Fig. 4

Cholesterol-induced EGFR/Src/Erk signaling reactivation sustains ERRα re-expression. a, b Western blot and RT-qPCR assay showed ERRα expression after treatment with inhibitors of mTOR, STAT3, NF-κB, Smad3, ROCK, EGFR, Erk or Src. c Cells were treated with 1 μM gefitinib or 0.1 μM osimertinib for 48 h, Western blot assay showed the proteins level. d Cells were treated with gefitinib, osimertinib, lapatinib, WH-4-023 or SCH772984 for 48 h, Western blot assay showed the proteins level. e Proteins in lipid rafts was extracted. Western blot assay showed EGFR and Src protein level in 20% non-lipid rafts fractions and lipid rafts fractions. f Cells were harvested after treatment with 10 mM MβCD for 45 min or 10 mM MβCD for 45 min + 10 μM cholesterol for another 2 h. Proteins in lipid rafts were extracted and EGFR, Src, Caveolin1 expression was detected. g Co-immunoprecipitation analysis showed the EGFR and Src interaction. h Cells were incubated with 1 μg/mL CTB for 1 h after treatment with MβCD or MβCD+ cholesterol. Confocal images showed the lipid rafts in green and the fluorescence intensity was analyzed. i Immunofluorescence staining of EGFR (green) and Src (red) markers in the indicated NSCLC cells after treatment with MβCD or MβCD + cholesterol. j Cells were treated with MβCD or MβCD + cholesterol then subsequently with gefitinib or osimertinib for 48 h, proteins levels were detected. Data are expressed as mean ± SEM (n = 3) *p < 0.05, **p < 0.01, ***p < 0.001 in a, b, c, d and e. Data are expressed as mean ± SEM (n = 3) **p < 0.01, ***p < 0.001 compared to control; ^$p < 0.05, ^$$p < 0.01, ^$$$p < 0.001 compared to MβCD in f, g, h and i. Data are expressed as mean ± SEM (n = 3) *p < 0.05, **p < 0.01, ***p < 0.001 compared to gefitinib or osimertinib; ^$p < 0.05, ^$$p < 0.01, ^$$$p < 0.001 compared to gefitinib + MβCD or osimertinib + MβCD in j