Extended Data Fig. 5: Regulation of cyclin D1 transcription requires CREB1/ATF1 and ETS1 transcription factors.
From: Cytosolic pH regulates proliferation and tumour growth by promoting expression of cyclin D1
(a–c) An elevated pHc is required for cyclin D1 expression. (a) HFF-1 cells were starved and cyclin D1 expression was determined by western blotting 6 hours after stimulation with the indicated conditions. (b) RPE-1 cells were grown and cyclin D1 and phosphorylated RB was determined by western blotting upon treatment with DMA at the indicated time points. (c) RPE-1 cells were subjected to siRNA against NHE1 and cyclin D1 abundance was determined after 72 hours by western blot (d and e) CDK4cyclin D1 activity is required for G1/S progression. Cells were treated as in Fig. 1f and cell-cycle progression was determined by (d) western blotting and (e) FACS analysis and upon treatment with the CDK4cyclin D1 inhibitor Ribociclib at the indicated times. (f) pHc regulates cyclin D1 transcription rather than mRNA stability. Cells were treated with the indicated inhibitors for 4 hours and relative mRNA levels of cyclin D1 were determined (mean ± S.E.M. of independent experiments N=4, two-sided t-Test) (g) CREB1, ATF1 and ETS1 are required for cyclin D1 expression. Cells were transduced with Lentiviral particles expressing shRNA constructs against the indicated genes and expression of transcription factors and cyclin D1 protein levels were determined by western blotting. For all western blots in this figure, blots representative of three independent experiments are shown.
