Fig 2: miR-148b-3p overexpression reversed H19-mitigated EndMT in ox-LDL-treated HUVECsCells were transfected with H19 plasmids or miR-148b-3p mimic and exposed to ox-LDL. (A–E) After that, the expression levels of CD31, VE-cadherin, α-SMA, and S100A4 in HUVECs were determined using western blot analysis. (F and G) Cell migration capacity was measured using wound-healing (F) and transwell assays (G). ∗p < 0.05, ∗∗p < 0.01 versus pLVX-NC+miR-NC group; #p < 0.05, ##p < 0.01 versus pLVX-H19+miR-NC group; n = 3. Measurement data are expressed as mean ± SD. Scale bars, 100 μm.

Fig 3: S100A4 expression enhanced AKT phosphorylation and pro‐MMP2 activation. (a) AKT, P‐AKT, ERK1/2 and P‐ERK1/2 protein levels in KO1, KO2 and EV cell lysates were determined by immunoblotting. Equal cell loading was confirmed by quantifying β‐actin (input control). Data are representative of three independent experiments. We quantified the results using ImageJ. P‐AKT/AKT and P‐ERK/ERK quotient of EV cells is expressed as 1. The relative quotient of KO1 and KO2 cells is presented as the ratio to that of EV cells. Results are shown as the mean ± SE. Statistical analysis was determined using Student's t‐test. *P < 0.05. (b) Amount of pro‐MMP2 (72 kDa) and MMP2 (64 kDa) in supernatant from KO1, KO2 and EV cells was evaluated by a gelatin zymography assay. Equal cell loading was confirmed by quantifying via immunoblotting β‐actin in cell lysates (input control). Data are representative of three independent experiments. We quantified the results using ImageJ. MMP2/actin quotient of EV cells is expressed as 1. The relative quotient of KO1 and KO2 cells is presented as the ratio to that of EV cells. Results are shown as the mean ± SE. Statistical analysis was determined using Student's t‐test. *P < 0.05.

Fig 4: S100a4 deletion enhances phagocytosis in myeloid cells and increases T cell activation.a A schematic summary of experimental design. S100a4 expressing GAMs (CD45hiCD11bhi) and CD4+ TILs (CD45+CD3+CD4+) were FACS sorted by GFP expression, and used in in vitro functional assays. b Fluorescence images showing phagocytosis of pHrodo™ labeled (red) nanoparticles in FACS-sorted GFP+CD45hiCD11bhi GAMs from S100a4 heterozygous or homozygous hosts. An average number of particles/cell were calculated (n = 4 tumors each). P-value from two-tailed student t test. Error bars represent SEM. Scale bar: 50 µm. c Fluorescence images showing phagocytosis of pHrodo™ labeled (red) nanoparticles in FACS sorted CD45hi CD11bhi tumor-infiltrating GAMs from B6 or S100a4−/− hosts. An average number of particles/cell were calculated (n = 3 tumors each). P-value from two-tailed student t test. Error bars represent SEM. Scale bar: 100 µm. d Box and whiskers plot showing IFN-γ levels measured by ELISA in FACS-sorted GFP+ CD45+CD3+CD4+ tumor-infiltrating T cells from S100a4 heterozygous or homozygous hosts. P-value represents two-tailed student’s t test (exact p-value = 1.8124E−12). Whiskers represent minimum and maximum values, the line inside the box represents the mean and the box extends from the 25th to 75th percentiles. n = 3 (heterozygous) and n = 5 (homozygous) gliomas, two experimental replicates from each tumor. e T cell proliferation assay using dye dilution. T cells were isolated from B6 spleens, labeled, and stimulated with CD3/Cd28 dynabeads then cocultured with FACS sorted GFP+CD45+CD3+CD4+ tumor-infiltrating T cells for 4-days. Dotted lines represent unstimulated T cells. P-value from two-tailed student t test. Whiskers represent minimum and maximum values, the line inside the box represents the mean and the box extends from the 25th to 75th percentiles. n = 3 (heterozygous) and n = 5 (homozygous) gliomas. Source data for b–e are provided as a Source Data file.

Fig 5: Ectopic expression of HIPK2 promotes EMT in HK-2 cells. (A) HK-2 cells were transfected with pcDNA3.1 empty vector or HIPK2 ORF. The cell lysates were immunoblotted with HIPK2 antibody as indicated, to confirm the expression of HIPK2. (B) The western blotting results in (A) were quantified and shown in a graph format. The intensities of the bands corresponding to HIPK2 were compared to those corresponding to β-actin. (C) HK-2 cells expressing miR-SCR or miR-141 were transfected with empty vector or 2 μg HIPK2 ORF. Post-transfection (48 h), the protein levels of the EMT markers were analyzed by immunoblotting. The figure is representative of three experiments with similar results. (D–F) The western blotting results in (C) were quantified to determine whether a statistically significant difference exists between the groups and they are shown in a graph format (P<0.05 compared to the control). The intensities of the bands corresponding to vimentin, FSP1 and E-cadherin were compared to those corresponding to β-actin. The expression of EMT was determined as a relative change from miR–SCR transfected with vector and shown as mean ± standard error. HIPK2, homeodomain-interacting protein kinase 2; EMT, epithelial-mesenchymal transition; FSP1, fibroblast-specific protein 1.