Figure 4: YAP1/TAZ induces TGF-β2 expression and directly upregulates TGF-βRII expression in vitro.
From: The Hippo-Salvador signaling pathway regulates renal tubulointerstitial fibrosis
(a) Western blot analysis of YAP1, TAZ, Sav1, CTGF, and SMAD4 expression in HK2 cells incubated with TGF-β1 after serum starvation for 18 h. (b) Western blot analysis of the indicated proteins in HK2 cells expressing control vectors or shRNA lentiviral vectors against Sav1. (c) qRT-PCR analysis of ANKRD1, CTGF, and NEGR1 expression in HK2 cells…P = 0.0073, 0.001236, 0.00061 (comparison between the 1st bar and 3rd bar) and 0.03576, 0.02104, 0.00083 (comparison between the 1st bar and 4th bar) for ANKRD1, CTGF and NEGR1, respectively. (d–f) Upregulation of TGF-β2 expression by YAP1/TAZ.. qRT-PCR analysis of TGF-β2 mRNA expression in HEK293 cells transduced with (d) Sav1-depleting shRNA vectors or control vectors and incubated with (dark gray bars) or without (light gray bars) TGF-β1 for 12 hours. P = 0.03522 (comparison between the 3rd bar and 4th bar). (e) TAZ or YAP1 expression plasmids and (f) TAZ-, YAP1- or TAZ/YAP1-depleting shRNA vectors. P = 0.00093 (comparison between the 1st bar and 4th bar). (g) The increase in TβRII mRNA expression elicited by TGF-β1 treatment in the Sav1 deficiency. qRT-PCR analysis of TβRII mRNA expression in Sav1 depleted-HK2 and control cells treated with or without TGF-β1. P = 0.03204 (comparison between the 3rd bar and 4th bar). (h) Downregulation of TβRII mRNA expression by deletion of both YAP1 and TAZ in HEK293 cells.. P = 0.0389 (comparison between the 1st bar and 4th bar). (i) Schematic of part of the TβRII genomic region containing a YAP1/TAZ-enriched site showing TEAD binding sequences (GGAATG/CATTCC) located between 30638606 and 30639826 in chromosome 3, a YAP1 or TAZ-enriched region (yellow box), and the TβRII transcription start site (TSS). (j) Luciferase activity assays of the TβRII genomic upstream region shown in (i). 293T cells transfected with an pGL3 vector containing a genomic fragment including the YAP1/TAZ-enriched region were co-transfected with YAP1 or TAZ expression plasmids or control vectors. (k) ChIP assay for TAZ-enriched regions. immunoprecipitated DNA with an anti-Flag antibody was amplified with specific primers covering potential TEAD binding sequences. All error bars represent SEMs. *P < 0.05; **P < 0.01; ***P < 0.005 by paired t-test (one-tailed).
