Fig. 7: FAM122A is required for cell cycle entry and progression through the G1 phase of the cell cycle following mitogen stimulation.

A FAM122A-WT and -KO T98G cells were serum starved for 60 h and re-stimulated with DMEM supplemented with 10% FBS and collected at the indicated time points and analyzed by PI/FACS. Nocodazole was added to prevent cells from progressing beyond mitosis. A >4 h delay is observed by the time the cells reach mitosis, but the delay is already noticeable at the G1/S border. B A serum starvation-restimulation experiment focused on the G1/S transition (1 h time points) that shows a delay in cells entering in S-phase. C T98G Parental and KO cells were serum-starved for 72 h and subject to serum-restimulation. 10 μM EdU was added 1 h prior to collection. The cells were fixed with 4% PFA in PBS, washed, and subject to saponin-based permeabilization followed by the Cy5-azide click-chemistry reaction and stained with PI/FACS analysis. The percentage of cells in G1 is indicated, suggesting delays prior to the onset of S-phase. D Delays in the expected modulation of pRB proteins and the expression of E2F-depedent gene products (Cyclins E and A and p107). The earliest defect is the limited expression of cyclin D1, whose expression is regulated by mitogens. Experiment performed in biological triplicate. E The effects of FAM122A KO in the activation/inactivation of key components of the major mitogenic signaling pathways regulating cyclin D1 expression in serum-starved and restimulated T98G during early signaling (AKT, ERKs, GSK3β and their relevant phosphoforms are indicated). Experiment performed in biological triplicate. Source data are provided as a Source Data file.