Fig. 2.

DPBA induces EGFR degradation in a lysosome-dependent manner. a DPBA did not reduce EGFR mRNA level. A549 and H1975 were treated with DPBA (6 μM) for 6, 12, and 24 h. EGFR mRNA level was measured by RT-PCR, n = 3. b DPBA enhanced EGFR half-time degradation. A549 and H1975 were treated with CHX (20 μM) in the presence or absence of DPBA (6 μM) for the indicated times. EGFR protein levels were measured by Western blot. c DPBA did not induce EGFR ubiquitination. A549 was treated with DPBA (4, 6, or 8 μM) for 3 h or EGF (100 ng/ml) for 5 min, and EGFR ubiquitination was measured by Western blot. d A549 and H1975 were treated with DPBA (6 μM) with or without MG132 (10 μM) or Baf A1 (200 nM) for 12 h. EGFR protein levels were measured by Western blot. e A549 and H1975 were treated with DPBA (6 μM) with or without leupeptin (100 μM), E-64 (200 μM), Ca074Me (10 μM), or pepstatin A (100 μM) for 12 h. EGFR protein levels were measured by Western blot. f A549 and H1975 were treated with DPBA (6 μM) in the presence or absence of Baf A1 (200 nM) for 24 h. Activation of EGFR pathway were measured by Western blot. g A549 and H1975 were treated with various concentrations of DPBA in the presence or absence of Baf A1 (200 nM) for 24 h. Cell viability was detected by the MTT assay, ***P < 0.001 vs. DPBA, n = 3. h DPBA induced EGFR perinuclear accumulation. A549 and H1975 were treated with DPBA (6 μM) for 6 h. EGFR sublocalisation was detected by immunofluorescence (magnification, ×200; scale bar, 50 μm). i DPBA induced surface EGFR endocytosis. A549 and H1975 were treated with DPBA (6 μM) for 3 and 6 h. Surface and intracellular EGFR were measured by biotinylation assay. TfR was the loading control for surface protein. j DPBA induced EGFR trafficking through endo-lysosome route. A549 was treated with DPBA (6 μM) for 3 and 6 h. Colocalisation between EGFR and LAMP1, Rab5, Rab7, or Rab11 was detected by immunofluorescence (magnification, ×630; scale bar, 10 μm). k DPBA activated Rab5 and Rab7, but not Rab11. A549 was treated with DPBA (6 μM) for 3, 6, and 12 h. Rab-GTP expression was measured with a Rab Activation Assay Kit