Fig 2: sFRP-4 inhibits HCC cell viability and proliferation, and promotes HCC cell apoptosis. (A) Transfection efficiency of sFRP-4-OE and si-sFRP-4 in HCCLM3 and Huh7 cells. (B) Effect of sFRP-4 on HCCLM3 and Huh7 cell viability, as evaluated by performing CCK-8 assays. (C) Effect of sFRP-4 on HCCLM3 and Huh7 cell proliferation, as evaluated by performing BrdU ELISA assays. Effect of sFRP-4 on HCCLM3 and Huh7 cell apoptosis was (D) evaluated via flow cytometry and (E) quantified. Data were analyzed using one-way ANOVA followed by Bonferroni's post hoc test. Data are presented as the mean ± SD from three independent experiments. *P<0.05 and **P<0.001 vs. CON. sFRP-4, secreted frizzled-related protein 4; HCC, hepatocellular carcinoma; OE, overexpression; si, small interfering RNA; CCK-8, Cell Counting Kit-8; CON, blank control; NC, negative control (empty vectors and si-NC); OD, optical density.

Fig 3: Expression of SLC20A1, Wif1, β-catenin and Ki67, assessed by tissue microarray in 52 somatotroph adenomas. (A) Immunohistochemistry analysis. Scale bar, 60 µm. (B) Western blotting showed higher Wif1 and sFRP4 protein levels and lower β-catenin level in non-invasive adenomas compared with invasive adenomas. *P<0.05, **P<0.01 vs. respective non-invasive group. SLC20A1, solute carrier family 20 member 1; Wif1, Wnt inhibitory factor 1; sFRP4, secreted frizzled-related protein 4.

Fig 4: iBAT paucity in Myf5Cre;Fstl1fl/fl mice(A) 3-day-old control and Myf5Cre;Fstl1fl/fl KO mice housed at room temperature (RT) and the size of iBAT.(B) Histology of iBAT of 3-day-old mice housed at RT.(C) Body surface temperature of newborn mice separated from dams at RT for 3 h. Periscapular skin temperature is quantified on the right (n = 5).(D) Survival curves of Myf5Cre;Fstl1fl/fl KO mice housed at 22°C (n = 8) or 30°C (n = 12).(E) Histology of iBAT from 2- and 12-day-old mice housed at 30°C.(F) Morphology of iBAT from mice housed at 30°C.(G) iBAT-to-body weight ratio of 4- to 5-day-old female mice housed at 30°C (n = 6–8).(H) Flow cytometry quantification of adipocyte progenitors (Lin−SFRP4+) and committed preadipocytes (Lin−SFRP4+ and Lin−PDGFRA+NRP1+) within the SVF of iBAT from 7-day-old mice housed at RT.(I) Fstl1fl/fl iBAT SVF cells were infected with lentiviruses expressing EGFP or Cre recombinase, followed by adipogenic differentiation. Expression of genes involved in adipogenesis and thermogenesis was determined (n = 3).(J–L) Generation of skeletal muscle-specific FSTL1 KO (J). iBAT morphology (K) and UCP1 protein expression (L) from adult mice were determined.(M–P) Fstl1CreERT2;Rosa26DTR mice were generated for DT-mediated cell ablation (M). Shown is expression of UCP1 protein in iBAT (N). Rectal temperature was determined during acute cold challenge (O). Body weight loss upon 6-h cold treatment was measured (P).Data are presented as mean ± SEM. *p < 0.05, **p < 0.01, ***p < 0.001 by unpaired Student’s t test (C, G, I, and P), two-way ANOVA (O), and Mantel-Cox test (D).

Fig 5: Effects of SFRP4 knockdown on the skin of aging mice. (A) Time course of the experiment. (B) RT-PCR analysis of SFRP4 and SASP gene expression in total skin extract. (C) Western blot analysis of collagen III and SFRP4 expression in whole skin extracts. SFRP4 siRNA treatment suppressed the expression of SASP factors and aging-related proteins, and increased collagen III. (D) H-E staining of skin from young (15 weeks old), control (PBS) aged, negative siRNA, and SFRP4 siRNA mice; thinning of subcutaneous adipose tissue and panniculus carnosus was observed in the control aged and negative siRNA groups. SFRP4 siRNA treatment improved these phenotypes. SF: Subcutaneous fat tissue; PC: Panniculus carnosus. Bar = 100 μm. (E) Skin in each intervention mouse; Masson’s trichrome staining; reduced thickness and density of collagen fibers and dermal connective tissue were observed in control aged mice and negative siRNA-treated mice, but the same improved with SFRP4 siRNA treatment. Collagen fibers are shown at higher magnification in the lower panel. Bar = 100 μm in the overall view, bar: 5 μm in magnified view. *; P < 0.05. Experiments were performed with n = 5 for each group. One-way ANOVA and Tukey’s post hoc test were used to compare differences between three groups.