Fig. 5: Amlodipine enhanced EGFR degradation through activating lysosomal pathway.

A After GSC23 and GSC11 cells were treated with amlodipine (varying from 0 to 25 μM, with 5 μM interval) for 48 h, mRNA level of EGFR was analyzed with qRT-PCR. B Western blot to detect the protein level of EGFR after GSC23 and GSC11 cells were treated with CHX (20 μM) alone or combined with amlodipine (15 μM) for the indicated times (0, 6, 12, 24, 36 and 48 h). C GSC23 and GSC11 cells were treated with amlodipine (15 μM), amlodipine(15 μM) plus MG132(10 μM), EGF (100 ng/ml), or EGF (100 ng/ml) plus MG132(10 μM), respectively for 12 h, then ubiquitination of EGFR was analyzed with Western blot. D GSC23 and GSC11 cells were treated with amlodipine (15 μM) alone, or combined either with MG132 (10 μM) or Baf A1 (200 nM), respectively for 48 h, then the protein level of EGFR was assayed with Western blot. E GSC23 and GSC11 cells were treated with amlodipine(15 μM) for 48 h, colocalization of EGFR (green) with LAMP1 (red) was detected by confocal immunofluorescence analysis, scale bar = 10 μm. F GSC23 and GSC11 cells were treated with amlodipine (15 μM) alone, or combined with Baf A1 (200 nM), respectively for 48 h, then the expression level of total EGFR, p-EGFR, Akt, p-Akt, mTOR, p-mTOR, ERK, p-ERK, STAT3 and p-STAT3 was evaluated with Western blot. G GSC23 and GSC11 cells were treated with indicated concentrations of amlodipine (varying from 0 to 25 μM, with 5 μM interval) alone, or combined with Baf A1 (200 nM), respectively for 48 h, then cell viability was detected by CCK8 assay. All data are presented as mean ± SD. n = 3 independent experiments. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001, compared with the control group.