FOS基因敲除Hela细胞

The stress-activated protein kinase (SAPK) family consists of three c-Jun N-terminal kinase (JNK) and four p38 members. To explore the isotype-specific or overlapping roles of SAPK members, HeLa-derived multiplex SAPK-KO cells, such as JNK1/2/3-triple KO, p38α/β/γ/δ-quadruple KO, and JNK1/2/3/p38α/β/γ/δ-septuple KO cells, were generated using the CRISPR-Cas9 method. Also, "sole survivor" (ss)-hextuple KO cells, in which only one of seven SAPK genes remains intact, were generated. Western blot analyses using phospho-specific antibodies for SAPK substrates showed that serum- or anisomycin-induced phosphorylation of MAPKAPK2, MSK1, Mnk1, and CREB (cyclic AMP response element-binding protein)/ATF-1 largely depended on p38, whereas anisomycin-induced phosphorylation of c-Jun/JunD depended on JNK. Similar analyses using the ss-hextuple KO cell lines revealed that JNK1 rather than JNK2 contributed to the c-Jun/JunD phosphorylation, whereas p38α was the primary species phosphorylating the examined p38 substrates. Expression analyses of three typical immediate-early genes, c-Jun, EGR1, and c-Fos, demonstrated that JNK1 and JNK2 are responsible for c-Jun expression induced by interleukin-1β, tumor necrosis factor-α, UV-C, and heat shock (HS), whereas p38 is predominant in EGR1 expression induced by UV and HS and in c-Fos expression induced by the cytokines, UV, and HS. On the other hand, neither JNK nor p38 contributed significantly to the cytokine-induced EGR1 expression, suggesting complicated SAPK-signaling mechanisms that regulate immediate-early gene expression. Together, these results demonstrate the utility of the comprehensive multigene KO and ss-KO strategy in dissecting intracellular signaling pathways consisting of multiple family members.