Journal: Nature communications

Article Title: RSPO2-LGR5 signaling has tumour-suppressive activity in colorectal cancer.

doi: 10.1038/ncomms4149

Figure Lengend Snippet: Figure 1 | RSPO2 expression is downregulated in CRC tumours. (a) Relative RSPO2 mRNA expression in CRC cell lines. RSPO2 mRNA levels were determined using qRT–PCR. Normal mucosa samples were used as a control (100%). Mean±s.d., data were calculated from triplicates. (b) qRT–PCR analysis of the relative RSPO2 mRNA expression in primary CRC tumours. The expression of RSPO2 was normalized to b-actin. Data were calculated from triplicates. Each bar represents the value of DDCt ¼ DCt[N] DCt[T]. [T] represents colorectal tumour, and [N] is the matched normal mucosa. The copy number variation (CNV) of RSPO2 within the concomitant tumour genome was determined with TaqMan copy number assay. (c) Western blotting for RSPO2 protein in CRC tumours. The results from five randomly selected pairs of CRC tumours (T) and matched adjacent colonic mucosa (N) are presented. (d) Immunohistochemical staining of RSPO2 in CRC tumours (T) and matched normal mucosa (N). The C þ panel is the negative control without the anti-RSPO2 antibody. Scale bar: 50 mm.

Article Snippet: ZNRF3 (sc-86958), Cyclin D1 (sc-20044) and RSPO2 (sc-74883) antibodies were purchased from Santa Cruz Biotechnology.

Techniques: Expressing, Quantitative RT-PCR, Control, TaqMan Copy Number Assay, Western Blot, Immunohistochemical staining, Staining, Negative Control

Journal: Nature communications

Article Title: RSPO2-LGR5 signaling has tumour-suppressive activity in colorectal cancer.

doi: 10.1038/ncomms4149

Figure Lengend Snippet: Figure 2 | The RSPO2 promoter is hypermethylated in CRC cells and tumours. (a) Schematic representation of the CpG island in the RSPO2 promoter region. Vertical bars indicate the location of the CpG sites. Arrows indicate the position of primers for MSP. Positions 78 to þ 336 indicate the region that was sequenced by bisulphite sequencing PCR. (b) RSPO2 expression reactivation. Cells were treated with 5 mM 5-aza-20-deoxycytidine (5-Aza-dC) for 3 days and/or 0.3 mM trichostatin A (TSA) for 1 day, and the RSPO2 mRNA level was determined using qRT PCR. Error bars indicate s.d. of three independent experiments. (c) Bisulphite sequencing of the RSPO2 promoter in HEK293 and CRC cell lines. Open and filled circles represent unmethylated and methylated CpG sites, respectively. Each row represents a single clone. (d) Methylation analysis of RSPO2 in CRC cells, primary tumours (T) and matched normal mucosa (N) by MSP using primers specific for methylated (M) or unmethylated (U) templates. (e) Bisulphite sequencing of primary CRC tumours (T) and matched normal mucosa (N). Error bars indicate the s.d.

Article Snippet: ZNRF3 (sc-86958), Cyclin D1 (sc-20044) and RSPO2 (sc-74883) antibodies were purchased from Santa Cruz Biotechnology.

Techniques: Bisulfite Sequencing, Expressing, Quantitative RT-PCR, Methylation

Journal: Nature communications

Article Title: RSPO2-LGR5 signaling has tumour-suppressive activity in colorectal cancer.

doi: 10.1038/ncomms4149

Figure Lengend Snippet: Figure 4 | RSPO2 suppresses Wnt/b-catenin signaling in CRC cells. (a) b-catenin reporter assay in three CRC cell lines following RSPO2 overexpression. Error bars indicate s.d. of three independent experiments. (b) b-catenin reporter assay in response to RSPO2 knockdown. Stable RSPO2-overexpressing clones of both LS174T and DLD1 cells were used for si-RSPO2 knockdown. A non-targeting siRNA was used as a control. Error bars indicate s.d. of three independent experiments. Statistical significance was determined by Student’s t-test. *Po0.05; **Po0.01. (c) The effect of RSPO2 on Wnt target gene expression. For RSPO2 overexpression (left panel), cells were transiently transfected with either the RSPO2 overexpression vector (p-R2) or the control vector pcDNA3.1 (Vec). For RSPO2 knockdown (right panel), stable clones R no. 1 of both LS174T and DLD1 in Fig. 3b were used for si-RSPO2 (si-R2) knockdown. A non-targeting siRNA was used as a control (C þ ). (d) The effect of RSPO2 protein exposure on phosphorylated LRP6 (pLRP6), c-Myc, CD44 and b-catenin accumulation. Cells were treated with 0, 10 or 30 ng ml 1 RSPO2 protein for 5 h. (e) RSPO2-induced b-catenin changes in CRC cells. Cells were stimulated without (Control) or with RSPO2 (40 ng ml 1) for 5 h. b-Catenin was detected using immunofluorescence staining and confocal microscopy. Scale bar: 20 mm. (f) b-catenin mRNA levels in response to RSPO2 treatment. Cells were stimulated with 40 ng ml 1 RSPO2, and b-catenin mRNA levels were determined using qRT–PCR. Error bars indicate s.d. of three independent experiments.

Article Snippet: ZNRF3 (sc-86958), Cyclin D1 (sc-20044) and RSPO2 (sc-74883) antibodies were purchased from Santa Cruz Biotechnology.

Techniques: Reporter Assay, Over Expression, Knockdown, Clone Assay, Control, Targeted Gene Expression, Transfection, Plasmid Preparation, Staining, Confocal Microscopy, Quantitative RT-PCR

Journal: Nature communications

Article Title: RSPO2-LGR5 signaling has tumour-suppressive activity in colorectal cancer.

doi: 10.1038/ncomms4149

Figure Lengend Snippet: Figure 5 | RSPO2 promotes Wnt signaling and tumour cell growth in CRC HT29 cells. (a) b-catenin reporter assay in HT29 cells with RSPO2 overexpression. Error bars indicate s.d. of three independent experiments. (b) b-catenin reporter assay in HT29 cells following RSPO2 knockdown. HT29 cells were transfected with either targeting siRNA (si-RSPO2) or non-targeting siRNA. Cells subjected to the transfection procedure in the absence of siRNA were set as mock-treated control (Mock). Error bars indicate s.d. of three independent experiments. **Po0.01 versus non-targeting group. (c) The effect of RSPO2 protein exposure on the accumulation of endogenous Wnt signaling proteins. HT29 cells were treated with 0, 10 or 30 ng ml 1 RSPO2 protein for 5 h. (d) RSPO2-induced cytosolic and nuclear b-catenin changes in HT29 cells. Cells were stimulated without (Control) or with RSPO2 (40 ng ml 1) for 5 h. b-Catenin was detected using immunofluorescence staining and confocal microscopy. Scale bar: 20 mm. (e) b-catenin reporter assay in nine CRC cell lines following RSPO2 overexpression. Data were calculated from triplicates. (f) RSPO2 promotes HT29 CRC tumour cell growth. The clonogenic assay was performed on two randomly selected stable RSPO2-overexpressing clones (R no. 1 and R no. 2) and an empty vector control (Vec). The RSPO2 protein level was determined using western blotting (top). Error bars indicate s.d. of three independent experiments. **Po0.01 versus Vec. (g) The effects of RSPO2 knockdown on HT29 cell proliferation. Data were calculated from triplicates. **Po0.01 versus non-targeting group. (b,f,g) Mean±s.d.; statistical significance was determined by Student’s t-test.

Article Snippet: ZNRF3 (sc-86958), Cyclin D1 (sc-20044) and RSPO2 (sc-74883) antibodies were purchased from Santa Cruz Biotechnology.

Techniques: Reporter Assay, Over Expression, Knockdown, Transfection, Control, Staining, Confocal Microscopy, Clonogenic Assay, Clone Assay, Plasmid Preparation, Western Blot

Journal: Nature communications

Article Title: RSPO2-LGR5 signaling has tumour-suppressive activity in colorectal cancer.

doi: 10.1038/ncomms4149

Figure Lengend Snippet: Figure 6 | RSPO2-induced Wnt signaling suppression is LGR5-dependent. (a) Time course of Wnt pathway protein levels following RSPO2 treatment. Cells were stimulated with RSPO2 (40 ng ml 1) for 0–6 h, and phosphorylated LRP6 (pLRP6), total LRP6 (tLRP6), c-Myc and cytosolic b-catenin were probed using western blotting. (b) LGR5 mRNA levels in response to RSPO2 treatment. Cells were stimulated with RSPO2 (40 ng ml 1) for 0 6 h. LGR5 mRNA levels were determined using qRT–PCR. Error bars indicate the s.d. of three independent experiments. (c) The effect of RSPO2 exposure on LGR5 protein expression. Cells were stimulated without (Control) or with RSPO2 (40 ng ml 1) for 80 min or 4 h. LGR5 protein was detected using immunofluorescence staining and confocal microscopy. Scale bar: 20 mm. (d) LGR5 knockdown attenuated RSPO2-induced Wnt signaling suppression in CRC cells. DLD1 cells were transfected with either non-targeting siRNA (C þ ) or siRNA targeting LGR4 (si-LGR4) or LGR5 (si-LGR5), and 2 days later, the cells were stimulated with RSPO2 (40 n ml 1) for 5 h. (e) Overexpression of LGR5 reversed RSPO2-induced Wnt signaling activation in HEK293 cells. HEK293 cells stably overexpressing LGR4, LGR5 or empty vector (Vec) were stimulated with RSPO2 (40 ng ml 1) for 0–6 h in DMEM with 1% FBS. (f) The effects of LGR5 and RSPO2 co-transfection on b-catenin reporter activity. A quantity of 100 ng of RSPO2 expression plasmid was used. Error bars indicate the s.d. of three independent experiments.

Article Snippet: ZNRF3 (sc-86958), Cyclin D1 (sc-20044) and RSPO2 (sc-74883) antibodies were purchased from Santa Cruz Biotechnology.

Techniques: Western Blot, Quantitative RT-PCR, Expressing, Control, Staining, Confocal Microscopy, Knockdown, Transfection, Over Expression, Activation Assay, Stable Transfection, Plasmid Preparation, Cotransfection, Activity Assay

Journal: Nature communications

Article Title: RSPO2-LGR5 signaling has tumour-suppressive activity in colorectal cancer.

doi: 10.1038/ncomms4149

Figure Lengend Snippet: Figure 7 | RSPO2 interacts with LGR5 to stabilize ZNRF3 in CRC cells. (a) The effect of LGR5 on RSPO2-induced Wnt signaling activation. HT29 cells stably overexpressing LGR5 (HT29-LGR5) or empty vector (HT29-Vec) were stimulated with RSPO2 (40 ng ml 1) for 0–6 h. (b) The effect of LGR5 on RSPO2-induced cell proliferation. HT29 cells stably expressing LGR5 (HT29-LGR5) or empty vector (HT29-Vec) were treated without or with 40 ng ml 1 RSPO2, and the cell numbers were determined by the MTT assay. The LGR5 protein level was determined using western blotting (Supplementary Fig. 12). Data were calculated from triplicates. *Po0.05. (c) Clonogenic assay in HT29 cells that stably overexpress LGR5. A stable LGR5-overexpressing clone (LGR5) and a control clone with empty vector (Vec) were used for experiments. Error bars indicate s.d. of three independent experiments. **Po0.01. (d) RSPO2 increases the level of membrane-associated ZNRF3 in CRC cells. Cells were stimulated with 40 ng ml 1 RSPO2 for 6 h, and the total cell lysate (TCL) and the membrane protein (avidin pull-down) were probed using western blotting. (e) LGR5 promotes RSPO2- induced membrane-associated ZNRF3 turnover. HT29 cells stably overexpressing LGR5 (HT29-LGR5) or empty vector (HT29-Vec) were stimulated with 40 ng ml 1 RSPO2 for 6 h. (b,c) Mean±s.d.; statistical significance was determined using Student’s t-test.

Article Snippet: ZNRF3 (sc-86958), Cyclin D1 (sc-20044) and RSPO2 (sc-74883) antibodies were purchased from Santa Cruz Biotechnology.

Techniques: Activation Assay, Stable Transfection, Plasmid Preparation, Expressing, MTT Assay, Western Blot, Clonogenic Assay, Control, Membrane, Avidin-Biotin Assay

Journal: Nature communications

Article Title: RSPO2-LGR5 signaling has tumour-suppressive activity in colorectal cancer.

doi: 10.1038/ncomms4149

Figure Lengend Snippet: Figure 8 | Correlation between RSPO2 and LGR5 expression in CRC primary tumours. (a) Correlation analysis of RSPO2 and LGR5 expression in human CRC tumour samples. There was a negative correlation between RSPO2 and LGR5 expression (n ¼ 80, Spearman’s Po0.001). (b) A mutation from G to A occurred within the TCF-binding site of the LGR5 promoter in Case no. 125 CRC tumour sample, as determined by Sanger sequencing. (c) Wnt target gene LGR5 and c-Myc expressions in response to Wnt3a stimulation. Cells were stimulated with Wnt3a (100 ng ml 1) for 12 h, LGR5 and c-Myc mRNA levels were determined using qRT–PCR. Error bars indicate s.d. of three independent experiments. (d) Scheme of the negative feedback loop of Wnt/b-catenin pathway induced by RSPO2 and LGR5.

Article Snippet: ZNRF3 (sc-86958), Cyclin D1 (sc-20044) and RSPO2 (sc-74883) antibodies were purchased from Santa Cruz Biotechnology.

Techniques: Expressing, Mutagenesis, Binding Assay, Sequencing, Quantitative RT-PCR