Figure 4.

Depletion of LRIG1 augments ErbB2 expression and enhances cellular proliferation. A, Lysates from MDA-MB-453 and BT474 cells transfected for 72h with RNAi oligos targeting LRIG1 (LRIG1-KD) or scrambled control (SC) were blotted with antibodies to p-ErbB2, ErbB2, LRIG1, and actin. B, 48h after transfecting with RNAi oligos, MDA-MB-453 cells were serum starved overnight and then treated with increasing concentrations of Nrg1 for 15 min. Whole cell lysates were blotted with antibodies to LRIG1, pAKT, tAKT, pERK, tERK, and tubulin as a loading control. The bottom panel plots the densitometic analysis of the average pAKT signal from three independent experiments for each Nrg1 concentration along with SE. C, 24h after transfecting cells with RNAi oligos as described above, MDA-MB-453 and BT474 cells were placed in serum starve media, in the absence or presence of 2.5 nM Nrg1, or complete media (10% FCS). After 48h, proliferation was measured by an MTT assay. Three independent experiments were conducted with a representative experiment shown. Error bars represent SE of four replicates within this experiment. D, 24h after transfecting cells with RNAi oligos as described above, MDA-MB-453 cells were placed in serum starve or complete media in the absence or presence of the EGFR/ErbB2 inhibitor 4557W. After 48h, proliferation was measured by an MTT assay, with basal proliferation being normalized as 100%. Additionally, whole cell lysates from serum starved cells were collected following treatment and blotted for p-ErbB2 and actin (top panel) to verify inhibition of ErbB2 by 4557W.