Extended Data Fig. 7: Variation in P5CS Expression Levels among Human Cancer Cell Lines under Glutamine Deprivation.
A) Bar plot illustrating the weights of subcutaneous xenograft tumors from NUGC2 cells (PLKO, shALDH18A1, and rescue with dox-inducible NUGC2). Statistical analysis was conducted using a one-way ANOVA followed by Tukey’s multiple comparison test. Data are presented as mean values ± SD. N = 8 independent animals. B) 15N-labeled ammonia (15NH4 + ) in NUGC2 gastric cancer cells with genetically downregulated P5CS. Data are represented as mean ± SD of three independent experiments. Statistical significance was determined using two-way ANOVA. C) Western blot of P5CS in human cancer cell lines that do not endogenously downregulate P5CS upon acute GLN starvation. Cells were cultured in the presence or absence of 2 mM GLN for 24 hrs, (n = 3). D) Comparison of P5CS levels in xenograft tumors derived from MKN45 cells. On the left, a western blot illustrates the relative P5CS protein levels in tumor extracts, while on the right, a bar graph represents the quantification of these levels. Statistical significance was determined using a one-tailed unpaired t-test with Welch’s correction for unequal variances. Data are presented as mean values ± SD. E) Additional representative immunofluorescence (IF) of a tumor section of uterine serous carcinoma, depicting staining by IF for P5CS (red), GS (green), and Dapi (blue). On the right, a selected area of the tumors demonstrates an inverse correlation between P5CS and GS expression, (n = 3). F) Total number of cells with partner P5CSlow/GShigh or vice-versa detected in five tumor sections of uterine serous carcinoma (USC). Statistical analysis was performed using an unpaired two-tailed Mann-Whitney test. Data are presented as mean values ± SD.
