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. 2021 May 24;10(7):e12109. doi: 10.1002/jev2.12109

FIGURE 6.

FIGURE 6

CRT blockade inhibits functional efferocytosis of MSC‐derived apoVs by T2D macrophages in vitro. (a) Representative confocal microscopy images showing uptake of apoVs (red) by bone marrow‐derived macrophages (BMDMs) (green) in vitro, counterstained by Hoechst (blue), and the corresponding quantification of fold changes of relative fluorescence intensity. apoV+CRT‐nAb, apoVs pre‐treated with CRT neutralizing antibody at 37℃ for 1 h. Scale bars, 25 μm. N = 5–6 per group. (b) Flow cytometric analysis showing uptake of apoVs by BMDMs in vitro, and the corresponding quantification of fold changes of mean fluorescence intensity (MFI). N = 6 per group. (C‐E) Quantitative real time polymerase chain reaction (qRT‐PCR) analysis of mRNA expression levels of tumor necrosis factor (Tnf) (c), interleukin 1 beta (Il1b) (d) and nitric oxide synthase 2, inducible (Nos2) (e) in cultured BMDMs, normalized to β‐actin (Actb), and quantification of fold changes over the Ctrl group. Ctrl, BMDMs derived from control mice; DIO, BMDMs derived from mice with diet‐induced obesity; apoV, BMDMs derived from DIO mice and treated with apoVs; apoV+CRT‐nAb, BMDMs derived from DIO mice and treated with apoVs which were pre‐treated with CRT‐nAb. N = 3–4 per group. (f) Enzyme‐linked immunosorbent assay (ELISA) analysis of TNF‐α in media from cultured BMDMs. N = 4 per group. (g‐i) qRT‐PCR analysis of mRNA expression levels of interleukin 10 (Il10) (g), mannose receptor, C type 1 (Mrc1) (h) and resistin like alpha (Retnla) (i) in cultured BMDMs, normalized to Actb, and quantification of fold changes over the Ctrl group. N = 3–4 per group. (j) ELISA analysis of IL‐10 in media from cultured BMDMs. N = 4 per group. (k) qRT‐PCR analysis of mRNA expression levels of chemokine (C‐C motif) ligand 2 (Ccl2) in cultured BMDMs, normalized to Actb, and quantification of fold changes over the Ctrl group. N = 3–4 per group. (l) ELISA analysis of CCL2 in media from cultured BMDMs. N = 4 per group. (m) Representative confocal microscopy images showing uptake of apoVs (red) by macrophages (green) in the liver, counterstained by Hoechst (blue). Scale bars, 25 μm. Data are presented as mean ± standard deviation (SD). Statistical analyses are performed by Student's t test with Welch correction (two‐tailed) for two group comparisons and One‐way ANOVA with Tukey's post hoc test for multiple group comparisons. *, P < 0.05; **, P < 0.01; ***, P < 0.001