Fig. 5. Activation of STAT3 in TRCs promotes PCK2 degradation.
a, Immunoblots showing the expression of PCK2 in HONE1 TRCs and bulk tumor cells transfected with indicated siRNAs. b, RT–qPCR analysis of PCK2 expression in HONE1 or A375 TRCs and bulk cells with STAT3 knockdown. c, Inhibition STAT3 with Stattic (5 µM) stabilized PCK2 protein in HONE1 TRCs with CHX treatment for indicated times. The immunoblot analysis of PCK2 protein level (left) and corresponding grayscale analysis (right) are shown. P = 0.00000042 and 0.000002. d, Immunoblot analysis showing PCK2 protein level in HONE1 TRCs and bulk cells treated with MG132 (10 μM), carfilzomib (100 nM), bafilomycin A1 (Baf A1, 0.2 μM) or chloroquine (CQ, 50 μM) for 6 h. e, HONE1 TRCs and bulk cells treated with or without Stattic (5 µM), after which the cell lysates were subjected to immunoprecipitation with anti-PCK2 antibody and immunoblotting with anti-ubiquitin (Ub) antibody. f, RT–qPCR analysis of HERC6 expression in HONE1 or A375 TRCs and bulk cells with STAT3 knockdown. P = 0.0000001, 0.0000003, 0.00000037, 0.000000003, 0.0000000067 and 0.0000000077. g, Immunoblots showing the expression of PCK2 in TRCs and bulk tumor cells with or without HERC6 knockdown. h,i, TRCs from STAT3-knockout HONE1 cells or parental cells and bulk HONE1 cells were subjected to indicated treatments (h). TRCs from HERC6-knockout HONE1 cells or parental cells and bulk HONE1 cells were subjected to indicated treatments (i). RSL3, 15 µM; radiation (IR), 8 Gy; cisplatin (Cis), 20 µM. The percentage of dead cells was measured. j, HONE1 cells were implanted subcutaneously into female BALB/c nude mice and were locally exposed to radiotherapy (8 Gy, arrow) or not. Mice were provided with Stattic (3 mg kg−1, every 2 d, starting from ‘arrowhead’ until the endpoint) or not. Tumor volumes for each group are shown (n = 5). P = 0.00002 and 0.0075. Data are shown as mean ± s.d. (b,f,h,i) or mean ± s.e.m. (j); one-way ANOVA (b,f,h,i) or two-way ANOVA (j). n = 3 independent experiments. One of three experiments is shown (a,c–e,g) CHX, cycloheximide.