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. 2022 Sep 5;13:932286. doi: 10.3389/fendo.2022.932286

Figure 4.

Figure 4

Effect of L2HGDH shRNA an L2HGDH siRNA on the intra- and extracellular L- and D-2Hydroxyglutarate Level. (A) Primary RPT cells were transfected with either L2HGDH Stealth siRNA or the corresponding Scrambled siRNA. The medium was changed on Days 1, 2 and 3 post-transcription. (A) Two hours after the medium change on Day 3 post-transfection, frozen cell pellets were prepared, and medium was collected. (B) Primary cultures were transfected either with L2HGDH Silencer Select siRNA or Negative Control siRNA. As in part A, the medium was changed on Days 1, 2 and 3 post-transfection. The medium that was changed on Day 3, however, was collected and frozen, while frozen pellets were prepared 2 h after the final medium change. The L- and D-2HG in each of the samples (i.e. cell pellets and medium) was derivatized, separated by GC/MS, and quantitated, as described in Materials and Methods. Values are averages (+/- SEM) of triplicate determinations. In part A, *p < 0.05 relative to intracellular L-2HG with Scr siRNA, while in part B, *p < 0.05 relative to Negative Control siRNA in the same condition (i.e. either intracellular L-2HG, or extracellular L-2HG). In the insets, L2HGDH mRNA levels were determined as described in Materials and Methods. (*) p < 0.05 relative to Control condition.