Figure 3. Senescence bypass by ATM knockdown correlates with an enhanced glucose and glutamine consumption and a metabolic shift towards the pentose phosphate pathway through an increase in G6PD activity. See also Figure S3.

(A) Primary IMR90 cells were infected with a shRRM2-expressing lentivirus alone or in combination with a shATM-expressing lentivirus, and glucose uptake was determined by incubating cells with a fluorescent glucose analog (2NBDG) followed by flow cytometry (n=3). Cells were gated for high glucose uptake based on fluorescence. (B–C) Same as (A). Media was harvested, and glucose consumption and lactate production (B) or glutamine consumption and glutamate production (C) were quantified (n=3). (D–G) Same as (A). Cells were subjected to the following analysis: liquid chromatography followed by mass spectrometry (LC-MS), and shown are the relative 6-phosphogluconate (6-PG) levels normalized to cell number (n=3) (D); [¹³C₆]-glucose labeling for 30min. Shown is the percent of ¹³C-labeled 6-PG (n=3) (E); glucose-6-phosphate dehydrogenase (G6PD) activity (n=3) (F); and immunoblotting of G6PD, p53 and β-actin (G). Error bars represent SD. (H) Melanoma cells with known p53 status were infected with a shRRM2-expressing lentivirus alone or in combination with a shATM (#1)-expressing lentivirus, and RRM2, ATM, G6PD, and β-actin protein expression was determined by immunoblotting. (I) Same as (H), but G6PD activity was determined (n=3). (J) Same as (H) but cells were examined for SA-β-Gal activity. Scale bars = 10μm. (K) Quantification of (J). 100 cells from each of the indicated groups were quantified for SA-β-Gal positivity (n=3). *p<0.05 shRRM2 vs. control; #p<0.05 shRRM2/shATM vs. shRRM2. Error bars represent SEM unless otherwise indicated.