Extended Data Fig. 6: PCK1-mediated INSIG1/2 phosphorylation is required for IGF1- induced SREBP activation for lipogenesis and does not affect the lipid depletion-induced translocation of SCAP from the ER to the Golgi apparatus.
From: The gluconeogenic enzyme PCK1 phosphorylates INSIG1/2 for lipogenesis
a, Parental Huh7 (left) and Hep3B (right) cells and the indicated clones with knock-in expression of PCK1(S90A) were stimulated with or without IGF1 (100 ng ml−1) for 16 h. Immunoblotting analyses were performed as indicated. b, Parental Huh7 cells and the indicated clones of Huh7 cells with knock-in expression of PCK1(S90A) were stimulated with or without IGF1 (100 ng ml−1) for 16 h. Immunofluorescence analyses were performed as indicated. Scale bar, 20 μm. c, Parental Huh7 cells and the indicated clones with knock-in expression of PCK1(S90A) were transiently transfected with vectors expressing β-galactosidase and an SRE-driven luciferase reporter and stimulated with or without IGF1 (100 ng ml−1) for 16 h. The relative SRE luciferase activity after normalization to β-galactosidase activity is shown (n = 6). Data are mean ± s.d. **P < 0.001 (two-tailed t-test). d, Parental Huh7 cells and the indicated clones with knock-in expression of PCK1(S90A) were stimulated with or without IGF1 (100 ng ml−1) for 16 h. The mRNA expression levels for SREBP target genes were measured using quantitative PCR (n = 6). Data are mean ± s.d. **P < 0.001 (two-tailed t-test). e, Parental Huh7 (left) and Hep3B (right) cells and the indicated clones with knock-in expression of PCK1(S90A) were stimulated with or without IGF1 (100 ng ml−1) for 16 h. Immunoblotting analyses were performed as indicated. f, Parental Huh7 cells and the indicated clones with knock-in expression of INSIG1(S207A)/INSIG2(S151A) double mutants (left) or PCK1(S90A) (right) were stimulated with or without IGF1 (100 ng ml−1) for 16 h. The mRNA expression levels for SREBF1 were measured using quantitative PCR (n = 6). Data are mean ± s.d. *P = 0.002, **P < 0.001 (two-tailed t-test). g, Parental Huh7 cells and the indicated clones with knock-in expression of PCK1(S90A) were stimulated with or without IGF1 (100 ng ml−1) for 16 h. The incorporation of 14C-glucose into triglycerides (left) and fatty acids (right) was measured (n = 6). Data are mean ± s.d. and were compared between groups using a two-tailed t-test. *P = 0.014, 0.012 (left to right); **P = 0.004, 0.001 (left to right). h, Endogenous PCK1-depleted CHL-1 human melanoma cells, U87 human glioblastoma cells and H1993 human non-small-cell lung cancer cells with reconstituted expression of shRNA-resistant wild-type HA–rPCK1 or HA–rPCK1(S90A) were stimulated with or without IGF1 (100 ng ml−1) for 16 h. The incorporation of 14C-glucose into fatty acids was measured (n = 6). Data are mean ± s.d. and were compared between groups using a two-tailed t-test. *P = 0.011, **P < 0.001. i, j, Parental Huh7 or Hep3B cells and the indicated clones with expression of PCK1(S90D) (i) or INSIG1(S207D)/INSIG2(S151D) double mutants (2S/D) (j) were collected for immunoblotting analyses as indicated. k, l, Parental Huh7 cells and the indicated clones with knock-in expression of PCK1(S90A) (k) or INSIG1(S207A)/INSIG2(S151A) (l) were stimulated with or without IGF1 (100 ng ml−1) for 16 h. Immunoblotting analyses were performed as indicated. m, n, Parental Huh7 cells and the indicated clones with knock-in expression of PCK1(S90A) (m) or INSIG1(S207A)/INSIG2(S151A) (n) were stimulated with or without IGF1 (100 ng ml−1) for 16 h. The mRNA expression levels for SREBP2 target genes were measured using quantitative PCR (n = 6). Data are mean ± s.d. **P < 0.001 (two-tailed t-test). o, p, Parental Huh7 cells and the indicated clones with knock-in expression of INSIG1(S207A)/INSIG2(S151A) double mutants were incubated with a lipid-depleted medium and treated with lovastatin (an inhibitor of HMGCR) to inhibit cholesterol synthesis for 16 h. The cells were subjected to homogenization and cell fractionation using gradient centrifugation. Immunoblotting analyses were performed as indicated (o). The relative distribution of each protein in different fractions was quantified by densitometric analysis of the blots (n = 3) (p). Data are mean ± s.d. q, r, Parental Huh7 cells and the indicated clones with knock-in expression of PCK1(S90A) were incubated with or without lipid-depleted medium for 16 h. The cells were subjected to homogenization and cell fractionation using gradient centrifugation. Immunoblotting analyses were performed as indicated (q). The relative distribution of each protein in different fractions was quantified by densitometric analysis of the blots (n = 3) (r). Data are mean ± s.d. s–x, Parental Huh7 cells and the indicated clones with knock-in expression of INSIG1(S207A)/INSIG2(S151A) double mutants (s, v) or PCK1(S90A) (t, w) were incubated with or without lipid-depleted medium for 16 h. Immunofluorescence analyses were performed as indicated. Scale bars, 20 μm. The white arrows indicate the Golgi-localized SCAP. The colocalization coefficients between SCAP and the ER marker calnexin (u) and the Golgi apparatus marker golgin-97 (x) are shown. At least n = 50 cells from each independent experiment were analysed and representative data are shown. Data are mean ± s.d. and were compared between groups using a two-tailed t-test. NS, not significant (P = 0.788, 0.514, 0.689, 0.693 (left to right) (u); P = 0.976, 0.606, 0.750, 0.940 (left to right) (x)). All experiments were repeated three times independently with similar results.
