: A Analysis of TFEB binding to DLEU2 and SMC4 promoters in human ECs. ChIP was performed using digested chromatin from control ECs and TFEBS142A ECs incubated with IgG (indicated in the bar graph as “+IgG”) or with Ab anti‐TFEB (indicated in the bar graph as “+Ab anti‐TFEB”), followed by qPCR for DLEU2 and SMC4 . Bar graph shows the percent enrichment ( n = 3, mean ± SD). B DLEU2 expression is regulated by TFEB. qPCR of DLEU2 in human scr‐shRNA, sh‐TFEB, or control and TFEBS142A ECs (left panel) and lung ECs obtained from control and Tfeb iEC−/− mice (right panel). Data are expressed as relative fold‐change compared with the expression in scr‐shRNA and control cells after normalization to the housekeeping gene TBP ( n = 3, mean ± SEM; * P < 0.01, and *** P < 0.0001 by Student's t‐ test). C Human miR‐15a‐5p and miR‐16‐5p are regulated by TFEB. qPCR of miR‐15a‐5p (left panel) and miR‐16‐5p (right panel) in sh‐TFEB or TFEBS142A ECs. Data are expressed as relative fold‐change compared with the expression in scr‐shRNA and control cells after normalization to the housekeeping gene RNU44 ( n = 3, mean ± SEM; *** P < 0.0001 by Student's t‐ test). D–G VEGFR2 expression is regulated by TFEB through a miR‐dependent mechanism. (D, F) qPCR of VEGFR2 in human scr‐shRNA and sh‐TFEB ECs (D, E) and in control and TFEBS142A ECs (F, G) treated with a specific miR‐control, miR‐15a‐5p, and miR‐16‐5p mimics or inhibitors. (D, F) Data are expressed as relative fold‐change compared with the expression in control cells after normalization to the housekeeping gene TBP ( n = 3, mean ± SEM; ** P < 0.001 and *** P < 0.0001 versus control or scr‐shRNA plus miR‐control and ## P < 0.001 and ## P < 0.0001 versus sh‐TFEB and TFEBS142A plus miR‐control by Student's t‐ test). (E, G) Representative Western blot of VEGFR2 expression under the same experimental conditions previously reported. The bar graph shows the densitometric analysis expressed as the ratio between VEGFR2 and α‐tubulin ( n = 3, mean ± SEM; ** P < 0.001 and *** P < 0.0001 versus control or scr‐shRNA plus miR‐control and # P < 0.01 and ## P < 0.001 versus sh‐TFEB and TFEBS142A plus miR‐control by Student's t‐ test). Source data are available online for this figure.

Article Snippet: Anti‐GFP and anti‐Ki67 (SP6) from Thermo Fisher Scientific; isolectin‐B4, anti‐MYO1C, and anti‐α‐tubulin (B‐5‐1‐2), anti‐LAMP1, anti‐PCNA (PC10) from Sigma‐Aldrich; anti‐CD31, anti‐FLK1‐APC‐conjugated (Avas12alpha1), anti‐CD71‐FITC‐conjugated (C2), anti‐CD117 (2B8)‐PE‐cy7‐conjugated, anti‐CD31‐FITC‐conjugated (Mec 13.3), anti‐CD102, anti‐IgG 2a k isotype ‐ APC‐conjugated (R3595), anti‐IgG2bk isotype‐PE‐Cy7 conjugated (2B8), anti‐CD326, anti‐Rab4, and anti‐Rab5 from BD Biosciences; anti‐VEGFR2 (55B11), anti‐p‐Tyr‐1175‐VEGFR2 (D5B11), anti‐PLCγ‐1, anti‐p‐Tyr‐783‐PLCγ‐1, anti‐ERK1/2, anti‐p‐ERK‐1/2 (T202/Y204; E10), anti‐Src (36D10), anti‐p‐Src (Tyr416; D49G4), anti‐CDK4 (D9G3E), anti‐Rb (4H1), anti‐p‐Rb (Ser780; C84F6), and anti‐p‐Rb (Ser807/811; D20B12), anti‐PCNA (PC10), anti‐ULK1 (D8H5), anti‐ATG9A (D409D) from Cell Signaling Technology; anti‐endomucin (V.7C7), anti‐Flk‐1 (A‐3), anti‐podocin (G‐20), anti‐caveolin‐1 (N‐20) and anti‐E2F2 (TFE‐25), anti‐E2F1 from Santa Cruz Biotechnology; anti‐human VEGFR2 (89109) from R&D System; anti‐TFEB from MyBiosource; hypoxyprobe‐1‐FITC‐conjugated antibody (Chemicon); anti‐LC3 from Novus Biologicals; anti‐cyclin D1 (SP4), anti‐E2F1, and anti‐TGN46 (2F7.1) from Abcam.

Techniques: Binding Assay, Control, Incubation, Expressing, shRNA, Western Blot