Extended Data Fig. 8: PCK1-mediated phosphorylation of INSIG1 Ser207 and INSIG2 Ser151 and SREBP1 activation are induced by oncogene- or growth-factor-mediated activation of AKT in several cancer types.

a, b, e–l, Cells were transfected with the indicated plasmids and immunoprecipitation or immunoblotting analyses were performed with the indicated antibodies. a, Endogenous PCK1-depleted HL7702 (left) and THLE-2 (right) cells with reconstituted expression of shRNA resistant wild-type HA–rPCK1 or HA–rPCK1(S90A) were treated with or without IGF1 (100 ng ml⁻¹) for 16 h. b, Endogenous PCK1-depleted HL7702 (left) and THLE-2 (right) cells with reconstituted expression of shRNA resistant wild-type HA–rPCK1 or HA–rPCK1(S90D) were analysed. c, C57BL/6J male mice (eight-week-old) fasted for 24 h were intraperitoneally injected with or without glucose (1 g per kg body weight). After 6 h, the mouse livers were dissected for immunoprecipitation and immunoblotting analyses. d, HL7702, THLE-2, Huh7 and Hep3B cells were analysed by Ni-NTA pull-down, immunoprecipitation and immunoblotting assays. e, Endogenous PCK1-depleted HL7702, THLE-2, Huh7 and Hep3B cells with reconstituted expression of shRNA resistant wild-type HA–rPCK1 or HA–rPCK1(S90A) were analysed. f, Parental Huh7 cells and the indicated clones with knock-in expression of PCK1(S90A) (left) or INSIG1(S207A)/INSIG2(S151A) double mutants (right) were transfected with or without Flag–KRAS(G12V). g, Endogenous PCK1-depleted CHL-1, U87 and H1993 cells with reconstituted expression of shRNA resistant wild-type HA–rPCK1 or HA–rPCK1(S90A) were transfected with the indicated plasmids. h, Parental Huh7 cells and the indicated clones with knock-in expression of PCK1(S90A) (left) or INSIG1(S207A)/INSIG2(S151A) double mutants (right) were transfected with or without constitutively active Flag–IGF1R(V922E). i, Endogenous PCK1-depleted CHL-1, U87 and H1993 cells with reconstituted expression of shRNA resistant wild-type HA–rPCK1 or HA–rPCK1(S90A) were transfected with the indicated plasmids. j, Endogenous PCK1-depleted CHL-1, U87 and H1993 cells with reconstituted expression of shRNA resistant wild-type HA–rPCK1 or HA–rPCK1(S90A) were transfected with the indicated plasmids and treated with or without PDGF (30 ng ml⁻¹) for 16 h. k, m, Endogenous PCK1-depleted Huh7, Hep3B, SNU-398 and SNU-475 cells with reconstituted expression of shRNA resistant wild-type HA–rPCK1 or HA–rPCK1(S90A) were analysed by immunoprecipitation and immunoblotting analyses with the indicated antibodies (k). The cells were plated and then collected and counted for 3 days (n = 6) (m). Data are mean ± s.d. **P < 0.001 (two-tailed t-test). l, n, Endogenous PCK1-depleted Huh7, Hep3B, SNU-398 and SNU-475 cells with reconstituted expression of shRNA resistant wild-type HA–rPCK1 or HA–rPCK1(S90A) were transfected with Flag–INSIG1 and His–INSIG2. After incubation with or without lipid-depleted medium for 16 h, the cells were collected for Ni-NTA pull-down, immunoprecipitation and immunoblotting analyses as indicated (l). Viable cells were counted for 3 days after lipid depletion (n=6) (n). Data are mean ± s.d. NS, not significant (P = 0.708, 0.619, 0.888, 0.901, 0.633, 0.788, 0.902, 0.764 (left to right)). All experiments were repeated three times independently with similar results.

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