HCQ and bafilomycin A1 sensitize gefitinib-treated cells to cell death.

<p>(<b>A</b>) BT474, JIMT-1 and MCF7-GFPLC3 cells were treated for 24 h, 72 h and 168 h, respectively, with vehicle (0 µM gefitinib) or increasing concentrations of gefitinib (Gef) in the absence or presence of HCQ (20 µM). The HCA (BT474 and JIMT-1) and flow cytometric data (MCF7-GFPLC3) are expressed as percentage of viable ETH- or PI-excluding cells, respectively, in the total population normalized to vehicle-treated cells expressed as 100%. (<b>B</b>) Western blot analysis of caspase activation in lysates derived from cells treated as indicated in (<b>A</b>). (<b>C</b>) SKBR3 cells were treated for 72 h and (<b>D</b>) MCF7-GFPLC3 cells were treated for 120 h with vehicle, 10 µM gefitinib (Gef), increasing concentrations of bafilomycin A1 (BAF) and the combination of gefitinib and bafilomycin A1 (Gef+BAF). Left graphs: absolute numbers of viable (trypan blue excluding) cells in cultures treated with the indicated agents relative to cultures treated with vehicle expressed as 100%. Middle graphs: flow cytometric analysis of the sub-G₀/G₁ cell fraction in cells treated with the indicated agents (mean±SD, n = 3). Right graphs: flow cytometric analysis of apoptosis and viability based on Annexin V-Alexa647 and PI staining. PI+AN-: necrotic cells; AN+: apoptotic cells; PI-AN-: viable cells. Each data point in (<b>A</b>), (<b>C</b>) and (<b>D</b>) represents a mean±SD from 3 replicate samples. Asterisks indicate a significant difference (p<0.05) between cells treated with gefitinib and the combination of gefitinib and HCQ in the same category. The results shown are representative of 2 experiments for each cell type. (<b>E</b>) Western blot analysis of caspase activation in lysates derived from SKBR3 and MCF7-GFPLC3 cells treated for 72 h and 120 h, respectively, with vehicle or gefitinib (Gef) in the absence or presence of 10 nM bafilomycin A1 (BAF).