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. 2021 Sep 28;10(10):1543. doi: 10.3390/antiox10101543

Figure 3.

Figure 3

Cystine and methionine deficiency induces BTG1 by activating ATF4. HepG2 and L-O2 cells were exposed to CST/Met (−) for 1–12 (a,d), or 24 h (b,c,e,f). (a) The level of BTG1 mRNA was determined by qPCR. Fer-1 (10 μM, 12 h) or ISRIB (1 μM, 12 h) was simultaneously applied to HepG2 cells under CST/Met (−) (left). (b) BTG1 protein. Open arrowheads and dashed lines in immunoblot images indicate nonspecific bands and cropped images of the same membrane, respectively (upper). (c) BTG1 transactivation. Schematic illustration of constructed reporter plasmids containing the human and murine BTG1 gene promoter (upper). An ATF4 expression plasmid was co-transfected with hpBTG1-luc, mpBTG1-luc, or mpBTG1-ΔA4RE-luc. pCDNA3.2/V5-DEST was used for mock transfection (lower). (d–f) Effect of siATF4 on BTG1 induction. A siRNA targeting human ATF4 (d,e) or murine ATF4 (f) was transfected into HepG2 cells, and expression of BTG1 (d,e) and transactivation (f) were determined by qPCR, immunoblot, and reporter gene assays, respectively. ** p < 0.01, * p < 0.05, versus control (a,b,d,e) or mock transfection (c,f); ## p < 0.01, # p < 0.05, versus HepG2 cells exposed to CST/Met (−) (a,d,e) or ATF4-transfected cells (c,f); †† p < 0.01, between basal level of ATF4 mRNA (d): A4RE, putative ATF4 response element; Con, control.