Figure 6. mTORC1 regulation of i-proteasome formation depends on PRAS40 and is important for cell survival against stress.

(A) PRAS40 KD enhances the mature form of β5i along with reduction of β5 in 3T3-L1 cells. 3T3-L1 cells were stably transduced by shRNA and treated with TNF-α (20 ng/mL) for 24 h.

(B) PRAS40 KD enhances the mature forms of iβ subunits in T24 cells.

(C) PRAS40 KD reduces the suppressive effect of Torin1 on β5i maturation. HCT116 cells stably transduced by shRNA were treated with IFN-γ (50 ng/mL) and Torin1 at 0, 20, 50, 100, and 250 nM for 16 h.

(D) PRAS40 KD makes cells to depend on i-proteasomes for viability under oxidative stress. KG1 cells transduced by shRNA were treated with H₂O₂ in the presence or absence of PR957 (PR, 100 nM) and ML604440 (ML, 500 nM) for 48 h. MTT assay was conducted for viability. Values are means ± SD (*, p<0.05; n=3).

(E) I-proteasome inhibition enhances oxidative stress-induced PARP1 cleavage to a greater extent when PRAS40 is silenced. KG1 cells transduced by shRNA were treated with TNFα (20 ng/mL), tunicamycin (1 μM), rotenone (1 μM) and/or H₂O₂ (5 μM) for 16 h in the presence (+PR/ML) or absence (−PR/ML) of i-proteasome inhibitors.

(F) PRAS40 phosphorylations at S183 and S221 are important for β5i maturation. Flag-tagged β5i was transiently expressed alone (−) or together with PRAS40 contructs in HEK293T cells.

(G) PRAS40 phosphorylations are important for β1i maturation and S6K1 phosphorylation. Myc-PRAS40 construct was stably reconstituted in PRAS40-silenced HCT116 cells.

(H) PRAS40 phosphorylations are important for cellular resistance to oxidative stress. HCT116 cells reconstituted with PRAS40 construct were treated with IFN-γ (1 ng/ml) and H₂O₂ (100 μM) for 24 h.

(I) PRAS40 phosphorylations are important for oxidative stress response and S6K1 phosphorylation. AA or EE HCT116 cells were exposed to IFN-γ (50 ng/mL) for 24 h then treated with H₂O₂ (100 μM), Torin1 (250 nM), PR957 (100 nM), and/or ML604440 (500 nM) for 24 h.

(J) AA or EE HCT116 cells were treated with IFN-γ (50 ng/mL) with or without H₂O₂ (100 μM) for 48 h, or with IFN-γ (50 ng/mL) for 24 h then starved of serum for 24 h. Values are means ± SD (*, p<0.05; **, p<0.01 vs EE; n=3).

(K) EE mutation induces PARP1 cleavage under serum-starved condition. HCT116 cells reconstituted with WT or mutant PRAS40 were treated with IFN-γ (0.1 ng/mL) for 4 days then starved of serum for 24 h.

See also Figure S6.