Figure 3
ATF4 induction is not due to an increase in ATF4 mRNA or ATF4 protein stability. (A) Structure of the human ATF4 gene. Large and small arrows indicate transcription and translation start sites, respectively. Functional ATF4 protein is translated from the small red arrow. Black, grey and slashed boxes indicate coding, intron and 5′ non-coding sequences, respectively. Arrowheads indicate positions of the primers used for RT-qPCR. (B) RT-qPCR analysis. Total RNA from HCT116 treated with 30 µM GSK-J4 or 2 µg/ml Tm for 30, 90 or 240 min were analyzed by RT-qPCR with biological triplicates (N = 3). Values were normalized against PPIA mRNA. Bars and asterisks indicate S.E.M. and significance, respectively, as in Fig. 1A. (C) CMV promoter-driven F-ATF4 was expressed from a lentivirus vector in HCT116. After 4 h exposure, the culture medium was replaced with fresh medium with or without 30 μM GSK-J4 and cultured for 20 h. ATF4 proteins were detected with α-ATF4 (upper panel) or α-Flag antibodies (middle panel). Endogenous and exogenous ATF4 are indicated by blue and red bars, respectively. (D) Effect of protein synthesis inhibition. HCT116 cells were treated at the indicated concentrations of cycloheximide (CHX) for 1 h prior to incubation with 30 μM GSK-J4 or 2 µg/ml Tm for 4 h.
