Fig. 5

SP1 transcribes ERRα directly and cholesterol/EGFR/Src/Erk axis regulates SP1 nuclear translocation. a, b RT-qPCR and Western blot assay showed SP1 or ERRα protein and mRNA level after treatment with SP1 inhibitor (plicamycin). c Cells were transfected with empty pCDNA3.1 or pCDNA3.1-SP1 for 48 h. The protein levels of SP1 and ERRα were determined by Western blot assay. d Cells were harvested after treatment with inhibitors of EGFR (gefitinib or osimertinib or lapatinib), Erk (SCH772984), Src (WH-4-023) for 48 h. Proteins in nucleus were extracted and SP1 expression was conducted by Western blot assay. e 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, then with 1 μM gefitinib or 0.1 μM osimertinib for 48 h. Proteins in nucleus were extracted and SP1 expression was conducted by Western blot assay. f Cells were transfected with empty pCDNA3.1 or pCDNA3.1-SP1 along with inhibitors of EGFR, Erk, Src for 48 h. The protein levels of ERRα were determined by Western blot assay. g, h Luciferase assay was performed to determine ERRα promotor activity. i Binding site of SP1 was at the promoter region − 1304 to − 1290 bp. j CHIP-qPCR analysis showed that the promoter amplicons in the SP1-binding site precipitated by anti-SP1 antibody and anti-IgG antibody in H1975 cells. k CHIP-qPCR analysis was performed after cells treated with osimertinib, SCH772984, WH-4-023 for 48 h in H1975 cells. Data are expressed as mean ± SEM (n = 3) *p < 0.05, **p < 0.01, ***p < 0.001 in a, b, c, d, g, h, j and k. Data are expressed as mean ± SEM (n = 3) *p < 0.05, **p < 0.01 compared to gefitinib or osimertinib; ^$p < 0.05 compared to gefitinib + MβCD or osimertinib + MβCD in e. Data are expressed as mean ± SEM (n = 3) *p < 0.05, **p < 0.01, ***p < 0.001 compared to pCDNA3.1; ^$p < 0.05, ^$$p < 0.01, ^$$$p < 0.001 compared to pCDNA3.1 + inhibitors in f