Extended Data Fig. 5: CLOCK-mediated PRPS1/2 stabilization promotes de novo nucleotide synthesis.

(g-l) Immunoprecipitation and immunoblotting with the indicated antibodies was performed. All experiments were repeated at least twice independently. (a-g) Data are the mean ± SD, n = 6, *P < 0.01; **P < 0.001; ***P < 0.0001; N.S., not significant by One-way ANOVA post hoc test. (a-f) The indicated cells expressing CLOCK shRNA with reconstituted expression of the indicated CLOCK proteins (a, b, e) or the indicated clones with knock-in expression of PRPS1/2 K29R (c, d, f) were transfected with or without active IGF1R-CA, followed by labeling with D-[6-¹⁴C] glucose for 30 min. The amounts of ¹⁴C-RNA (a, c) and ¹⁴C-DNA (b, d) were measured. The ¹³C-labeled PRPP, IMP, AMP, GMP, UMP, and CMP were measured by LC/MS-MS (e, f). (g) Purified GST–CLOCK was incubated with or without His-CK2α in the presence of ATP for an in vitro kinase assay, followed by incubation with or without the indicated His-PRPS1/2 proteins in the presence of acetyl-CoA. The PRPS1/2 activity was measured. (h, i) Huh7 cells expressing CLOCK shRNA with reconstituted expression of the indicated Flag-rCLOCK were serum-starved for 12 h in the presence of CQ and then treated with or without EGF (h) or FGF1 (i) for 1 h. Cytosolic and whole cell lysates were harvested. (j) Huh7 cells were stimulated with EGF (100 ng/ml) or FGF1 (25 ng/ml) for the indicated time. (k) Huh7 cells stably transfected with constitutively active EGFR-vIII or FGFR1-CA were harvested. (l) Huh7 cells expressing CLOCK shRNA and constitutively active EGFR-vIII or FGFR1-CA with reconstituted expression of the indicated Flag-rCLOCK were treated with CHX for the indicated time. The quantification of PRPS1/2 protein levels is shown. Data are the mean ± SD, **P < 0.001 by One-way ANOVA post hoc test.

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