To prepare total cell extracts, cells were washed with 1X PBS containing 1X protease- and phosphatase-inhibitor cocktails (Roche), scratched and collected by centrifugation at low speed (140×g) for 5 min, lysed in 200 μl of SDS sample buffer (62.5 mM Tris-HCl, pH 6.8, 10% glycerol, 2% SDS and 5% 2-mercaptoethanol), and boiled for 5 min. Fifteen μg of total cell proteins were separated on SDS-PAGE and transferred to nitrocellulose membranes (Millipore). The primary antibodies used were anti-Renilla Luciferase rabbit polyclonal (1:3000, Code No. PM047, MBL), anti-H-Ras mouse monoclonal (1:250, clone F235, sc-29, Santa Cruz Biotechnology, CA, USA), anti-phospho-H3S10 rabbit polyclonal (1:1000, #06–570, Millipore, Billerica, MA, USA), anti-phospho-RBS795 rabbit polyclonal (1:500, #9301, lot: 13, Cell Signaling), anti-RB mouse monoclonal (1:1000, clone 4H1, #9309, Cell Signaling Technology), anti-MCM6 rabbit polyclonal (1:1000, A300-194A, Bethyl Laboratories), anti-p53 mouse monoclonal (1:1000, clone DO-1, sc-126, Santa Cruz Biotechnology), anti-p21 (1:500, 556431, BD Pharmingen), anti-RNR (1:500, sc-398294, Santa Cruz Biotechnology), anti-phospho-γH2A.XS139 mouse monoclonal (1:500, JBW-301, lot: 2552645, Millipore, Billerica, MA), anti-c-Myc rabbit polyclonal (1:1000, clone A-14, sc-789, Santa Cruz Biotechnology) and anti-α-tubulin mouse monoclonal (B-5-1-2, 1:20000, Sigma-Aldrich). Signals were revealed after incubation with goat anti-mouse IgG (1:3000, #170-6516, Bio-Rad, Mississauga, ON, Canada) or goat anti-rabbit IgG (1:3000, #170-6515, Bio-Rad) secondary antibodies, and by using enhanced chemiluminescence (ECL, Amersham) or Lumi-LightPLUS (Roche).

Immunofluorescence were done as described (Igelmann et al., 2021) with the following modifications for the AGO1x staining. For blocking 10% goat serum (#16210072, Life Technologies), and 1% BSA (BioShop Canada) in PBS for 30 min. Primary antibodies were AGO1x (1:50, #RBP 1510, Lucerna-Chem.) and KI67 (1:200, Cat# RM9106, ThermoFisher Scientific). Cells were analyzed using upright microscope Zen Imager with 20X air objective. Images were processed using Image J and quantification was done using region of interest (ROI) mean intensity staining. To determine ROI, DAPI staining was used.