Extended Data Fig. 8. STAT3 promotes PCK2 degradation via HERC6- dependent proteasome pathway in TRCs.
a, The indicated mRNA expression was analyzed by qRT-PCR for genes knockdown efficiency assessment in HONE1 cells. P = 0.0000005, 0.0000006, 0.0000005, 0.00000058, 0.00000028, 0.00000016, 0.000000025 and 0.000000019. b, Immunoblots showing the expression of PCK2 in HONE1 cells treated with or without STAT3 inhibitor (Stattic). c, Volcano plot showing upregulation of seven E3 ligases in HONE1 TRCs. d, Quantitative RT-PCR analysis of the relative mRNA expression of indicated genes in HONE1 TRCs and bulk cells treated with or without Stattic. P = 0.168, 0.168, 0.00000057, 0.0000016, 0.267, 0.987, 0.999, 0.818, 0.536, 0.872, 0.581, 0.913, 0.000006, 0.00002. e, STAT3-binding elements on the promoters of HERC6 and TRIM47 genes were predicted by JASPAR. f, qRT-PCR analysis of TRIM47 expression in HONE1 or A375 TRCs and bulk cells with STAT3 knockdown. P = 0.0000000047, 0.000000017, 0.000000014, 0.00000011, 0.00000034 and 0.00000029. g, Immunoprecipitation and immunoblot analysis showing the binding of PCK2 to HERC6 in HEK293T cells transfected with Flag- PCK2 and HA-HERC6. h, Immunoprecipitation and immunoblot analysis showing that PCK2 does not bind to TRIM47 in HEK293T cells that were transfected with Flag-PCK2 and HA-TRIM47. i, HERC6-knockout HONE1 cells (sg HERC6) or parental HONE1 cells (sg GFP) were cultured in 3D fibrin gels, after which the cell lysates were subjected to immunoprecipitation with anti-PCK2 antibody and immunoblotting with anti-ubiquitin (Ub) antibody. j, Immunoblots showed that knocking out HERC6 stabilized PCK2 protein in HONE1 TRCs with CHX treatment for indicated times. k, The HONE1 cells were implanted subcutaneously into female BALB/c nude mice to construct xenograft growth models. Mice were provided with Stattic (3 mg/kg, every two days, starting from ‘arrowhead’, until the endpoint) or not. Cisplatin or vehicle was intraperitoneally injected (arrow). Tumors volumes for each group were shown (n = 5). Data are shown as mean ± SD (a, d, f) or mean ± SEM (k), one- way ANOVA (a, d, f) or two-way ANOVA (k). n = 3 independent experiments. One of three experiments is shown (b, g-j).