Journal: Journal of Cellular and Molecular Medicine

Article Title: Mesenchymal stem cell–secreted extracellular vesicles carrying TGF‐β1 up‐regulate miR‐132 and promote mouse M2 macrophage polarization

doi: 10.1111/jcmm.15860

Figure Lengend Snippet: Primer sequences for RT‐qPCR

Article Snippet: Thereafter, the membrane was probed with primary antibodies against inducible nitric oxide synthase (iNOS; 1:100, ab15323), CD86 (1:100, ab112490), CD206 (1:5000, ab125028), arginase‐1 (Arg‐1; 1:100, ab91279), TGF‐β1 (1:200, ab92486), TSC2 (1:1000, ab166790), GAPDH (1:5000, ab8245) (from Abcam) and Mycbp2 (1:2000, 12022‐1‐AP; ProteinTech Group).

Techniques: Sequencing

Journal: Journal of Cellular and Molecular Medicine

Article Title: Mesenchymal stem cell–secreted extracellular vesicles carrying TGF‐β1 up‐regulate miR‐132 and promote mouse M2 macrophage polarization

doi: 10.1111/jcmm.15860

Figure Lengend Snippet: miR‐132 targets Mycbp2 in RAW264.7 cells. A, Putative miR‐132 binding sites in the 3’UTR of Mycbp2 mRNA in the online prediction software starbase. B, Binding of miR‐132 to Mycbp2 confirmed by dual‐luciferase reporter gene assay in 293T cells. C, miR‐132 expression and Mycbp2 mRNA expression were determined by RT‐qPCR in the co‐culture system of RAW264.7 cells with MSCs‐EVs treated with si‐TGF‐β1, si‐TGF‐β1 + miR‐132 mimic or miR‐132 inhibitor, normalized to U6 and GAPDH, respectively. The data of the two groups were analysed by unpaired t test, while the data of multiple groups were analysed by one‐way ANOVA with Tukey's post hoc test. Data are shown as mean ± SD of three technical replicates. *, P value between groups was less than 0.05

Article Snippet: Thereafter, the membrane was probed with primary antibodies against inducible nitric oxide synthase (iNOS; 1:100, ab15323), CD86 (1:100, ab112490), CD206 (1:5000, ab125028), arginase‐1 (Arg‐1; 1:100, ab91279), TGF‐β1 (1:200, ab92486), TSC2 (1:1000, ab166790), GAPDH (1:5000, ab8245) (from Abcam) and Mycbp2 (1:2000, 12022‐1‐AP; ProteinTech Group).

Techniques: Binding Assay, Software, Luciferase, Reporter Gene Assay, Expressing, Quantitative RT-PCR, Co-Culture Assay

Journal: Journal of Cellular and Molecular Medicine

Article Title: Mesenchymal stem cell–secreted extracellular vesicles carrying TGF‐β1 up‐regulate miR‐132 and promote mouse M2 macrophage polarization

doi: 10.1111/jcmm.15860

Figure Lengend Snippet: miR‐132 targets Mycbp2 in macrophages to promote M2 polarization of macrophages. A, miR‐132 expression and Mycbp2 mRNA expression were determined by RT‐qPCR in RAW264.7 cells treated with miR‐132 mimic, Mycbp2 or both, normalized to U6 and GAPDH, respectively. B, Representative Western blots of Mycbp2 protein and its quantitation in RAW264.7 cells treated with miR‐132 mimic, Mycbp2 or both, normalized to GAPDH. C, Quantitative analysis of cell ratio of CD86 + and CD206 + in F4/80 cells by flow cytometry in the presence of miR‐132 mimic, Mycbp2 or both. D, Representative Western blots of iNOS and Arg‐1 proteins and their quantitation, normalized to GAPDH. E, Expression of inflammatory factors was measured by ELISA in cell supernatant in the presence of miR‐132 mimic, Mycbp2 or both. The data of multiple groups were analysed by one‐way ANOVA with Tukey's post hoc test. Data are shown as mean ± SD of three technical replicates. *, P value between groups was less than 0.05

Article Snippet: Thereafter, the membrane was probed with primary antibodies against inducible nitric oxide synthase (iNOS; 1:100, ab15323), CD86 (1:100, ab112490), CD206 (1:5000, ab125028), arginase‐1 (Arg‐1; 1:100, ab91279), TGF‐β1 (1:200, ab92486), TSC2 (1:1000, ab166790), GAPDH (1:5000, ab8245) (from Abcam) and Mycbp2 (1:2000, 12022‐1‐AP; ProteinTech Group).

Techniques: Expressing, Quantitative RT-PCR, Western Blot, Quantitation Assay, Flow Cytometry, Enzyme-linked Immunosorbent Assay

Journal: Journal of Cellular and Molecular Medicine

Article Title: Mesenchymal stem cell–secreted extracellular vesicles carrying TGF‐β1 up‐regulate miR‐132 and promote mouse M2 macrophage polarization

doi: 10.1111/jcmm.15860

Figure Lengend Snippet: MSCs‐EVs carrying TGF‐β1 up‐regulate miR‐132 expression and thus inhibit MYCBP2 expression, polarizing macrophages towards M2 phenotype in vitro. A, mRNA expression of Mycbp2 and TGF‐β1 was determined by RT‐qPCR in the co‐culture system of RAW264.7 cells with MSCs‐EVs treated with si‐TGF‐β1 or in combination with sh‐Mycbp2, normalized to GAPDH. B, Representative Western blots of Mycbp2 protein and its quantitation in the co‐culture system of RAW264.7 cells with MSCs‐EVs treated with si‐TGF‐β1 or in combination with sh‐Mycbp2, normalized to GAPDH. C, Quantitative analysis of cell ratio of CD86 + and CD206 + in F4/80 cells detected by flow cytometry in the co‐culture system of RAW264.7 cells with MSCs‐EVs treated with si‐TGF‐β1 or in combination with sh‐Mycbp2. D, Representative Western blots of iNOS and Arg‐1 proteins and their quantitation in the co‐culture system of RAW264.7 cells with MSCs‐EVs treated with si‐TGF‐β1 or in combination with sh‐Mycbp2, normalized to GAPDH. E, Expression of inflammatory factors was measured by ELISA in cell supernatant in the co‐culture system of RAW264.7 cells with MSCs‐EVs treated with si‐TGF‐β1 or in combination with sh‐Mycbp2. The data were conducted by one‐way ANOVA with Tukey's post hoc test. Data are shown as mean ± SD of three technical replicates. *, P value between groups was less than 0.05

Article Snippet: Thereafter, the membrane was probed with primary antibodies against inducible nitric oxide synthase (iNOS; 1:100, ab15323), CD86 (1:100, ab112490), CD206 (1:5000, ab125028), arginase‐1 (Arg‐1; 1:100, ab91279), TGF‐β1 (1:200, ab92486), TSC2 (1:1000, ab166790), GAPDH (1:5000, ab8245) (from Abcam) and Mycbp2 (1:2000, 12022‐1‐AP; ProteinTech Group).

Techniques: Expressing, In Vitro, Quantitative RT-PCR, Co-Culture Assay, Western Blot, Quantitation Assay, Flow Cytometry, Enzyme-linked Immunosorbent Assay

Journal: Journal of Cellular and Molecular Medicine

Article Title: Mesenchymal stem cell–secreted extracellular vesicles carrying TGF‐β1 up‐regulate miR‐132 and promote mouse M2 macrophage polarization

doi: 10.1111/jcmm.15860

Figure Lengend Snippet: Mycbp2 ubiquitinates and degrades TSC2 in macrophages. A, Representative Western blots of Mycbp2 and TSC2 proteins and their quantitation in RAW264.7 cells treated with oe‐Mycbp2, normalized to GAPDH. B, Representative Western blots of Mycbp2 and TSC2 proteins and their quantitation in RAW264.7 cells treated with oe‐Mycbp2, MG132 or both, normalized to GAPDH. C, Co‐IP detection of the interaction between Mycbp2 and TSC2 in RAW264.7 cells. D, Effect of Mycbp2 on ubiquitination of TSC2 protein in 293T cells. E, Effect of Mycbp2 on ubiquitination of TSC2 protein in RAW264.7 cells. F, Quantitative analysis of cell ratio of CD86 + and CD206 + in F4/80 cells by flow cytometry in oe‐TSC2‐treated RAW264.7 cells. G, Representative Western blots of iNOS and Arg‐1 proteins and their quantitation in oe‐TSC2‐treated RAW264.7 cells. H, Expression of inflammatory factors was measured by ELISA in cell supernatant upon oe‐TSC2 treatment. The comparison between two groups was analysed using unpaired t test, while comparisons among multiple groups were conducted by one‐way ANOVA with Tukey's post hoc test. The data at different time‐points were compared by two‐way ANOVA followed by Dunnett's correction. Data are shown as mean ± SD of three technical replicates. *, P value between groups was less than 0.05

Article Snippet: Thereafter, the membrane was probed with primary antibodies against inducible nitric oxide synthase (iNOS; 1:100, ab15323), CD86 (1:100, ab112490), CD206 (1:5000, ab125028), arginase‐1 (Arg‐1; 1:100, ab91279), TGF‐β1 (1:200, ab92486), TSC2 (1:1000, ab166790), GAPDH (1:5000, ab8245) (from Abcam) and Mycbp2 (1:2000, 12022‐1‐AP; ProteinTech Group).

Techniques: Western Blot, Quantitation Assay, Co-Immunoprecipitation Assay, Ubiquitin Proteomics, Flow Cytometry, Expressing, Enzyme-linked Immunosorbent Assay, Comparison

Journal: Journal of Cellular and Molecular Medicine

Article Title: Mesenchymal stem cell–secreted extracellular vesicles carrying TGF‐β1 up‐regulate miR‐132 and promote mouse M2 macrophage polarization

doi: 10.1111/jcmm.15860

Figure Lengend Snippet: EVs from MSCs can carry TGF‐β1 to promote M2 polarization of macrophages through the miR‐132/Mycbp2/TSC2 axis. A, Expression of TGF‐β1, miR‐132, Mycbp2 and TSC2 was determined by RT‐qPCR in the co‐culture system of RAW264.7 cells with MSCs‐EVs treated with si‐TGF‐β1, normalized to U6 and GAPDH, respectively. B, Quantitative analysis of cell ratio of CD86 + and CD206 + in F4/80 cells by flow cytometry in the co‐culture system of RAW264.7 cells with MSCs‐EVs treated with si‐TGF‐β1. C, Representative Western blots of iNOS and Arg‐1 proteins and their quantitation in the co‐culture system of RAW264.7 cells with MSCs‐EVs treated with si‐TGF‐β1, normalized to GAPDH. D, Expression of inflammatory factors was measured by ELISA in cell supernatant in the co‐culture system of RAW264.7 cells with MSCs‐EVs treated with si‐TGF‐β1. The data of multiple groups were analysed by one‐way ANOVA with Tukey's post hoc test. Data are shown as mean ± SD of three technical replicates. *, P value between groups was less than 0.05

Article Snippet: Thereafter, the membrane was probed with primary antibodies against inducible nitric oxide synthase (iNOS; 1:100, ab15323), CD86 (1:100, ab112490), CD206 (1:5000, ab125028), arginase‐1 (Arg‐1; 1:100, ab91279), TGF‐β1 (1:200, ab92486), TSC2 (1:1000, ab166790), GAPDH (1:5000, ab8245) (from Abcam) and Mycbp2 (1:2000, 12022‐1‐AP; ProteinTech Group).

Techniques: Expressing, Quantitative RT-PCR, Co-Culture Assay, Flow Cytometry, Western Blot, Quantitation Assay, Enzyme-linked Immunosorbent Assay

Journal: Journal of Cellular and Molecular Medicine

Article Title: Mesenchymal stem cell–secreted extracellular vesicles carrying TGF‐β1 up‐regulate miR‐132 and promote mouse M2 macrophage polarization

doi: 10.1111/jcmm.15860

Figure Lengend Snippet: The mechanistic diagram depicts that MSC‐derived EVs carrying TGF‐β1 promote macrophage M2 polarization through the miR‐132/Mycbp2/TSC2 axis. In brief, MSC‐derived EVs carry TGF‐β1 into macrophages, promote the expression of miR‐132 and then inhibit the expression of Mycbp2 which promotes ubiquitination and degradation of TSC2 protein, ultimately polarizing macrophages towards M2 phenotype

Article Snippet: Thereafter, the membrane was probed with primary antibodies against inducible nitric oxide synthase (iNOS; 1:100, ab15323), CD86 (1:100, ab112490), CD206 (1:5000, ab125028), arginase‐1 (Arg‐1; 1:100, ab91279), TGF‐β1 (1:200, ab92486), TSC2 (1:1000, ab166790), GAPDH (1:5000, ab8245) (from Abcam) and Mycbp2 (1:2000, 12022‐1‐AP; ProteinTech Group).

Techniques: Derivative Assay, Expressing, Ubiquitin Proteomics