Fig 1: Midkine inhibition prevents immunosuppressive MDSCs infiltrating into HCC tumors.Hepa 1-6 cells transduced with Sh-MDK-1, Sh-MDK-2 or Sh-NC lentivirus were orthotopically implanted into the liver of C57BL/6 mice, then treated with sorafenib (Sor) or vehicle control (Veh) for three weeks. A protein expression of midkine in midkine-depleted orthotopic Hepa 1-6 tumors was validated by western blot. B, C Ly6CintLy6G+ PMN-MDSCs and Ly6ChighLy6G- M-MDSCs in intratumoral CD11b+Gr-1+ subset of midkine-depleted orthotopic Hepa 1-6 tumors were evaluated by flow cytometry. D–F Arg1+ cells in infiltrating CD11b+Gr-1+ subset (D–E) and MDSCs subset (F) of midkine-depleted orthotopic Hepa 1-6 tumors were evaluated by flow cytometry. G, H infiltrating PMN-MDSCs and M-MDSCs of midkine-depleted orthotopic Hepa 1-6 tumors were used for T-cell suppression assay. Ki67+ (G) and Granzyme B+ (H) cytotoxic T cells were evaluated by flow cytometry. All assays were done with at least three repeats. Data were shown as mean ± s.d., *P < 0.05.
Fig 2: LRP1 knockdown in fibroblasts reverses the pulmonary fibrosis and ferroptosis induced by MDK in vivo(A–C) Representative images of H&E (A), Masson staining (B), and IHC of hydroxyproline (C) (n = 6 for each group; Scale bars: 200 μm).(D) The concentration of hydroxyproline and the Ashcroft score evaluating pulmonary fibrosis of mouse lungs. (n = 6 for each group; mean ± SD; one way ANOVA; ∗p < 0.05).(E–H) Western blotting of the protein levels of hub ferroptosis regulators (E), the level of lipid oxidation (F and G), and the GSH/GSSG ratio and MDA levels (H) in fibroblasts extracted from mice. (n = 6 for each group; mean ± SD; one way ANOVA; ∗p < 0.05; Scale bars: 50 μm).
Fig 3: AT2 secretes MDK to activate LRP1 in CTHRC1+ fibroblasts and inhibits fibrotic ferroptosis(A) Overview of the cell-cell crosstalk and communications between AT2 cells and CTHRC1+ fibroblasts in GEO: GSE135893.(B) The differences in expression levels of MDK and LRP1 between normal and IPF samples as evaluated by RT-qPCR and western blotting. (n = 4 for each group; mean ± SD; Student’s t test; ∗p < 0.05).(C and F) After co-culturing AT2 and CTHRC1+ fibroblasts (C), the protein levels of hub ferroptosis regulators in CTHRC1+ fibroblasts were evaluated by western blotting (D), and the levels of lipid oxidation were measured by flow cytometry and confocal microscope (E and F) (n = 3 for each group; Scale bars: 50 μm).(G) After the ferroptosis inhibitor Lip–1 was added in the co-culturing cell model, the protein levels of hub ferroptosis regulators were evaluated in CTHRC1+ fibroblasts. (n = 3 for each group).(H and I) the levels of lipid oxidation were measured by flow cytometry and confocal microscope (n = 3 for each group).(J) IP and co-IP assays of LRP1 and MDK in CTHRC1+ fibroblasts.(K) Subcellular localization of MDK and LRP1 in CTHRC1+ fibroblasts. (n = 3 for each group; Scale bars: 50 μm).
Fig 4: LRP1 upregulates OTUB1 to reduce ferroptosis in CTHRC1+ fibroblasts(A) Changes in LRP1 gene and protein levels after stimulation with different concentrations of MDK. (n = 3 for each group; mean ± SD; Student’s t test; ∗p < 0.05).(B and C) Heatmap and volcano plot analysis of differentially expression genes.(D) Normalized gene counts of transcript factors related to LRP1 upregulation in RNA-seq. (n = 3 for each group).(E) GO analysis of differentially expression genes.(F and G) The expression levels of OTUB1 after LRP1 silencing in CTHRC1+ fibroblasts using western blotting and immunofluorescence assay (n = 3; Scale bars: 50 μm).(H–J) The protein levels of hub ferroptosis regulators (H), the GSH/GSSG ratio and MDA level (I), and the level of lipid oxidation (J) in OTUB1 knockdown CTHRC1+ fibroblasts (n = 3). (n = 3 for each group; Scale bars: 50 μm).
Supplier Page from Abcam for Mouse Midkine ELISA Kit