Fig. 6. Inflammatory factor-stimulated AKT activation mediates CMA inhibition.

a MSCs were treated with TNF-α plus IFN-γ (10 ng/mL each) and mRNA, and protein were collected at 0 h, 2 h, 4 h, 6 h, 12 h, and 24 h after treatment. AKT and AKT phosphorylation at Ser 473 and Thr 308 were analyzed by immunoblotting. b, c After pretreatment of MSCs with DMSO or MK2206 (10 μM) for 6 h, MSCs were treated with or without TNF-α plus IFN-γ (10 ng/mL each) for 24 h. Protein and mRNA were collected to detect the expression of L2A by immunoblotting b and quantitative real-time PCR c, respectively. d MSCs stably expressing KFERQ-PA-mCherry-C1 were photoactivated by a 405-nm light and then pretreated with DMSO or MK2206 for 6 h prior to treatment with TNF-α plus IFN-γ (10 ng/mL each) for 24 h. The images were collected by a Zeiss fluorescence microscope. The number of red fluorescent puncta per cell was quantified. e The level of NFAT1 was detected by immunoblotting using the proteins in b, and the quantitative analysis result is shown. f Lysosomes were isolated from MSCs that were pretreated with DMSO or MK2206 for 6 h and then treated with or without TNF-α plus IFN-γ (10 ng/mL each) for 24 h. Protein was extracted from the lysosomes, and the level of PPCA was measured by immunoblotting. The relative level of PPCA to LAMP-1 was calculated. g SCR-MSCs and L2A-KD-MSCs were treated with or without TNF-α plus IFN-γ (10 ng/mL each) for 24 h. Protein was collected, and AKT and phosphorylation of AKT at Ser 473 and Thr308 were analyzed by immunoblotting. h SCR-MSCs and L2A-KD-MSCs were pretreated with MK2206 or DMSO for 6 h and then treated with mitomycin C (50 ng/mL) for 4 h prior to coculture with CFSE-labeled splenocytes activated by anti-CD3/CD28 antibodies for 3 days at a ratio of 1:20. CD8⁺ and CD4⁺ T cells were stained for proliferation analysis by flow cytometry at the end of coculture, and the percentage of nonproliferating T cells is shown. Scale bar: 100 μm. The results are representative of three to six independent experiments and are presented as the mean ± s.e.m. Significant differences were analyzed by one-way ANOVA a, h or two-way ANOVA b–g, and are expressed as *P < 0.05, **P < 0.01, ***P < 0.001, and n.s., no significance.