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. 2021 Nov 5;12(11):1052. doi: 10.1038/s41419-021-04329-9

Fig. 4. STEAP3 regulates RAC1 to activate ERK-STAT3/JNK-STAT6 signaling axes to promote cell proliferation in HCC.

Fig. 4

A Phosphorylation levels of STAT3 and STAT6 were examined by western blot. B Nuclear translocation of p-STAT3 Ser727 and p-STAT6 Tyr641 was verified by IF in PLC/PRF/5-STEAP3 and control cells. Nuclei were visualized by DAPI (blue). Scale bar, 10 μm. C PLC/PRF/5-STEAP3 and control cells were starved for 24 h, then supplemented with serum and administrated with different doses (2.5, 5, 7.5, 10 μM) of STAT3 and STAT6 inhibitor, C188-9 and AS1517499, respectively, for 2 h. Scale bar, 200 μm. Cells were fixed, and visualized by crystal violet. D Cell viability was examined by CCK8 assays in PLC/PRF/5-STEAP3 treated with C188-9 (0.5 μM) and AS1517499 (1 μM), respectively. Cells administrated with DMSO were used as a control. Data are presented as means ± SD of three independent experiments. **p < 0.01. E Phosphorylation levels of STAT3 Ser727 and STAT6 Tyr641 were examined by western blot after U0126 and SP600125 administration for 0.5 and 1 h, respectively, in PLC/PRF/5-STEAP3. F mRNA levels of NANOG, MCL1, IL-8 and IL-18, were examined by real-time RT-PCR in PLC/PRF/5-STEAP3 after administration with C188-9 and AS1517499, respectively, and presented as means ± S.D. of three independent experiments. G Expression levels of Rho GTPase family members (RAC1/2/3, RAC1, RhoA, RhoB, and RhoC) were examined by western blot in PLC/PRF/5-STEAP3 and control cells. H Phosphorylation levels of ERK1/2, JNK, STAT3 Ser727, and STAT6 Tyr641 were examined by western blot in PLC/PRF/5-STEAP3 after RAC1 inhibition by EHop-016 (5 μM) for 0.5 and 1 h.