Fig 2: Endothelial-specific Klf3 knockout mice show increased CD31hiEMCNhi vessels and accelerated bone regeneration. A and B, Representative images (A) and quantification (B) of CD31 (green) and EMCN (red) immunostaining in bone regeneration area after femoral trabecular bone ablation of 3-month-old mice. Nuclei, DAPI (blue). Scale bar, 100 µm. C and D, Representative images (C) and quantification (D) of alkaline phosphatase (ALP, green) immunostaining in bone regeneration area of 3-month-old mice. Nuclei, DAPI (blue). Scale bar, 100 µm. E and F, Immunohistochemical staining (E) and quantification (F) of osteocalcin positive cells (OCN+, brown) in bone regeneration area of 3-month-old mice. Red arrows point at positive cells. Scale bar, 100 µm. G and H, Representative images (G) and quantification (H) of TRAP (red) staining in bone regeneration area of 3-month-old mice. Scale bar, 100 µm. I, Representative images of HE staining in distal femora of 3-month-old mice. Scale bar, 200 µm. J and K, Representative µCT images (J) and quantitative µCT analysis (K) of bone regeneration after femoral trabecular bone ablation of 3-month-old mice. Selected areas for the measurements of bone volume (BV)/tissue volume (TV) were indicated with a yellow square. Data are shown as mean ± SD. (n = 6 in B, D, F, H and K). *P < .05; **P < .01 by two-tailed Student's t test

Fig 3: The GST-KLF1 DBD protein displaces endogenous KLF1 at a number of KLF1 target genes in vivo. (A) Schematic depicting the experimental design of the ChIP experiment. (B) ChIPs were performed using a-KLF1, a-GST and a-IgG in both in MEL + GST and MEL + GST-KLF1 DBD clones, (n = 4 for each group per IP). Data are represented as the fold-change enrichment of IP over input, where the negative control for each IP, the Klf1 promoter for GST, and the Klf8 +33 kb locus for both KLF1 and IgG, was set to 1. The Klf3 1b and Klf8 promoters have been included as positive controls while the Klf1 promoter, and Klf3 +10 kb and Klf8 +33 kb genomic regions are shown as negative controls. Error bars shown represent standard error of the mean, p values indicate the difference between the means, *p < 0.05, **p < 0.01 (paired Student’s two-tailed t test).

Fig 4: No differences were detected in energy intake or output between Klf3+/+ and Klf3-/- mice. A: Food intake was measured daily over 3 days in male 12-week Klf3+/+ (n = 7–9) and Klf3-/- (n = 5–6) mice fed either chow or high-fat diet. B: Energy in feces was determined by bomb calorimetry and data corrected for daily fecal output. Klf3+/+, n = 10; Klf3-/-, n = 7–8. C and D: Total energy expenditure determined from Vo2 and expressed as kJ/day per mouse and per gram of lean body mass (n = 7–10 per group). Data are means ± SEM. **P < 0.01, ***P < 0.001, chow vs. high-fat diet by ANCOVA. E and F: Relationship between individual energy expenditure data calculated as heat production and lean body mass; diet, but not genotype, significantly affects energy expenditure.

Fig 5: Klf3−/− mice are lean and resistant to diet-induced obesity. A: Body length of 9-week-old male mice on chow diet (Klf3+/+, n = 8; Klf3−/−, n = 5). Data are means ± SEM. ***P < 0.001. B and C: Male body weight with respect to age. Mice were placed on chow diet (Klf3+/+, n = 48; Klf3+/−, n = 57; and Klf3−/−, n = 11) (B) or high-fat diet (C) from age 3–12 weeks (Klf3+/+, n = 20; Klf3+/−, n = 24; and Klf3−/−, n = 6). D–G: Tissue weights as determined by dissection and individual weighing of tissues in chow diet (Klf3+/+, n = 31; Klf3+/−, n = 53; and Klf3−/−, n = 11) (D and F) and high-fat diet (Klf3+/+, n = 25; Klf3+/−, n = 20; and Klf3−/−, n = 6) (E and G). BAT, brown adipose tissue; EPI, epididymal; ING, inguinal subcutaneous; RP, retroperitoneal. Data are means ± SEM. *P < 0.05, **P < 0.01, ***P < 0.001, Klf3+/+ vs. Klf3−/−; #P < 0.05, ##P < 0.01, ###P < 0.001, Klf3+/− vs. Klf3−/−. H: Representative photographs of hematoxylin-eosin stained epididymal WAT (×20 magnification) from Klf3+/+ and Klf3−/− male mice fed either chow or high-fat diet. I: Size distribution of epididymal adipocytes. Left panel: Adipocytes from male mice on chow diet. Right panel: Adipocytes from male mice on high-fat diet. Klf3+/+, n = 2–7; Klf3−/−, n = 3–6 (200–300 cells measured per mouse).