Fig. 3. The combination of berberine and osimertinib augments Bim elevation and Mcl-1 reduction (A) through modulating their degradation rates (B and C), which critically mediates the enhanced apoptosis by the combination (D-G), in MET-amplified osimertinib-resistant cells.

A, HCC827/AR were treated with DMSO, 200 nM osimertinib (Osim), 100 μM berberine (BBR) or their combination (Comb) for varied times as indicated. Western blotting was used to detect the proteins of interest. B and C, HCC827/AR cells were exposed to DMSO, 200 nM Osimertinib, 100 μM berberine or the combination of osimertinib and berberine for 12 h followed by the addition of 10 μg/ml CHX. The cells were then harvested at the indicated times for preparation of whole cell-protein lysates and subsequent detection of the tested proteins by Western blotting. NIH image J software was used to quantify band intensities. Bim and Mcl-1 levels are shown as percentage of levels at 0 time post CHX treatments. D-G, The indicated cell lines with Bim knockout (D and E) and Mcl-1 overexpression (F and G) were exposed to DMSO, 200 nM osimertinib, 100 μM berberine or osimertinib plus berberine for 48 h. The cells were then harvested for preparation of whole-cell lysates and subsequent Western blotting to detect the indicated proteins (D and F) and for detection of apoptotic cells with annexin V/flow cytometry (E and G). The data are means ± SDs of duplicate determinations.