FIGURE 3. NRG1 activates endogenous ERBB4 to induce YAP target genes.
(A) RT-PCR of CTGF in T47D cells starved for 3 hours in Opti-MEM then treated with NRG1 (50 ng/ml) for 1 hour with or without an hour pre-treatment with lapatinib (1 μM). Data are means (SD) from 3 experiments.
(B) RT-PCR (left) of CTGF in T47D cells expressing pLKO encoding ERBB4 shRNA (sh1 or sh3) or scrambled control (scr). Cells were treated as in (A). Immunoblot (right) showing ERBB4 protein knockdown. RT-PCR data are means (SD) from 3 experiments. Blots are representative of 3 experiments.
(C) RT-PCR of (left to right) YAP, CTGF, CYR61, and ANKRD1 in T47D cells expressing pINDUCER10 encoding YAP shRNA. Cells were treated with DOX (1 μg/ml) for 5 days then starved overnight (Opti-MEM, +DOX) followed by 1 hour NRG1 (50 ng/ml) treatment. Data are means (SD) from 3 technical replicates, representative of 3 experiments.
(D) Immunoblot showing time-course of NRG1-induced Tyr1284 phosphorylated ERBB4, ERBB4 ICD, Ser127 phosphorylated YAP, and CTGF abundance in T47D cells. Cells were starved overnight in Opti-MEM then treated with NRG1 (50 ng/ml) for 0, 15, 30, 60, 120, or 240 minutes. Blots are representative of 3 experiments.
(E) Quantification of the ratio of phosphorylated YAP at Ser127 to total YAP protein levels from immunoblots in (D). Data are normalized to t=0 minutes. Data are means (SD) from 3 experiments. n.s., not significant; *p<0.05; **p<0.01, ***p<0.001, ****p<0.0001.