cd81 boster  (Boster Bio)

Journal: Bioscience Reports

Article Title: Exosomes derived from circRNA Rtn4-modified BMSCs attenuate TNF-α-induced cytotoxicity and apoptosis in murine MC3T3-E1 cells by sponging miR-146a

doi: 10.1042/BSR20193436

Figure Lengend Snippet: ( A ) Western blot analysis showed that BMSCs-Exos were positive for CD63, CD9, CD81, and Alix. ( B ) The exosome uptake assay was performed to assess the uptake of PKH26-labeled exosomes into recipient MC3T3-E1 cells. Red: PKH26-labeled BMSCs-Exos. Blue: nuclei. Scale bar = 20 μm. ( C ) MC3T3-E1 cells were treated with TNF-α (5 ng/ml) and BMSCs-Exos (0, 25, 50 and 100 μg/ml) and then subjected to cell viability testing. Results showed that BMSCs-Exos dose-dependently blocked TNF-α-induced inhibition of cell viability. ( D ) Flow cytometry analysis of MC3T3-E1 cells treated with TNF-α and BMSCs-Exos. The results showed that BMSCs-Exos dose-dependently mitigated TNF-α-induced increase in cell apoptosis. ( E ) qRT-PCR analysis showed that TNF-α-induced increase in miR-146a expression was blocked when MC3T3-E1 cells were co-cultured with BMSCs-Exos. ( F ) Western blot analysis showed that BMSCs-Exos dose-dependently blocked TNF-α-induced cleaved caspase-3 and Bax expression. ( G ) ELISA data showed that BMSCs-Exos inhibited TNF-α-induced caspase-3 activity. All experiments were independently repeated three times. The caspase-3 activity and MTT assays were performed in triplicate. The differences among multiple groups were determined using one-way ANOVA test. n =3. * P <0.05.

Article Snippet: After blocking with 5% skim milk, the membranes were probed with primary antibodies against caspase-3 (Cell Signaling Technology), cleaved caspase-3 (Cell Signaling Technology), Bcl-2-associated X protein (Bax; Cell Signaling Technology), CD9 (Boster, Wuhan, China), CD81 (Boster), CD9 (Boster), Alix (Boster), and β-actin (Cell Signaling Technology), followed by incubation with horseradish peroxidase–conjugated secondary antibody (Boster).

Techniques: Western Blot, Labeling, Inhibition, Flow Cytometry, Quantitative RT-PCR, Expressing, Cell Culture, Enzyme-linked Immunosorbent Assay, Activity Assay

Journal: Bioscience Reports

Article Title: Exosomes derived from circRNA Rtn4-modified BMSCs attenuate TNF-α-induced cytotoxicity and apoptosis in murine MC3T3-E1 cells by sponging miR-146a

doi: 10.1042/BSR20193436

Figure Lengend Snippet: BMSCs were transfected with NC or pcDNA-circ-Rtn4, and their exosomes were isolated. ( A,B ) The expression of circ-Rtn4 was measured in NC- or pcDNA-circ-Rtn4-transfected BMSCs and their exosomes using qRT-PCR. ( C ) MC3T3-E1 cells were co-cultured with Rtn4-Exos or NC-Exos, and tested for circ-Rtn4 expression using qRT-PCR. ( D,E ) Evaluation of miR-146a expression in NC- or pcDNA-circ-Rtn4-transfected BMSCs and their exosomes using qRT-PCR. ( F ) qRT-PCR analysis of miR-146a expression in MC3T3-E1 cells treated with Rtn4-Exos or NC-Exos. ( G ) MC3T3-E1 cells were treated with TNF-α, followed by co-culture with Rtn4-Exos or NC-Exos. The viability of MC3T3-E1 cells was evaluated using MTT assay. ( H ) Flow cytometry analysis to evaluate cell apoptosis in MC3T3-E1 cells treated with TNF-α and exosomes from different sources. ( I ) The protein expression levels of caspase-3, cleaved caspase-3, and Bax were determined using Western blotting. ( J ) Caspase-3 activity was measured in MC3T3-E1 cells treated with TNF-α and exosomes from different sources using ELISA. ( K ) Western blot analysis of surface markers (CD63, CD81, CD9, and Alix) in exosomes. All experiments were independently repeated three times. The caspase-3 activity and MTT assays were performed in triplicate. The differences among multiple groups were determined using one-way ANOVA test. n =3. * P <0.05.

Article Snippet: After blocking with 5% skim milk, the membranes were probed with primary antibodies against caspase-3 (Cell Signaling Technology), cleaved caspase-3 (Cell Signaling Technology), Bcl-2-associated X protein (Bax; Cell Signaling Technology), CD9 (Boster, Wuhan, China), CD81 (Boster), CD9 (Boster), Alix (Boster), and β-actin (Cell Signaling Technology), followed by incubation with horseradish peroxidase–conjugated secondary antibody (Boster).

Techniques: Transfection, Isolation, Expressing, Quantitative RT-PCR, Cell Culture, Co-Culture Assay, MTT Assay, Flow Cytometry, Western Blot, Activity Assay, Enzyme-linked Immunosorbent Assay