FIGURE 3.

TP53-silenced Hke3 cells with a competent KRAS display higher basal respiration and tightly-coupled mitochondria. (A) Total ATP concentration normalized to micrograms of proteins for each cell line after 80 min of nutrients starvation. All data are expressed as mean ± SEM from experimental triplicates and three independent cultures. *p ≤ 0.05 compared to WT control cells; #p ≤ 0.05 between cell lines (ANOVA, post-hoc Tukey). (B–F) Cellular oxygen consumption rate (OCR) and extracellular acidification rate (ECAR) were measured in Hke3 and Hke3 p53 KD cultured cells in ambient O₂ concentrations using a Seahorse XF96 Extracellular Flux Analyzer. Sequential injection of substrate (10 mM glucose, G) and metabolic inhibitors Oligomycin (O, 3 μM), FCCP (F, 0.5 μM) and Antimycin/Rotenone (A/R, 1 μM/1 μM) was performed at the indicated times (dashed lines) and enabled determination of bioenergetic parameters. Bioenergetic profiles (B) and basal OCR and ECAR (C) are represented. ATP synthesis and proton (H⁺) leak parameters were calculated (D) and the ratio represented as respiratory control rate (RCR; E). Basal respiration was calculated subtracting non-mitochondrial respiration from OCR after the initial stabilization (third measurement), and it was considered 100%. The percentage of maximal uncoupled respiration (MUR; K) in basal state (non-stimulated conditions) were calculated in relation to basal OCR and determined in the presence of glucose. All data represent mean values ±SEM from four independent experiments from a total of 26–52 wells. OCR and ECAR were normalized to micrograms of protein in each monitored well. *p < 0.05 compared to Hke3 cells (Student’s t test). (G,H) Cell net growth (calculated as the ratio of the number of living/proliferating cells (Hoechst 33,588) and dead cells (PI) for Hke3 (in blue) and Hke3 p53 KD (in grey) cultured cells in RPMI with 5 mM (G) and 11 mM (H) glucose. (p ≥ 0.05; ANOVA, post-hoc Tukey).