Fig. 1. The inflammatory factors TNF-α plus IFN-γ induce CMA inhibition in MSCs.

a MSCs stably expressing KFERQ-PA-mCherry-C1 were photoactivated by a 405-nm light and then treated 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. Scale bar: 100 μm. b, c 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. Expression of LAMP-2A (L2A) was detected at the protein and mRNA levels using immunoblotting and quantitative real-time PCR, respectively. d Lysosomes from MSCs treated with/without TNF-α plus IFN-γ (10 ng/mL) for 24 h were isolated, and proteins were extracted for immunoblot analysis of all forms of LAMP-2 (upper) and PPCA (lower). The arrowhead indicates a previously identified truncated form of LAMP-2 lacking the cytosolic/transmembrane region. LAMP-1 was used as a control for normalization. The relative levels of the truncated forms of LAMP-2 and PPCA were quantified. The results are representative of three to six independent experiments and are presented as the mean ± s.e.m. Significant differences were analyzed by Mann–Whitney U test a, d or one-way ANOVA b, c, and are expressed as: *P < 0.05, **P < 0.01, and ***P < 0.001.