Fig 2: FZD5 promotes G1/S transition and DNA replication.A Cell cycle of MDA-MB-231 cells was analyzed by flow cytometry. B CDK2, Cyclin E2, Cyclin A2, and PCNA expression in MDA-MB-231 cells was detected by Western blot. C DNA replication of MDA-MB-231 cells was analyzed by EDU staining. Scalebar: 200 μm, mean ± SD, n = 3. *P < 0.05, **P < 0.01, vs. shCtrl. D Cell cycle of Hs-578t cells was analyzed by flow cytometry. E CDK2, Cyclin E2, Cyclin A2, and PCNA expression in Hs-578t cells was detected by Western blot. F DNA replication of Hs-578t cells was analyzed by EDU staining. Scale bar: 200 μm, mean ± SD, n = 3. *P < 0.05, **P < 0.01, vs. OE Ctrl.

Fig 3: FOXM1 acts as a downstream effecter of FZD5.A CCLE database was interrogated for FZD5, FOXM1, BRCA1, and BIRC5 expression. Correlation between two genes in a total of 57 breast cancer cell lines was analyzed by Pearson statistics. B FOXM1, BRCA1, and BIRC5 expression in MDA-MB-231 cells was detected by Western blot. C FOXM1, BRCA1, and BIRC5 expression in Hs-578t cells was detected by Western blot. D Binding sites for FOXM1 on BRCA1 and BIRC5 promoters were shown. E Binding of FOXM1 to BRCA1 and BIRC5 promoters in MDA-MB-231 cells was analyzed by ChIP and real-time PCR, mean ± SD, n = 3. **P < 0.01, ***P < 0.001, vs. IgG. NC negative control. F FOXM1, BRCA1, and BIRC5 expression in Hs-578t cells was detected by Western blot. G Active β-catenin expression in MDA-MB-231 cells was detected by Western blot. H FOXM1 expression in MDA-MB-231 cells was detected by Western blot.

Fig 4: FZD5 induces cell growth in vitro and in vivo.A FZD5 expression in MDA-MB-231 cells stably transfected with shCtrl or shFZD5-1/2 was detected by Western blot and real-time PCR. B Viability of MDA-MB-231 cells was analyzed by CCK8. C Growth of MDA-MB-231 cells was determined by Colony formation, mean ± SD, n = 3. **P < 0.01, ***P < 0.001, vs. shCtrl. D FZD5 expression in Hs-578t cells stably transfected with OE Ctrl or OE FZD5 was detected by Western blot and real-time PCR. E Viability of Hs-578t cells was analyzed by CCK8. F Growth of Hs-578t cells was determined by colony formation, mean ± SD, n = 3. *P < 0.05, **P < 0.01, ***P < 0.001, vs. OE Ctrl. G Growth curves of xenograft tumors from MDA-MB-231 cells were shown. H Phosphorylated histone 3 (p-H3) expression in xenograft tumors was detected by immunohistochemistry. Scalebar: 25 μm, mean ± SD, n = 5. **P < 0.01, ***P < 0.001, vs. shCtrl.

Fig 5: TRP120 directly interacts with Fzd5. (A) rTRP120-FL interacts with rFzd2, 4, 5, 7, 9, and 10 ECD truncate proteins. To measure direct binding of TRP120 and Fzd proteins, recombinant Fzd extracellular domain (ECD) truncated proteins dissolved in PBS, or a blank, were adsorbed onto an ELISA plate and rTRP120-FL (gray) or rWnt5a (white) was added as an interacting protein. Positive interactions were detected by incubation with α-TRP120-I1 or α-Wnt5a followed by alkaline phosphatase-conjugated secondary antibodies. Data are an average from three independent experiments, and values represent the mean plus or minus standard deviation. (B) THP-1 cells were treated with rTrx- or rTRP120-FL-coated microspheres for 1 h. Cells were dual stained for coated microspheres (“MS,” tetramethylrhodamine isothiocyanate [TRITC], red) or Fzd (fluorescein isothiocyanate [FITC], green), and nuclei were stained with DAPI (blue). Arrow represents dimensions of line profile graph of FITC channel (black line) and TRITC channel (dashed line). Scale bar = 10 μm. Images are representative of three independent experiments. (C) THP-1 cells were infected with E. chaffeensis (MOI 100), and whole-cell lysate was harvested 70 hpi. Lysate was run over immobilized α-TRP120, and the input lysate as well as the immunoprecipitated eluates was probed for Fzd5 and Fzd8 by immunoblotting. (D to F) One hundred nanomolar rTRP120-FL (D and F) or rTRP120-TRD (E) was immobilized on a Ni-nitrilotriacetic acid (NTA) sensor chip, and 2-fold dilutions (1,600 nM to 50 nM) of solubilized rFzd5-ECD (D and E) or rFzd8-ECD (F) were sequentially run over the chip to determine binding affinity as analyzed by Biacore T100. Sensorgrams are representative of two independent experiments. (G) Binding affinities are reported.