Journal: Biomaterials Research
Article Title: Mesenchymal Stem Cell-Derived Exosomes Inhibit Stim1–Orai1 Signaling and Calcium Overload-Induced Mitochondrial Damage of Follicular Helper T Cells in Lupus
doi: 10.34133/bmr.0255
Figure Lengend Snippet: MSC-Exos regulate calcium homeostasis in Tfh. (A) Volcano diagram of DEGs of Tfh between control group and Exo group ( n = 3). (B) KEGG enrichment analysis of Tfh between control group ( n = 3) and Exo group ( n = 3). These pathways were significant differences in the diagram. (C) GO enrichment analysis of DEGs between Tfh and naïve CD4 + T cells in C57 based on the GSE157648 dataset. (D) Fluo-4 AM-loaded Th0 and Tfh treated with/without MSC-Exos in HBSS medium for 30 min, and intracellular Ca 2+ levels were detected by flow cytometry using Fluo-4 AM as the probe ( n = 5). (E) mRNA expression levels of Stim1, Stim2, and Orai1 in Tfh with PBS or MSC-Exos coculture were detected by RT-qPCR. (F) WB analysis of Stim1 and Orai1 protein expression in Th0 and Tfh after treatment with MSC-Exos or PBS. (G) WB analysis of Stim1 and Orai1 protein expression in Tfh treated with/without MSC-Exos, and ionomycin (5 μM) was used to elevate intracellular Ca 2+ levels. (H) RT-qPCR analysis of NFATc1, NFATc2, and CaN mRNA expression levels in Tfh after treatment with MSC-Exos or PBS. (I) WB analysis of CaN and NFATc2 protein expression in Tfh treated with/without MSC-Exos. (J) WB analysis of IκB, p-IκB, P65, and p-P65 protein expression in Tfh treated with/without MSC-Exos. Bars indicate the means ± SEM. * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001 determined by one-way ANOVA with Tukey’s multiple comparisons test.
Article Snippet: These membranes were blocked with 5% nonfat milk at 25 °C for 1 h. Primary antibodies targeting CaN (Proteintech, 13422-1-AP, China), NFATc2 (Proteintech, 22023-1-AP, China), P65 (Affinity, AF5006, USA), p-P65 (Affinity, AF2006, USA), IκB (Abmart, T55026 , China), p-IκB (Abmart, TA2002, China), Stim1 (Proteintech, 11565-1-AP, China), Orai1 (Proteintech, 28411-1-AP, China), and MCU (Abways, BY0101, China) were incubated overnight at 4 °C.
Techniques: Control, Flow Cytometry, Expressing, Quantitative RT-PCR