FIGURE 7.

Dynamic ATF4 protein degradation in adaptive chemotherapy. (A) The process of drug withdraw and re‐addition for resistant cells maintaining was shown. Generally, the resistant cells were maintained in DDP free medium for 2–3 weeks to release the stress of DDP, which was named as SGC‐R wash and BGC‐R wash cells. Then DDP containing medium was used to re‐stress SGC‐R wash and BGC‐R wash cells for additional 2–3 weeks, and these cells were named as SGC‐R wash+DDP and BGC‐R wash+DDP. The sensitivity (B) and apoptosis (C) of different medium maintaining resistant cells as indicated to DDP for 24 h was measured. (D) Expression of ATF4 in resistant cells cultured with different medium as indicated was detected by western blotting. (E) Expression of ATF4 in sensitive cells (left for SGC7901 and right for BGC823) with time‐course DDP (1.2 μg/ml) treatment was determined by western blotting. (F) Interaction of Myc‐ATF4 with Flag‐βTrCP1 in HEK293T with/without DDP treatment for 12 h was determined by anti‐Flag co‐IP, and followed with anti‐Myc and anti‐Flag immunoblot. (G) The ubiquitination level of ATF4‐mediated by βTrCP1 with/without DDP treatment for 12 h was measured by in vitro ubiquitination assay. (H) Interaction of ATF4 with βTrCP1 and CK1δ in SGC7901 cells with/without DDP treatment for 12 h was determined by anti‐ATF4 co‐IP, then immunoblot with anti‐βTrCP1 and anti‐CK1δ