(A) Potential activated kinases predicted by a kinase-substrate phosphorylation network are depicted. Dots represent previously unstudied kinases in ESCC, and triangles represent kinases implicated in ESCC based on previous literature. The color denotes the calculated P value for each kinase, and the size of the shapes corresponds to their enrichment ratios. (B) Kinase-phosphosubstrate regulation networks are depicted for selected kinases and their substrates, with orange circles representing kinases and blue circles representing their corresponding substrates. (C) Cell proliferation at 72 hours was evaluated by MTT assay after knockdown of the indicated kinases in KYSE30 cell (n = 6 for each group). (D and E) Cell proliferation measured by MTT assay (n = 6 for each group) (D) and soft agar assay (n = 12 for each group) (E) after CSNK2A1 knockdown. Scale bar: 200 μm. (F and G) Cell proliferation measured by MTT (n = 6 for each group) (F) and soft agar (n = 12 for each group) (G) assays after CSNK2A1 overexpression. Scale bar: 200 μm. (H–K) KYSE30 and KYSE450 cells stably expressing scramble or shCSNK2A1 were s.c. injected into the right flank of each mouse (KYSE30: scramble, n = 12; sh1, n = 10; sh2, n = 10; KYSE450: scramble, n = 12; sh1, n = 11; sh2, n = 10). Tumors were excised at the end of the experiment. Images of xenograft tumors are shown in H, while I presents xenograft tumor growth curves data from mice experiments; J displays tumor weights; and K represents tumor inhibition ratios. In all statistical plots, data were expressed as the mean ± SD. Two-tailed Student’s t test (F and G) and 1-way ANOVA (C–E, I, and J) were used to determine statistical significance. *P < 0.05, **P < 0.01, ***P < 0.001. Representative results from at least 3 independent biological replicates (C–G) are shown.