Extended Data Fig. 6: Overexpressed CK2α in HCC cells is critical for CLOCK-enhanced PRPS1/2 stability.
(a, b, d-l) Immunoprecipitation and immunoblotting with the indicated antibodies was performed. All experiments were repeated three times independently. (c, d, g, i) Data are the mean ± SD. n = 6, *P < 0.01; **P < 0.001; ***P < 0.0001 by One-way ANOVA post hoc test. (a, b) The indicated cells expressing CLOCK shRNA with reconstituted expression of the indicated shRNA-resistant CLOCK were constructed (a) and serum-starved for 12 h in the presence of CQ before treatment with or without IGF1 for 1 h (b). Cytosolic and whole cell lysates were harvested. (c) The indicated cells with reconstituted expression of the indicated CLOCK proteins were treated with or without IGF1 for 12 h, followed by labeling with D-[6-14C] glucose for 30 min. The amounts of 14C-RNA and 14C-DNA were measured. (d) The indicated cells expressing CLOCK shRNA and IGF1R-CA with reconstituted expression of Flag-rCLOCK were treated with CHX for the indicated time. The quantification of PRPS1/2 levels is shown. (e, f) The indicated cells were treated with or without IGF1 for 1 h. Whole cell lysates were harvested. (g) L02 cells transfected with the indicated plasmids were treated with CHX for the indicated time in the presence or absence of IGF1. The quantification of PRPS1/2 levels is shown. (h) Huh7 cells transfected with the indicated shRNA were treated with or without IGF1 for 1 h. (i) Huh7 cells transfected with the indicated shRNA were treated with CHX for the indicated time in the presence or absence of IGF1. The quantification of PRPS1/2 levels is shown.
