Fig 2: Expression of Wnt5a suppresses tumor formation and increases latency in MMTV-Wnt1 mice.(A) Frequency of tumor formation in 6-month-old MMTV-Wnt1 and MMTV-Wnt1;MMTV-Wnt5a mice (P-value = 0.001). (B) Kaplan-Meier-type curve to measure tumor latency. MMTV-Wnt1;MMTV-Wnt5a mice display a 6- to 7-week delay in tumor onset (P-value = 0.0045; n = 22 MMTV-Wnt1, n = 25 MMTV-Wnt1;MMTV-Wnt5a). (C–E) Tumor proliferation. Sections from MMTV-Wnt1 (C) and MMTV-Wnt1;MMTV-Wnt5a (D) mammary tumors stained with anti-Ki-67 antibody (Ki-67, green; nuclei, blue). No statistical difference (T-test) of Ki-67 staining was observed (E). Values are shown as means +/- standard error (n = 6 MMTV-Wnt1 tumors, 3 fields per tumor; n = 5 MMTV-Wnt1;MMTV-Wnt5a tumors, 3 fields per tumor).

Fig 3: YAP activation in the adipocytes of HFD fed mice.A–D Subcutaneous WAT (sWATs) and epididymal WATs (eWATs) of male C57BL/6 J mice fed ND or HFD for 35 weeks from 8 weeks old. A, B Confocal microscopic images of cellular FABP (green) and nuclei (blue) in subcutaneous and epididymal WATs, respectively (left panel). C Cell sizes (µm2) were determined from cellular FABP areas using ImageJ. 52~78 cells were observed for each diet. Scale bars indicate 50 µm. D Correlation between cell sizes and Wnt5a mRNA levels. The x-axis indicates Wnt5a mRNA levels relative to endogenous 18 S rRNA and the y-axis indicates adipocyte sizes (µm2) as observed in sWATs after 35 w of the HFD (?), eWATs after 35 w of the HFD (?), sWAT after 35 w of the ND (¦), and eWAT after 35 w of the ND (?) mice (diet programs are shown in Fig. 1B). E Western blot analyses of Wnt5a, YAP, and 14-3-3? proteins in sWATs of four male C57BL/6 J mice fed the ND or the HFD for 35 weeks from 8 weeks old. Relative intensities of YAP proteins in sWATs were assessed by western blot (right graphs). F, G qRT-PCR of Ctgf and Ankrd1 mRNA levels in sWATs and adipocytes isolated from the sWATs of mice fed the ND or the HFD for 35 weeks from 8 weeks old. mRNA levels were normalized versus 18 S rRNA levels. qRT-PCR data are presented as means ± SEs. All experiments were conducted independently at least twice. The significances of differences were determined using a two-tailed paired Student’s t-test. *p < 0.05, **p < 0.01, ***p < 0.001; ns, not significant.

Fig 4: Wnt5a expressing tumors demonstrate a decrease in markers of the basal tumor subtype.(A) Western blot using protein lysates isolated from the epithelium of MMTV-Wnt1 and MMTV-1;MMTV-Wnt5a mammary tumors. The expression of molecular markers of basal and luminal tumor subtypes were compared. Keratin 6 and Keratin 5 were strongly down-regulated in Wnt5a expressing tumors. Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) was used as a loading control. (B–D) Immunostaining for K6. Sections from MMTV-Wnt1 (B) and MMTV-1;MMTV-Wnt5a (C) tumors were stained with anti-Keratin 6 antibody using immunofluorescence (K6 = green; nuclei = blue). The percentage of cells expressing K6 was determined and graphed (D). Values are means +/- standard error (n = 6 MMTV-Wnt1, 3 fields per tumor; n = 5 MMTV-Wnt1;MMTV-Wnt5a, 3 fields per tumor). MMTV-Wnt1;MMTV-Wnt5a tumors demonstrated a significant decrease in K6-expressing cells as measured by T-test (* = p<0.05).

Fig 5: Description of histological subtypes in MMTV-Wnt1 and MMTV-Wnt1;Wnt5a tumors.Sections from tumors were stained with H&E and the percentage in each tumor of 4 different histologic patterns: cribriform (A), papillary (B), solid (C), and squamous metaplasia (D) was determined and graphed (E). Values are shown as means +/- standard error (n = 6 MMTV-Wnt1, n = 5 MMTV-Wnt1;MMTV-Wnt5a separate tumors). Both MMTV-Wnt1 and MMTV-Wnt1;MMTV-Wnt5a mice formed tumors with a mixed histology. Although a trend toward less solid and squamous tumors was observed in tumors containing Wnt5a, the changes did not reach statistical significance as determined by Mann-Whitney test for non-parametric data.