Fig 2: Enhancement of Wnt5a upregulation by gp120 and morphine in the SDH. (a) Immunoblots of Wnt5a, Wnt3a, and p-JNK. (b to d) Quantitative summary of panel (a). Potentiation of gp120 and morphine on Wnt5a and p-JNK but not Wnt3a (**p < 0.01; ***p < 0.001; one-way analysis of variance with Tukey post hoc tests, n = 6). The same animals used for Figure 1 were analyzed. JNK: c-Jun N-terminal kinase.

Fig 3: WNT5A expression in human RMS. WNT5A expression level (A) of ARMS and ERMS from two publicly available microarray datasets [see (22) and (3) for datasets 1 and 2, respectively] and (B) of 10 ARMS, 10 ERMS and five normal skeletal muscle samples in the validation data set from the CWS study group measured by reverse transcription-quantitative PCR. (C) Immunohistochemical analysis of human RMS showing low or high expression of WNT5A when using the WNT5A monoclonal antibody (3D10) from Thermo Fisher Scientific, Inc (magnification, 50×). (D) WNT5A mRNA expression (three independent experiments each measured in duplicates) of the human ARMS cell lines CRL2061 and RH30 and ERMS cell lines RD and TE671 in comparison with normal skeletal muscle, which was set=1. (E) Western blot analysis of WNT5A protein levels in the supernatant (top left; please note the quantification of secreted WNT5A using HSC70, which is occasionally secreted by tumor cells (58), was not possible because RMS cell lines apparently do not secrete HSC70) and on another gel in the respective whole cell lysates (bottom left) of RMS tumor cell lines. ß-Actin detection served as loading control for whole cell lysates (bottom) and the right panel shows the respective quantification. Horizontal lines in (A-B) are mean ± SD; bars in (D) are also ± SD. Statistical analyses in (A-B) were performed by non-parametric Mann-Whitney tests and that in (D) by one-way ANOVA. *P<0.05, **P<0.01, ****P<0.0001. RMS, rhabdomyosarcoma; ARMS, alveolar RMS; ERMS, embryonal RMS.

Fig 4: SCRG1 regulated chondrogenesis of UCMSCs through Wnt5a signaling pathway. (a) Regulating pluripotency of stem cell signaling pathway was the most significantly enriched KEGG pathway based on GSEA, wnt5a was negatively correlated with this pathway after SCRG1 overexpression. (b, c) Western blot analysis showed that Wnt5a decreased after shSCRG1 transfection and was restored after administration of rhSCRG1 (450 ng/mL); protein levels of ß-catenin exhibited the opposite trend to Wnt5a. (d, e) Protein levels of COL2A1 and ACAN were detected in UCMSCs by IHC. The results showed that the protein levels of COL2A1 and ACAN were reduced following SCRG1 knockdown and restored after treated with rhWnt5a (300 ng/mL) and rhSCRG1 (450 ng/mL) protein at 24 h. Scale bar = 100 µm. Experiments were repeated three times, *P < 0.05 (t-test).

Fig 5: ABCG2 and Wnt5a are expressed in human pancreatic cancer tissue. (A) ABCG2 and Wnt5a IHC staining of PDAC and adjacent healthy tissue samples. n=24. Magnification, ×10. (B) IHC scores for ABCG2 and Wnt5a expression in PDAC and paracarcinoma tissues. ***P<0.001. (C) Correlation analysis of Wnt5a and ABCG2 IHC scores. n=24. (D) Survival analysis of patients with PDAC according to ABCG2 expression. (E) Correlation analysis of ABCG2 IHC scores and survival time in deceased patients. n=14. ABCG2, ATP binding cassette subfamily G member 2; PDAC, pancreatic ductal adenocarcinoma; IHC, immunohistochemistry.