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. Author manuscript; available in PMC: 2022 Oct 1.

Published in final edited form as: J Mol Cell Cardiol. 2021 Jun 15;159:62–79. doi: 10.1016/j.yjmcc.2021.06.004

Figure 5. — C57BL/6J mice were subjected to sham or LAD ligation surgeries. Twenty four hours after the MI procedure, mice were randomly divided into groups that received subcutaneous injection of vehicle or rapamycin at 0.25mg/kg/day. Cardiac immune cells were isolated from pooled infarct tissues from three mice (n=3 MI hearts from each group) 7-day after MI for flow cytometry. Cardiac tissues from the same cohorts were subjected to immunoblot analysis to detect the phosphorylated and total Akt. A. The immune cells isolated from DMSO- or rapamycin-treated hearts were labeled with CD45, CD11b, CCR2, Ly6G, CD206, CD64, F4/80, Ly6C, and MHCII. After forward and side scatter selection, cMPs were gated as CD45+CD11b+Ly6Glow cells. The selected cMP population was gated further with CD206 and CD64 and is presented individually as DMSO or rapamycin group or with the two group over-layed. The percentage of CD206highCD64low or CD206lowCD64high were indicated. B. Positive correlation F4/80 with CD206. C. The CD45+CD11b+Ly6Glow population was further gated based on Ly6C and F4/80. Percentage of Ly6ClowF4/80high, Ly6CmidF4/80mid, and Ly6ChighF4/80mid were 11.5%, 33.2%, and 9.2% for DMSO-treated hearts and 52%, 12.8%, and 0.4% for rapamycin-treated group. D. The landscape changes of cMPs. E. Heat map of the median fluorescent intensity (MFI) of the inflammatory cell surface markers (CCR2, Ly6C, CD64), antiinflammatory markers (CD206, F4/80), macrophage maturation marker (MHCII) and myeloid marker (CD45) of cMPs from DMSO- or rapamycin-treated hearts. F. Expression of IL1β or TNFα from cMPs isolated from DMSO- or rapamycin-treated MI hearts at indicated time points. n=6 to 8 MI hearts (some data points are from pooled MI tissue). G. IL6 expression from cMPs isolated from DMSO- or rapamycin-treated hearts 7-day after MI. H. Protein samples extracted from hearts 7-day after MI were subjected to immunoblot analysis. Individual lanes represent different hearts. The activation of Akt was determined by the levels of p-AktSer473 and p-AktThr308. I and J. Summarized data from experiments performed as in H. n=4–6 MI hearts. * p<0.05 ** p<0.01.

Figure 5. — C57BL/6J mice were subjected to sham or LAD ligation surgeries. Twenty four hours after the MI procedure, mice were randomly divided into groups that received subcutaneous injection of vehicle or rapamycin at 0.25mg/kg/day. Cardiac immune cells were isolated from pooled infarct tissues from three mice (n=3 MI hearts from each group) 7-day after MI for flow cytometry. Cardiac tissues from the same cohorts were subjected to immunoblot analysis to detect the phosphorylated and total Akt. A. The immune cells isolated from DMSO- or rapamycin-treated hearts were labeled with CD45, CD11b, CCR2, Ly6G, CD206, CD64, F4/80, Ly6C, and MHCII. After forward and side scatter selection, cMPs were gated as CD45+CD11b+Ly6Glow cells. The selected cMP population was gated further with CD206 and CD64 and is presented individually as DMSO or rapamycin group or with the two group over-layed. The percentage of CD206highCD64low or CD206lowCD64high were indicated. B. Positive correlation F4/80 with CD206. C. The CD45+CD11b+Ly6Glow population was further gated based on Ly6C and F4/80. Percentage of Ly6ClowF4/80high, Ly6CmidF4/80mid, and Ly6ChighF4/80mid were 11.5%, 33.2%, and 9.2% for DMSO-treated hearts and 52%, 12.8%, and 0.4% for rapamycin-treated group. D. The landscape changes of cMPs. E. Heat map of the median fluorescent intensity (MFI) of the inflammatory cell surface markers (CCR2, Ly6C, CD64), antiinflammatory markers (CD206, F4/80), macrophage maturation marker (MHCII) and myeloid marker (CD45) of cMPs from DMSO- or rapamycin-treated hearts. F. Expression of IL1β or TNFα from cMPs isolated from DMSO- or rapamycin-treated MI hearts at indicated time points. n=6 to 8 MI hearts (some data points are from pooled MI tissue). G. IL6 expression from cMPs isolated from DMSO- or rapamycin-treated hearts 7-day after MI. H. Protein samples extracted from hearts 7-day after MI were subjected to immunoblot analysis. Individual lanes represent different hearts. The activation of Akt was determined by the levels of p-AktSer473 and p-AktThr308. I and J. Summarized data from experiments performed as in H. n=4–6 MI hearts. * p<0.05 ** p<0.01.