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. Author manuscript; available in PMC: 2023 Jun 22.
Published in final edited form as: Oncogene. 2023 Apr 10;42(21):1763–1776. doi: 10.1038/s41388-023-02686-7

Figure. 2: Induction of UBXN2A interferes with the mTORC2 pathway but not the mTORC1 signaling pathway.

Figure. 2:

Tet-On inducible HCT-116 cells were incubated with DOX to express GFP or GFP-UBXN2A and analyzed by flow cytometry using Alexa Fluor pAKT-Ser473 and pAKT-T308. Induction of UBXN2A significantly decreased pAKT-Ser473 and pAKT-Thr308 (A-E). Similarly, UBXN2A significantly decreased pAKT-Ser473 in SW480 cells. However, UBXN2A induction showed no effect on pAKT-Thr308 in SW480, suggesting UBXN2A affects mTORC2 in a cell-dependent manner (F). Xenograft mice carrying Tet-On inducible HCT-116 cells were fed doxycycline (G), followed by tumor extraction and WB experiments (H).Representative WB of three mice with similar results (one GFP-empty tumor and one GFP-UBXN32A tumor per mouse) confirmed UBXN2A decreases the level of pAKT-S473/T308 in xenograft tumors (I). PDX tissues were subjected to WB followed by quantitation of their corresponding bands. Results revealed that the high level of UBXN2A in PDXs significantly decreases Rictor protein (J-K), and it changes Rictor’s downstream target proteins, including AKT-473, VEGF, and E-Cadherin (L). ELISA assays (M) and WB of phospho-p70 S6 kinase (T389) in HCT-116 cells (N and O) combined with flow-cytometry analysis of the total level of p70 S6 kinase (P) revealed that elevated UBXN2A has no significant effect on phosphorylation and activation of phospho-p70 S6 kinase (T389), which is located downstream of mTORC1 (n=3, * p< 0.05, **** p< 0.0001, mean +/− SD).