Fig. 4
Transcriptional activation of c-Myc by ACYP2 in glioma cells through regulating intracellular Ca2+ homeostasis and calpain activity. a, Western blot analysis was carried out to determine the effect of ectopic expression of ACYP2 in SHG44 and A172 cells on the levels of transcriptionally active c-Myc and its downstream target NCL. Tubulin and GAPDH were used as loading controls. Cells transfected with the indicated shRNAs were treated with the vehicle or 10 μM calpeptin for 10 h. b, Western blot analysis was performed to evaluate their effect on protein expression of c-Myc and its target NCL. c, qRT-PCR assay was used to evaluate their effect on mRNA levels of ACYP2 and NCL. GAPDH was used as a loading control in western blot analysis. 18S rRNA was used as a reference gene in qRT-PCR assay. d, Cells transfected with the indicated shRNAs were treated with the vehicle or 5 μM BAPTA-AM for 6 h. Western blot analysis was then performed to investigate their effect on the expression of c-Myc and its downstream target NCL. GAPDH was used as a loading control. e, The indicated cells were treated with the vehicle or 100 μM 10,058-F4 for 48 h, and the MTT assay was then used to evaluate their effect on cell proliferation. The data were presented as mean ± SD (n = 3). **, P < 0.01; ***, P < 0.001