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. 2018 Dec 27;38(3):e98250. doi: 10.15252/embj.201798250

Figure 8. Indirect regulation of VEGFR2 via miR‐15a/16 by TFEB in ECs.

Figure 8

  • 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.