Figure 1. EGFR but Not Its Kinase Activity Is Required for Autophagy.

(A) Knockdown of EGFR inhibits basal and serum starvation induced LC3-II (autophagosome marker) turnover in MDA-MB-231 cells. Cells transfected with indicated siRNAs were cultured in normal medium (N) or serum free starvation medium (S) for 24 h, and then treated with 80 μM chloroquine (CQ) for 2 h to induce LC3-II accumulation. Whole cell lysates were harvested for Western blot analysis of LC3-II.

(B) Knockdown of EGFR by two distinct siRNAs inhibits LC3-II generation. MDA-MB-231 cells were cultured in normal medium. Unless otherwise indicated, the EGFR siRNA #1 was used in all the remaining experiments.

(C) Quantification of LC3-II levels in (B); mean + SD, n = 3, *** P < 0.001.

(D) CQ induces accumulation of LC3 puncta (autophagosomes) in control cells but not EGFR knockdown cells. Control or EGFR siRNA transfected cells in normal medium were pretreated with CQ for 3 h and fixed for immunostaining of endogenous LC3 (red) and EGFR (green). DAPI was used to stain the nuclei; Boxes indicate selected regions for magnified new; Bar, 10 μm.

(E) Quantification of LC3 puncta in (D); mean + SD, n = 3, *** P < 0.001.

(F) Left: Re-expression of wild type (WT) or the K721A kinase dead (KD) EGFR rescues LC3-II turnover in EGFR-knockdown cells. Right: Gefitinib (2 μM) treatment enhances autophagy rescue by EGFR-WT. SiRNA resistant EGFR-WT or -KD was expressed in MDA-MB-231 cells by lentivirus mediated infection. Endogenous EGFR was knocked down by siRNA #1, and cells were cultured in normal medium and treated with CQ for 2 h before whole cell lysate harvest for Western analysis. EV, empty vector.

(G) Quantification of LC3-II levels in (F); mean + SD, n = 3, ** P < 0.01, *** P < 0.001.

(H) Representative images of serum starvation induced EGFP-LC3 puncta in MDA-MB-231 cells treated with indicated siRNAs with or without re-expression of EGFR-WT or EGFR-KD. EGFP-LC3 was expressed by lentivirus infection and a monoclonal cell line stably expressing low levels of EGFP-LC3 was selected. Endogenous EGFR was knocked down by siRNA, and siRNA-resistant WT or KD EGFR was re-expressed using lentivirus. Cells were serum starved and fixed for fluorescence microscopy. Bar, 10 μm.

(I) Quantification of the number of EGFP-LC3 puncta in (H); mean + SD, n = 3, *** P < 0.001. See also Figure S1.