Figure 5. High l-2HG causes exogenous serine dependency in RCC cell lines.

(A) LC-MS analysis of serine synthesis (m+3) from labeled glucose (U-¹³C). n = 6 biological replicates. (B) Proliferation of OS-RC-2 cells stably transduced with the indicated vector (left, control; right, L2HGDH) in media with or without SerGly. n = 3 biological replicates. Repeated-measures analysis was conducted, and P values reflect Tukey’s post hoc comparisons. (C and D) Relative growth ratio (–SerGly/+SerGly) at day 4 in RCC cells transduced with the indicated vector. n = 3 biological replicates. *P < 0.05. (E) Immunoblot of PHGDH from 769p cells stably expressing control vector or PHGDH cDNA. (F and G) Proliferation of 769p cells transduced with control vector (F) or PHGDH (G) in media with or without SerGly. n = 3 biological replicates of each group. Repeated-measures analysis was conducted, and P values reflect Tukey’s post hoc comparisons. (H) Immunoblot of Sn12pm6 cells stably expressing the indicated shRNA. (I) Relative l/d-2HG levels in Sn12pm6 cells transduced with the indicated shRNAs (shC, control). n = 3 biological replicates. (J and K) Cell proliferation (by connectivity index) was measured from Sn12pm6-Scr (J) and Sn12pm6-shL2HGDH (K) cells grown with or without SerGly. n = 4 biological replicates of each group. Repeated-measures analysis was conducted, and P values reflect Tukey’s post hoc comparisons. (L) Left: After subcutaneous implantation of OS-RC-2 cells, mice were randomly distributed and fed chow with or without SerGly (n = 10 per group) for 4 weeks. Right: Tumor volume at day 28. Repeated-measures analysis was performed followed by Tukey’s post hoc comparisons at each time point. (M) 786-O xenografts were established in flanks of nude mice. Left: After the average tumor size reached 100 mm³, mice were fed chow with or without SerGly (n = 7 per group) and followed over time (left). Right: Xenograft size (day 40). Repeated-measures analysis was performed followed by Tukey’s post hoc comparisons at each time point Data are shown as (A–D, F, G, J, and K) mean ± SD and (I, L, and M) mean ± SEM.