Figure 6.

The Ras/SOCS/YAP/AREG feedback loop

1. Upregulation of AREG and survivin in BJ^{T/p53KD/p16KD/ST} cells transduced to express Ras^V12, two independent shRNAs targeting SOCS5/6, or YAP^S127A. RNA was isolated from a subset of the cells prepared for the colony formation assay in Fig3A. The immunoblot control for SOCS6 depletion is shown in Fig3A. Transcript levels were measured by quantitative PCR. Data: average of three independent experiments ± SD. GAPDH was used as a control.
2. Immunoblot showing the effect of YAP depletion on AREG protein levels. BJ^{T/p53KD/p16KD/ST} cells were transduced to express two independent shRNAs targeting YAP or TEAD-DN. Blots were probed with antibody to YAP and AREG. Actin served as a loading control.
3. Immunoblot showing the effect of AREG depletion on EGFR activity in BJ^{T/p53KD/p16KD/ST/Rasv12} cells. Cells were transduced to express two independent shRNAs targeting AREG. Blots were probed with antibody to Y1068 phosphorylated EGFR to monitor EGFR activity, and with antibody to EGFR and AREG. Actin served as a loading control.
4. Effect of AREG depletion on soft agar colony formation by fully transformation-competent BJ^{T/p53KD/p16KD/ST}/Ras^V12 cells. Cells were transduced to express two independent shRNAs targeting AREG or with empty vector as a control. Lower left: average colony number from three independent experiments ± SD shown as % of vector control. P < 0.05 comparing each siRNA to control. Lower right: AREG mRNA levels were measured by quantitative PCR to monitor shRNA efficacy.
5. Effect of AREG depletion on soft agar colony formation by YAP1^S127A-expressing cells. BJ^{T/p53KD/p16KD/ST}/ER-Ras^v12 cells were transduced to express YAP1^S127A and with shRNA targeting AREG or with empty vector as a control. Below: average colony number from three independent experiments ± SD, normalized to YAP1^S127A-expressing cells. P < 0.05 comparing each siRNA to it respective control.

Source data are available online for this figure.