Fig. 6: PCYT2 is less active and dephosphorylated in senescent cells.
a, Schematic overview of the phosphatidylethanolamine pathway highlighting pEtN and the enzymes involved in the pathway. b, Curves of decay of labelled pEtN or CDP-Etn in WI38 fibroblasts proliferating or undergoing DDIS (day 10), after a pulse of 1 h followed by a chase for the indicated times. n = 3 biological replicates. c, Representative western blots of a Phos-tag gel (top) and a conventional gel (bottom) showing the indicated protein levels in extracts from WI38 fibroblasts proliferating, undergoing DDIS (14 days) or RAS OIS (7 days). The phosphatase-treated extract is from proliferating cells. Loading control (actin) was migrated into the same gel as RB. d, Representative western blots of a Phos-tag gel (top) and a conventional gel (bottom) showing the indicated protein levels in extracts from WI38 fibroblasts proliferating or undergoing RAS OIS and non-transfected (-) or transfected with a non-silencing siRNA or an siRNA targeting the p53 mRNA for 3 days. The phosphatase-treated extract is from proliferating cells. Dashed lines indicate the cropping of two lanes. Sample processing control (actin) was migrated into a different gel than p21. The experiment was repeated independently twice with similar results. e, Fold change of pEtN:CDP-Etn ratio in WI38 fibroblasts undergoing RAS OIS and infected with shCtrl-OE or shp53-OE adenoviruses for 7 days, relative to the value of non-infected cells (Prolif.). n = 3 biologically independent experiments. f, Representative western blots of a Phos-tag gel (top) and a conventional gel (bottom) showing the indicated protein levels in extracts from WI38 fibroblasts treated by Nutlin-3 (10 µM) for the indicated times. The phosphatase-treated extract is from proliferating cells. The experiment was repeated independently twice with similar results. g, FC of pEtN:CDP-Etn ratio in WI38 fibroblasts treated by Nutlin-3 (10 µM) for 7 days. n = 3 biologically independent experiments. h, Representative western blots of a Phos-tag gel (top) and a conventional gel (bottom) showing the indicated protein levels in extracts from WI38 fibroblasts treated with BisIndo.I at the indicated concentrations for 16 h. Dashed lines indicate the cropping of one lane. For b,e,g, data are presented as mean ± s.d. All the indicated P values were calculated using an unpaired two-sided Student’s t-test. For c,h, the experiment was repeated independently three times with similar results.
