Fig 2: LRP5 gene expression of hPDLSCs after interference with shRNA plasmid and β-catenin protein level after disturbance with shRNA plasmids. a LRP5 gene expression after interference of different concentrations of shRNA plasmid. b LRP5 protein level after disturbance with 1.5 μg/μl shRNA plasmids. c β-catenin protein detection after shRNA plasmid and/or AuNP treatment. *p < 0.05. AuNP gold nanoparticle, a.u. arbitrary units, shRNA short hairpin RNA

Fig 3: MANF interacts with LRP5.A Immunofluorescence shows the co-localization of LRP5 (green) and MANF (red) in the liver tissues after 2/3 PH. DAPI was used to stain the nuclei (blue). Scale bar = 20 μm. B The interaction of MANF and LRP5 was detected by Co-IP in the liver tissues after 2/3 PH. The tissues were lysed for Co-IP assay and precipitated by using anti-LRP5 antibody. The isotype IgG was used as a negative control. The bound proteins were blotted by using the antibodies as indicated. IgG (H) and IgG (L) represent the heavy chain and light chain of IgG, respectively. C Schematic representation of LRP5’s fragments. Two truncated forms of LRP5 include LRP5-N and LRP5-C. Numbers indicate the amino acid position. TM: transmembrane domain. D–G The interaction of MANF and LRP5 was detected by GST pull-down assay. GST-MANF, His-LRP5-C, His-LRP5-N, His-MANF, GST-LRP5-C, and GST-LRP5-N fusion proteins were purified. GST protein was used as a negative control. The proteins were detected by Coomassie blue staining and immunoblotting, respectively. H Schematic diagram of MANF in regulating Wnt/β-catenin pathway.

Fig 4: The expression levels of Wnt signaling pathway-related genes and proteins were decreased in Epi-GMSCs. (A) RT-qPCR analysis of the expression of LRP5, LRP6, COL1A1, β-catenin, RUNX2, WNT3, WNT5A, WNT11, FZD5, FZD7, and TCF7L2 after days 3, 9, and 15 of incubation in GECCM (n = 3, ****P < 0.0001). (B) WB and (C) quantitative analysis of the expression of LRP5, COL1A1, β-catenin, and RUNX2 after days 3, 9, and 15 of incubation in GECCM. Full-length blots/gels are presented in Supplementary Fig. 4. (n = 3, *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001). (D) Quantitative analysis of β-catenin immunofluorescence intensity using ImageJ. (n = 3, *P < 0.05). (E) Representative confocal fluorescence images showing the subcellular localization of β-catenin (red). Cytoskeleton analysis was performed by immunofluorescence staining with F-actin (green). (n = 3)

Fig 5: Myeloid-specific LRP5, but not LRP6, KO, leads to increased myocardial ischemia-reperfusion injury and NET formation.Mice lacking LRP5 (Lrp5M, (a)) or LRP6 (Lrp6M, (b)) in myeloid cells and their corresponding wildtype (WT) littermate control mice were subjected to myocardial ischemia-reperfusion injury. Infarction area (I) normalized against risk area (R) and Ejection Fractions before and after myocardial ischemia-reperfusion injury are shown. The ratios of risk area (R) to total area (T) are also shown. Each datum point represents one mouse (n = 8 for WT and n = 10 for Lrp5M in (a). n = 7 for WT and n = 7 for Lrp6M in (b)). Data were combined from three independent experiments. c Representative heart section images stained with triphenyl tetrazolium chloride from mice subjected to myocardial ischemia-reperfusion injury for (a, b). d Neutrophil presence in injured hearts determined by flowcytometry. Each datum point represents one mouse (n = 10 for WT and n = 8 for Lrp5M). Data were combined from three independent experiments. e Pearson’s coefficients of co-localization of MPO and citrullinated histone (c-His) in the injured heart sections. Each datum point is one section. Three independent sections per mouse and five mice per group were analyzed. f Representative images of heart sections stained with anti-MPO and anti-citrullinated histone (Cit-His). g, h LRP5-deificient (KO) and wildtype (WT) neutrophils were subjected to Western analysis to examine citrullinated histone or ELISA to detect extracellular DNA. Each datum point represents one mouse (n = 3 for WT and n = 3 for KO). Data were combined from three independent experiments. Data in this figure are all presented as mean ± sem with p values (Student’s t-test, Two-tailed, unpaired).