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Fig. 2 | Molecular Cancer

Fig. 2

From: Cholesterol promotes EGFR-TKIs resistance in NSCLC by inducing EGFR/Src/Erk/SP1 signaling-mediated ERRα re-expression

Fig. 2

Cholesterol-mediated ERRα overexpression is responsible for EGFR-TKIs resistance. a, b Results of RT-qPCR and Western blot assay showed ERRα mRNA and protein level in indicated cells. c IHC staining detected ERRα expression in tumors. d Cells were treated with 2.5 mM MβCD, 10 µM cholesterol or 2.5 mM MβCD +10 µM cholesterol for 6h, then ERRα mRNA level in indicated cells was determined by RT-qPCR assay. e Cells were treated with 2.5 mM MβCD, 10 µM cholesterol or 2.5 mM MβCD +10 µM cholesterol for 24h, then ERRα protein level in indicated cells was determined Western blot assay. f Cells were treated with gefitinib, osimertinib alone or combined with XCT790 for 48 h. Then MTT assay was conducted. g Cells were cultured in medium with XCT790 for 48 h, then fixed with cold ethanol overnight, incubated with PI/RNase staining buffer (550825, BD, Franklin Lake, NJ, USA) for 15 min. Cell cycle distribution was analyzed by flow cytometry. h Cells were treated with XCT790 for 48 h and then incubated with DCFH-DA (S0033S, Beyotime Biotechnology, Shanghai, China) for 30 min. Confocal images showed the ROS in green and the fluorescence intensity was analyzed. i Cells were treated with XCT790 for 48 h and then fixed and incubated with 5 μM MitoSOX Red Mitochondrial Superoxide Indicator (40778ES50, Yeasen, Shanghai, China) for 10 min. Confocal images showed the MitoSOX in green and the fluorescence intensity was analyzed. j, k Flow cytometry analysis was conducted to determine the ROS and MitoSOX accumulation in indicated cells. Data are expressed as mean ± SEM (n = 3) *p < 0.05, **p < 0.01, ***p < 0.001 in a, b, h, i, j and k. Data are expressed as mean ± SEM (n = 3) *p < 0.05, **p < 0.01, ***p < 0.001 compared to control; $p < 0.05, $$p < 0.01, $$$p < 0.001 compared to MβCD in d and e

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