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. 2018 Jul 8;18(15):1700388. doi: 10.1002/pmic.201700388

Figure 6.

Figure 6

Overexpression of HMAG2 or knocking down of ALOX5 suppresses gefitinib resistance by inhibiting autophagy. A) WB validation of HMAG2 overexpression in PC9/GR cells stably expressing the control (Control OE, #1 and #2) or HMGA2 overexpression (HMGA2 OE, #1 and #2) plasmids. The ratio of band intensity of HMGA2 versus GAPDH (HMGA2/GAPDH) was shown underneath each condition. B) Comparison of gefitinib‐resistant colony formation between PC9/GR cells stably expressing the control or HMGA2 overexpression plasmid. PC9/GR cells were cultured in 5 or 10 μm gefitinib. The number of colonies is quantified in the bar graph on the right side. Each condition was repeated in triplicates. This is true for all the rest of the experiments. C) Quantification of HMAG2 knockdown level in PC9/GR cells stably expressing the control (sh‐Control) or shRNA targeting HMGA2 (sh‐HMGA2, #1 and #2) by RT‐qPCR. D) Comparison of gefitinib‐resistant colony formation between PC9/GR cells stably expressing sh‐Control or sh‐HMGA2 #1, cultured in 5 or 10 μm gefitinib. E) WB validation of flag tagged ALOX5 overexpression in PC9/GR cells stably expressing the control (Control OE) or ALOX5 overexpression (Flag‐ALOX5 OE) plasmid. F) Comparison of gefitinib‐resistant colony formation between PC9/GR cells stably expressing the control or ALOX5 overexpression plasmid. G) Quantification of ALOX5 knockdown level in PC9/GR cells stably expressing the control (sh‐Control) or shRNA targeting ALOX5 (sh‐ ALOX5, #1 and #2) by RT‐qPCR. H) Comparison of gefitinib‐resistant colony formation between PC9/GR cells stably expressing sh‐Control or sh‐ ALOX5 #1. I) Comparison of gefitinib‐resistant colony formation between PC9/GR cells treated with the control (DMSO) or 200 um Zileuton. J) WB detection of LC3B II (autophagy marker) expression in PC9/GR cells overexpressing control (Control OE) or HMGA2 (HMGA2 OE) plasmid at 1 or 4 days. K) WB detection of LC3B and ALOX5 expression in PC9/GR cells overexpressing the control shRNA (sh‐Control) or shRNA targeting ALOX5 (sh‐ALOX5, #1 and #2).