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. 2020 Dec 29;22(2):e50967. doi: 10.15252/embr.202050967

Figure 5. HAT1 succinylates PGAM1 to enhance glycolysis.

Figure 5

  1. PGAM1 was immunoprecipitated from HepG2, HAT1 KO Clone 1 HepG2, and HAT1 KO Clone 1 HepG2 cells reconstituted with either wild‐type HAT1 or mutant HAT1 (T188A). The levels of succinylation, PGAM1, HAT1, and β‐actin were analyzed by Western blot analysis in the cells.
  2. Relative PGAM1 activity was measured by PGAM1 activity assays in the indicated tumor cells. N = 3 biological replicates. Data are presented as mean ± SD. Student's t‐test, **P < 0.01.
  3. The decrease of 2‐PG and increase of 3‐PG were measured by ELISA assays in HepG2, HAT1 KO HepG2, and HAT1 KO HepG2 cells reconstituted with either wild‐type HAT1 or mutant HAT1 (T188A). N = 3 biological replicates. Data are presented as mean ± SD. Student's t‐test, **P < 0.01.
  4. HAT1‐catalyzed PGAM1 succinylation was analyzed by mixing purified HAT1, PGAM1, and succinyl‐CoA/acetyl‐CoA in the in vitro succinylation assays. Western blot analysis was performed with indicated antibodies.
  5. Purified HAT1, PGAM1 or PGAM1 mutant, and succinyl‐CoA were mixed in the in vitro succinylation assays and relative PGAM1 activity was measured by PGAM1 activity assays in vitro. N = 3 biological replicates. Data are presented as mean ± SD. Student's t‐test, **P < 0.01.
  6. The decrease of glucose and increase of lactate in the culture medium and the relative amounts of 2‐PG and 3‐PG were analyzed by ELISA assays in HepG2 cells depleted endogenous PGAM1 and reconstituted expression of PGAM1 or PGAM1 mutant (K99R). N = 3 biological replicates. Data are presented as mean ± SD. Student's t‐test, **P < 0.01.