Figure 2. YAP and TEAD directly control mechanical cue‐dependent Skp2 transcription.

• A
  Relative mRNA levels for the indicated genes in MDA‐MB‐231 cells transfected with either control siRNAs or YAP‐specific siRNAs, as measured by qPCR (N = 3).
• B
  Relative Skp2 mRNA expression in MDA‐MB‐231 cells transduced with vector or 5SA‐YAP retroviruses (N = 6).
• C
  Immunoblots of MDA‐MB‐231 cells transfected with the indicated siRNAs.
• D
  Schematic depicting the human Skp2 promoter, which contains two TEAD‐binding motifs (TB1, TB2). White boxes indicate exons (Ex).
• E
  ChIP‐seq of the human Skp2 promoter. (Above) ChIP‐seq using a Flag‐specific antibody on Flag‐5SA‐YAP‐expressing MCF10A cells. Flag‐5SA‐YAP binds to TB1 and TB2 in the Skp2 promoter. (Below) ChIP‐seq using YAP‐ or TEAD1‐specific antibodies on the SF268 human glioblastoma cell line (Stein et al, 2015). Endogenous YAP and TEAD1 bind TB2 in the Skp2 promoter.
• F
  Enrichment of endogenous YAP at TB2 in the Skp2 promoter, as determined by ChIP‐qPCR. The genomic region 3′ of the CTGF or Skp2 genes were used as negative controls (neg. ctl; N = 3).
• G
  Enrichment of Flag‐tagged 5SA‐YAP or 5SA/94A‐YAP at TB2 in the Skp2 promoter, as determined by ChIP‐qPCR. The CTGF promoter (prmt) and the genomic region 3′ of the CTGF gene were used as positive (pos.ctl) and negative controls (neg.ctl), respectively (N = 3).
• H
  Luciferase reporter assays with a wild‐type TB2 sequence‐containing Skp2 promoter (TB2‐WT) or a mutant TB2‐containing promoter (CATTCC‐>AGAAAA; TB2‐Mut) in MCF10A cells stably expressing vector, 5SA‐YAP, or 5SA/94A‐YAP (N = 4).
• I
  MDA‐MB‐231 cells were transduced with the indicated retroviruses and suspended on poly‐HEMA‐coated dishes. Then, qPCR (above, N = 3) or immunoblotting (below) was performed at the indicated times.
• J
  MDA‐MB‐231 cells expressing vector or 5SA‐YAP were transfected with either control siRNAs or TEAD1/3/4‐specific siRNAs. After 2 days, the cells were suspended for the indicated times and harvested for qPCR analyses (N = 3).
• K
  Relative Skp2 and CTGF mRNA levels, as measured by qPCR, in MCF10A cells transduced with either vector or 5SA‐YAP retroviruses and then either non‐treated or treated with 5 μM Lat. B for 14 h (N = 3).
• L, M
  Enrichment of endogenous YAP at TB2 in the Skp2 promoter in MCF10A cells treated with either DMSO or Lat. B for 14 h (L) or suspended for 2 h (M), as determined by ChIP‐qPCR. The CTGF promoter (prmt) and the genomic region 3′ of the CTGF gene were used as positive (pos.ctl) and negative controls (neg.ctl), respectively.

Data information: All error bars indicate standard error of the mean (s.e.m.) from N independent experiments. Statistical significance, as determined by a two‐tailed t‐test, is indicated above each bar.