Fig 1: IL-6 Up-regulates MMP12 in Macrophages. A Schematic diagram of tumor cells (MC38/CT26 cell lines) co-cultured with macrophage cells (RAW264.7 cell lines). All quantifications were performed by image J software for gray scale statistics. B, C Representative western blots showing the secreted MMP12 protein levels from RAW264.7 cell lines (1-2 × 105) cultured alone or co-cultured with MC38 cell lines (control, 1 × 104, 3 × 104, 5 × 104). β-Actin as the internal control. D, E Representative western blots showing the secreted MMP12 protein levels from RAW264.7 cell lines (1-2 × 105) cultured alone or co-cultured with CT26 cell lines (control, 1 × 104, 3 × 104, 5 × 104). GAPDH as the internal control. F Schematic diagram of IL-6 treated macrophages. RAW264.7 cells incubated with fresh media were served as untreated negative controls. Western blot was used to detect MMP12 in RAW 264.7 cells and GAPDH as the internal control. G, H RAW264.7 cells (1-2 × 105) were seeded in 6-well plates and treated with increasing doses of IL-6 (0, 2, 5, 10, 30 ng/mL) for 72 h. I Immune Gene data (https://www.immgen.org/ImmGenApps.html) suggest that IL-6 receptor expresses on F480+ macrophages. The colored bars refer to the expression level of IL-6 receptors on macrophages. Red represents high expression of IL-6 receptors, and green represents low expression of IL-6 receptors
Fig 2: Knockout of MMP12 in ApcMin/+ Mice Prevents Weight and Muscle Loss. A Plots of the body weight of wild-type (WT), ApcMin/+, ApcMin/+; MMP12−/− and MMP12−/− mice from 5 to 24 weeks (n = 6 per group). B The ratio of inguinal white adipose tissue to body weight (***P < 0.001; **P < 0.01, n = 5). C The ratio of skeletal muscle to body weight (*P < 0.05, n = 5). D Hematoxylin and eosin staining of inguinal white adipose tissue in WT, ApcMin/+, ApcMin/+; MMP12−/− and MMP12−/− mice at 24 weeks old. Scale bars: 5 μm. E Evaluation of the inguinal white adipose tissue across 4 groups by ImageJ software (40X) (***P < 0.001; **P < 0.01, Each cell area are shown as means ± SD; n = 5 mice per group). F Hematoxylin and eosin staining of muscle in ApcMin/+ and ApcMin/+; MMP12−/− at 24 weeks old. Scale bars: 5 μm. G Evaluation of the cross-sectional area of the gastrocnemius from ApcMin/+ mice and ApcMin/+; MMP12−/− mice by ImageJ software (40X) (*P < 0.05; data are shown as means ± SD; n = 6 mice per group)
Fig 3: MMP12 Is Up-regulated in Muscle Tissues and Macrophages of ApcMin/+ Mice. A MMP12 antibody immunostaining in the muscle of healthy individuals. Scale bar: 2 μm. B Immunostaining of MMP12 in muscle tissue of WT mice and ApcMin/+ mice. Scale bar: 5 μm. C Quantification of MM12 expression in gastrocnemius tissue was performed by ImageJ software (40X) (*P < 0.05, data are shown as means ± SD; n = 9 per group). D Representative images of dual immnofluorescent staining of macrophages (F4/80 in green) and MMP12 (in red) in WT mice are shown. The yellow areas in the merged images indicate overlapping localization of the red and green signals, indicated by the white arrows. Scale bars: 20 μm. E Quantification of MMP12 mRNA level in peritoneal macrophages isolated from WT mice and ApcMin/+ mice by qPCR (*P < 0.05; data are shown as the means ± SD; n = 6 per group). F The serum MMP12 levels detected in WT and ApcMin/+ mice at 9, 15, and 24 weeks old by ELISA (P > 0.05; data are shown as means ± SD; n = 6 per group)
Fig 4: Inhibiting MMP12 in ApcMin/+ Mice Slows Down Weight Loss. A Schematic diagram of the administration process of 17-week-old ApcMin/+ mice. The drugs were given every 2 days (MMP408: 5 mg/kg, 5-FU: 30 mg/kg). The saline group was used as a control. B Percentage of weight gain compared to the basal weight after administration of drugs in ApcMin/+ mice (**P < 0.01, data are shown as means ± SD; n = 5 per group)
Fig 5: MMP12 Degrades Insulin and Insulin-like Growth Factor-1. A Representative images of the peak were detected at 488 nm after the insulin peptide was degraded by normal mice serum. The insulin peptide was labeled with FAM (488 nm) and DABCLY (quenching fluorescence). When the insulin peptide was broken, FAM was then observed at 488 nm (resonance energy transfer, FRET). B The synthetic insulin (or IGF-1) peptide was labeled with FAM and DABCLY as shown. The insulin peptide was labeled with FAM (488 nm) and DABCLY (quenching fluorescence). When the insulin (IGF-1) peptide was broken, FAM was then observed at 488 nm. Fluorescence intensity of the peak were detected at 488 nm after the insulin (IGF-1) peptide was degraded by MMP12 using a fluorescence microplate reader. Electrospray Ionization Mass Spectrometry (IMS) was used to detect its characteristic peaks. C The MMP12 and insulin (or IGF-1) peptide interaction led to appearance of a fluorescence signal on dose-dependent (***P < 0.001, **P < 0.01; data are shown as means ± SD)
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