Research Paper Volume 13, Issue 6 pp 8989—9010

Primary cilia regulate gastric cancer-induced bone loss via cilia/Wnt/β-catenin signaling pathway

Abnormal overexpression of cilia caused by gastric cancer activates the Wnt/β-catenin signaling pathway in MSCs. (A) Analysis of primary cilia in MSC, MSC+HGC27, MSC+ HGC27+DKK1 and MSC+HGC27+chloral hydrate by immunofluorescence. Primary cilia were stained with anti-acetylated α-tubulin (axoneme, green) and anti-γ-tubulin (basal body, red) antibodies. Nuclei were stained with DAPI (blue). White arrows indicate cilia. Scale bars, 20 μm. (B) Western blot analysis of acetylated α-tubulin and γ-tubulin expression in MSC, MSC+HGC27 and MSC+HGC27+DKK1. (C) Quantitative analysis of protein levels in (B). Protein levels were normalized to GAPDH. (D) Analysis of β-catenin (red) expression by immunofluorescence in MSC, MSC+HGC27, and MSC+HGC27+DKK1 groups. Nuclei were stained with DAPI (blue). Scale bars, 20 μm. (E) MSC, or MSC co-cultured with HGC27 with or without DKK1 treatment were analyzed for Wnt3a (red) expression. Nuclei were stained with DAPI (blue). Scale bar, 100 μm. (F) Quantitative analysis of the fluorescence intensity in (E). (G) Western blot analysis comparing Naked1, Axin1, or β-catenin expression in the MSC, MSC+HGC27, and MSC+HGC27+DKK1 groups. (H) Quantitative analysis of protein levels in (G). The protein levels were normalized to GAPDH. (I) Western blot analysis comparing Naked1, Axin1, or β-catenin expression in the MSC, MSC+HGC27 and MSC+HGC27+chloral hydrate groups. (J) Quantitative analysis of protein levels in (I). The protein levels were normalized to GAPDH. (K) qRT-PCR results showing Wnt3a, β-catenin, TCF-1, and GSK-3β transcription levels in MSC, MSC+HGC27, MSC+HGC27+DKK1 on day 3 following OS induction. The gene expression levels were normalized to GAPDH expression. Data are shown as mean ± SEM. Statistical differences were obtained using One-way ANOVA with post-hoc testing, *, p

Figure 6. Abnormal overexpression of cilia caused by gastric cancer activates the Wnt/β-catenin signaling pathway in MSCs. (A) Analysis of primary cilia in MSC, MSC+HGC27, MSC+ HGC27+DKK1 and MSC+HGC27+chloral hydrate by immunofluorescence. Primary cilia were stained with anti-acetylated α-tubulin (axoneme, green) and anti-γ-tubulin (basal body, red) antibodies. Nuclei were stained with DAPI (blue). White arrows indicate cilia. Scale bars, 20 μm. (B) Western blot analysis of acetylated α-tubulin and γ-tubulin expression in MSC, MSC+HGC27 and MSC+HGC27+DKK1. (C) Quantitative analysis of protein levels in (B). Protein levels were normalized to GAPDH. (D) Analysis of β-catenin (red) expression by immunofluorescence in MSC, MSC+HGC27, and MSC+HGC27+DKK1 groups. Nuclei were stained with DAPI (blue). Scale bars, 20 μm. (E) MSC, or MSC co-cultured with HGC27 with or without DKK1 treatment were analyzed for Wnt3a (red) expression. Nuclei were stained with DAPI (blue). Scale bar, 100 μm. (F) Quantitative analysis of the fluorescence intensity in (E). (G) Western blot analysis comparing Naked1, Axin1, or β-catenin expression in the MSC, MSC+HGC27, and MSC+HGC27+DKK1 groups. (H) Quantitative analysis of protein levels in (G). The protein levels were normalized to GAPDH. (I) Western blot analysis comparing Naked1, Axin1, or β-catenin expression in the MSC, MSC+HGC27 and MSC+HGC27+chloral hydrate groups. (J) Quantitative analysis of protein levels in (I). The protein levels were normalized to GAPDH. (K) qRT-PCR results showing Wnt3a, β-catenin, TCF-1, and GSK-3β transcription levels in MSC, MSC+HGC27, MSC+HGC27+DKK1 on day 3 following OS induction. The gene expression levels were normalized to GAPDH expression. Data are shown as mean ± SEM. Statistical differences were obtained using One-way ANOVA with post-hoc testing, *, p<0.05, **, p<0.01, ***, p<0.001. NS, not statistically significant, n=3, per-group. Ace-tubulin, acetylated α-tubulin.