Figure 5. Rescue of reduced 2-PG levels due to PGAM1 attenuation results in decreased 3-PG levels by activating PHGDH.

(A) 3-PG levels in diverse cancer cells with stable knockdown of PGAM1 were determined in the presence and absence of methyl-2-PG.

(B–C) Enzyme activity of PHGDH in PGAM1 knockdown H1299 (B; left) or 212LN (B; right) cell lysates and recombinant PHGDH (rPHGDH) (C) were determined in the presence of increasing concentrations of 2-PG. Relative enzyme activity was normalized to the control samples without 2-PG treatment. 2-PG levels in control H1299 cells with empty vector and PGAM1 knockdown cells are 46.2±10.2μM and 15.0±14.1μM, respectively, while 2-PG levels in 212LN cells with empty vector and stable knockdown of PGAM1 are 58.3±20.1μM and 17.8±14.4μM, respectively.

(D) Serine biosynthesis rate of H1299 cells with stable knockdown of PGAM1 was determined by measuring ¹⁴C incorporation into serine from ¹⁴C-glucose in the presence and absence of methyl-2-PG. Relative serine biosynthesis was normalized to control cells harboring an empty vector without methyl-2-PG treatment.

(E) Western blot result shows shRNA-mediated knockdown of PHGDH in H1299 cells with stable knockdown of PGAM1 in the presence or absence of methyl-2-PG treatment.

(F) 2-PG (left) and 3-PG (right) levels in PGAM1 knockdown cells upon PHGDH knockdown were determined in the presence and absence of methyl-2-PG.

(G–H) PGAM1 stable knockdown cells treated with or without shRNA targeting PHGDH were tested for PPP flux (G) as well as biosynthesis of serine, lipids and RNA (H; left, middle and right, respectively) in the presence and absence of methyl-2-PG.

The error bars represent mean values +/− SD from three replicates of each sample (*: 0.01<p<0.05; **: 0.01<p<0.01; ***: p<0.001; n.s.: not significant).