PP2A reduces ATP consumption and inhibits growth of TSCs during hypoxic conditions.

<p>(A) Effect of PP2A inhibition on ATP consumption of TSCs during normoxia and hypoxia. Intracellular ATP was measured in TSCs 1 hour following substrate depletion (D5030 media) with our without PP2A inhibition. PP2A inhibition leads to significantly higher ATP consumption during hypoxic conditions. The ATP content was measured and normalized to the lysate protein content. Bars represent the mean values of 8 independent experiments ± SEM. (B) 24 hours following plating, TSCs are exposed to CoCl₂ and/or OA for additional 24 hours. Hypoxia leads to G1/S phase growth inhibition in TSCs. Representative examples of cell cycle analysis experiments. (C) Summary of six independent experiments reveals that hypoxia-mediated G1/S phase arrest is partially reversed by OA in a dose-dependent fashion. Accordingly, PP2A inhibition allows for significantly more hypoxic TSCs to progress into the S-phase. (D) In line with pharmacological inhibition of PP2A, depletion of PP2A by shRNA alleviates G1/S cell cycle arrest in TSCs propagated in CoCl₂. Given the longer duration of PP2A inhibition in PP2A-depleted cells (48 h) compared to OA-treated cells (24 h [C]), more PP2A-depleted cells progress to G2 phase. Increasing PP2A activity by depletion of the endogenous PP2A inhibitor PME-1 has the opposite effect. Western blots reveal depletion of PP2A-C and PME-1 in TSCs by shRNA.