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R&D Systems human rspo4 recombinant protein

Identification of <t>RSPO4</t> as a methylated target gene with clinical significance. (A) MeDIP-chip study identified RSPO4 as a methylated target in CRC and NPC cell lines and primary tumors (C18, OCT83 and NH18). RSPO4 gene structure, promoter and exon 1 (UCSC Genome Browser NCBI36/hg18) are shown on the top panel. e1: exon 1. Positive methylation signal peak (blue) in HCT116 was identified by methylated DNA immunoprecipitation (MeDIP)-chip. Promoter CpG methylation of RSPO4 was also identified by double-enzyme reduced representation bisulfite sequencing (dRRBS) in HCT116 and its double knock-out of DNMT1 and DNMT3A (DKO) cells (bottom panel). (B) β-value as the indicator of methylation level of RSPO4 in cancer tissues and the normal control in TCGA datasets, as retrieved from DNMIVD. HNSC, head and neck squamous carcinoma; LUAD, lung adenocarcinoma; ESCA, esophageal carcinoma; COAD, colon adenocarcinoma; N, normal control; T, tumor. (C) RSPO4 mRNA expression levels in different cancer types in TCGA datasets, as retrieved from SangerBox. LUSC, lung squamous cell carcinoma; STES, stomach and esophageal carcinoma; READ, rectum adenocarcinoma. (D) Analyses of TCGA datasets reveal an inverse correlation between mRNA expression level and promoter methylation level of RSPO4 in ESCA and LUAD, as retrieved from DNMIVD. Each green circle represents a single clinical sample. Pearson correlation coefficient analysis is used. (E) Kaplan-Meier curve analyses show the association between RSPO4 mRNA expression and overall survival of patients with LUAD in TCGA datasets, as retrieved from KM-plotter, and CRC as retrieved from PrognoScan. CRC, colorectal carcinoma. (F) RT-PCR detected RSPO4 mRNA expression in a panel of human normal adult and fetal tissues. (G) Western blot detected RSPO4 protein level in a panel of human normal adult and fetal tissues. 293T cell line ectopically expressing RSPO4 was used as a positive control. (H) Schematic structure of RSPO4 promoter. The primers for RT-PCR and multiplex DNA PCR are indicated with arrows. Exon 1, CpG sites (short vertical lines), MSP sites and BGS region analyzed are shown. RT-PCR and MSP detected RSPO4 mRNA expression and promoter CpG methylation in cancer cell lines and non-transformed epithelial cell lines (NP69, Het-1A, NE1 and NE3), respectively. M, methylated; U, unmethylated. Ca, carcinoma; NPC, nasopharyngeal carcinoma; ESCC, esophageal squamous cell carcinoma. (I) BGS analysis of RSPO4 promoter in non-transformed epithelial cell line (NP69) and cancer cell line (C666-1). Each row of circles represented an individual promoter allele. Filled circle, methylated CpG site; open circle, unmethylated CpG site; open triangle, SNP rs6077512 (C/G). (J) RSPO4 mRNA expression in methylated/silenced cancer cell lines was detected by RT-PCR after pharmacologic demethylation treatment with Aza combined with TSA (A+T). (K) MSP analysis of RSPO4 methylation in different types of primary tumor tissues. Representative samples are shown. (L) BGS analysis of RSPO4 methylation pattern in representative primary tumor tissues and normal tissues. (M) Kaplan Meier analysis shows the association between RSPO4 promoter methylation at specific CpG sites and overall survival of patient with READ from TCGA datasets, as retrieved from MethSurv. The methylation patient groups are dichotomized by higher (β > cut-off) and lower (β < cut-off), according to a best cut-off point in MethSurv.

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1) Product Images from "RSPO4 exerts tumor suppression through antagonizing canonical and non-canonical Wnt signaling"

Article Title: RSPO4 exerts tumor suppression through antagonizing canonical and non-canonical Wnt signaling

Journal: International Journal of Biological Sciences

doi: 10.7150/ijbs.124734

Identification of RSPO4 as a methylated target gene with clinical significance. (A) MeDIP-chip study identified RSPO4 as a methylated target in CRC and NPC cell lines and primary tumors (C18, OCT83 and NH18). RSPO4 gene structure, promoter and exon 1 (UCSC Genome Browser NCBI36/hg18) are shown on the top panel. e1: exon 1. Positive methylation signal peak (blue) in HCT116 was identified by methylated DNA immunoprecipitation (MeDIP)-chip. Promoter CpG methylation of RSPO4 was also identified by double-enzyme reduced representation bisulfite sequencing (dRRBS) in HCT116 and its double knock-out of DNMT1 and DNMT3A (DKO) cells (bottom panel). (B) β-value as the indicator of methylation level of RSPO4 in cancer tissues and the normal control in TCGA datasets, as retrieved from DNMIVD. HNSC, head and neck squamous carcinoma; LUAD, lung adenocarcinoma; ESCA, esophageal carcinoma; COAD, colon adenocarcinoma; N, normal control; T, tumor. (C) RSPO4 mRNA expression levels in different cancer types in TCGA datasets, as retrieved from SangerBox. LUSC, lung squamous cell carcinoma; STES, stomach and esophageal carcinoma; READ, rectum adenocarcinoma. (D) Analyses of TCGA datasets reveal an inverse correlation between mRNA expression level and promoter methylation level of RSPO4 in ESCA and LUAD, as retrieved from DNMIVD. Each green circle represents a single clinical sample. Pearson correlation coefficient analysis is used. (E) Kaplan-Meier curve analyses show the association between RSPO4 mRNA expression and overall survival of patients with LUAD in TCGA datasets, as retrieved from KM-plotter, and CRC as retrieved from PrognoScan. CRC, colorectal carcinoma. (F) RT-PCR detected RSPO4 mRNA expression in a panel of human normal adult and fetal tissues. (G) Western blot detected RSPO4 protein level in a panel of human normal adult and fetal tissues. 293T cell line ectopically expressing RSPO4 was used as a positive control. (H) Schematic structure of RSPO4 promoter. The primers for RT-PCR and multiplex DNA PCR are indicated with arrows. Exon 1, CpG sites (short vertical lines), MSP sites and BGS region analyzed are shown. RT-PCR and MSP detected RSPO4 mRNA expression and promoter CpG methylation in cancer cell lines and non-transformed epithelial cell lines (NP69, Het-1A, NE1 and NE3), respectively. M, methylated; U, unmethylated. Ca, carcinoma; NPC, nasopharyngeal carcinoma; ESCC, esophageal squamous cell carcinoma. (I) BGS analysis of RSPO4 promoter in non-transformed epithelial cell line (NP69) and cancer cell line (C666-1). Each row of circles represented an individual promoter allele. Filled circle, methylated CpG site; open circle, unmethylated CpG site; open triangle, SNP rs6077512 (C/G). (J) RSPO4 mRNA expression in methylated/silenced cancer cell lines was detected by RT-PCR after pharmacologic demethylation treatment with Aza combined with TSA (A+T). (K) MSP analysis of RSPO4 methylation in different types of primary tumor tissues. Representative samples are shown. (L) BGS analysis of RSPO4 methylation pattern in representative primary tumor tissues and normal tissues. (M) Kaplan Meier analysis shows the association between RSPO4 promoter methylation at specific CpG sites and overall survival of patient with READ from TCGA datasets, as retrieved from MethSurv. The methylation patient groups are dichotomized by higher (β > cut-off) and lower (β < cut-off), according to a best cut-off point in MethSurv.

Figure Legend Snippet: Identification of RSPO4 as a methylated target gene with clinical significance. (A) MeDIP-chip study identified RSPO4 as a methylated target in CRC and NPC cell lines and primary tumors (C18, OCT83 and NH18). RSPO4 gene structure, promoter and exon 1 (UCSC Genome Browser NCBI36/hg18) are shown on the top panel. e1: exon 1. Positive methylation signal peak (blue) in HCT116 was identified by methylated DNA immunoprecipitation (MeDIP)-chip. Promoter CpG methylation of RSPO4 was also identified by double-enzyme reduced representation bisulfite sequencing (dRRBS) in HCT116 and its double knock-out of DNMT1 and DNMT3A (DKO) cells (bottom panel). (B) β-value as the indicator of methylation level of RSPO4 in cancer tissues and the normal control in TCGA datasets, as retrieved from DNMIVD. HNSC, head and neck squamous carcinoma; LUAD, lung adenocarcinoma; ESCA, esophageal carcinoma; COAD, colon adenocarcinoma; N, normal control; T, tumor. (C) RSPO4 mRNA expression levels in different cancer types in TCGA datasets, as retrieved from SangerBox. LUSC, lung squamous cell carcinoma; STES, stomach and esophageal carcinoma; READ, rectum adenocarcinoma. (D) Analyses of TCGA datasets reveal an inverse correlation between mRNA expression level and promoter methylation level of RSPO4 in ESCA and LUAD, as retrieved from DNMIVD. Each green circle represents a single clinical sample. Pearson correlation coefficient analysis is used. (E) Kaplan-Meier curve analyses show the association between RSPO4 mRNA expression and overall survival of patients with LUAD in TCGA datasets, as retrieved from KM-plotter, and CRC as retrieved from PrognoScan. CRC, colorectal carcinoma. (F) RT-PCR detected RSPO4 mRNA expression in a panel of human normal adult and fetal tissues. (G) Western blot detected RSPO4 protein level in a panel of human normal adult and fetal tissues. 293T cell line ectopically expressing RSPO4 was used as a positive control. (H) Schematic structure of RSPO4 promoter. The primers for RT-PCR and multiplex DNA PCR are indicated with arrows. Exon 1, CpG sites (short vertical lines), MSP sites and BGS region analyzed are shown. RT-PCR and MSP detected RSPO4 mRNA expression and promoter CpG methylation in cancer cell lines and non-transformed epithelial cell lines (NP69, Het-1A, NE1 and NE3), respectively. M, methylated; U, unmethylated. Ca, carcinoma; NPC, nasopharyngeal carcinoma; ESCC, esophageal squamous cell carcinoma. (I) BGS analysis of RSPO4 promoter in non-transformed epithelial cell line (NP69) and cancer cell line (C666-1). Each row of circles represented an individual promoter allele. Filled circle, methylated CpG site; open circle, unmethylated CpG site; open triangle, SNP rs6077512 (C/G). (J) RSPO4 mRNA expression in methylated/silenced cancer cell lines was detected by RT-PCR after pharmacologic demethylation treatment with Aza combined with TSA (A+T). (K) MSP analysis of RSPO4 methylation in different types of primary tumor tissues. Representative samples are shown. (L) BGS analysis of RSPO4 methylation pattern in representative primary tumor tissues and normal tissues. (M) Kaplan Meier analysis shows the association between RSPO4 promoter methylation at specific CpG sites and overall survival of patient with READ from TCGA datasets, as retrieved from MethSurv. The methylation patient groups are dichotomized by higher (β > cut-off) and lower (β < cut-off), according to a best cut-off point in MethSurv.

Techniques Used: Methylation, Methylated DNA Immunoprecipitation, Immunoprecipitation, CpG Methylation Assay, Methylation Sequencing, Knock-Out, Control, Expressing, Reverse Transcription Polymerase Chain Reaction, Western Blot, Positive Control, Multiplex Assay, Transformation Assay

RSPO4 encodes a secreted protein which inhibits tumor cell clonogenicity, migration, invasion and stemness. (A) Subcellular localization by immunofluorescence showed that RSPO4 protein co-localized with the endoplasmic reticulum (ER). Original magnification, ×400. Scale bar, 200μm. (B) RSPO4 protein can be detected in conditioned medium after 24 hrs posttransfection. CM, serum-free conditioned medium; TCL, total cell lysates. (C) Monolayer CFA in KYSE150, A549 and HCT116 cells. 5,000 cells were seeded in each well and colonies were counted after 2 weeks. (D) Anchorage-independent soft agar assay on KYSE150 and HCT116 cells. 5,000 cells were seeded in each well and colonies were counted after 4 weeks. (E) Flow cytometry analysis of apoptosis by Annexin V-FITC/PI staining of A549 and KYSE150 cells. Both early and late apoptotic cells (Annexin V-positive) were counted. (F) Western blot detected the protein level of cleaved caspase 3, 7, 9 and PARP in cancer cells transfected with vector- and RSPO4 . (G) In vivo tumor formation ability of LoVo cells transduced with lentivirus encoding RSPO4 or empty vector, then injected subcutaneously into BALB/c nude mice. (H) Transwell migration and invasion assay of HONE1 cells transfected with empty vector and RSPO4 plasmid. (I) Morphological changes in RSPO4 -transfected cancer cells compared with vector control after genecitin selection for 2 weeks. Original magnification, ×400. Scale bar, 200μm. (J) Indirect immunofluorescence staining of E-cadherin and vimentin in empty vector- and RSPO4 -transfected KYSE150 and A549 cells, respectively. Original magnification, ×400. Scale bar, 200μm. (K) Western blot detected the expression levels of E-cadherin, vimentin and fibronectin in RSPO4 -stably ( Left ) and transiently expressed cancer cells ( Right ). (L) Western blot detected the protein level of vimentin, N-cadherin and fibronectin in H1299 cells with knockdown of RSPO4 by siRNAs. (M) Effect of ectopic RSPO4 expression on cytoskeletal structures of A549 cells. Red, Rhodamine-labeled phalloidin; Blue, DAPI. Original magnification, ×400. Scale bar, 20μm. (N) Sphere-forming assays evaluated the stemness of cancer cells transfected with empty vector and RSPO4 plasmid. Scale bar, 100 μm. For C, D, E, F, H and N, n = 3 biologically independent replicates were examined over three independent experiments with similar results. Data are presented as mean values ± SD. For C, D, E, H and N, Student's test was performed to obtain the P values. For G, n=5 mice were used for RSPO4 and vector control, and one-way ANOVA was performed to obtain the P values.

Figure Legend Snippet: RSPO4 encodes a secreted protein which inhibits tumor cell clonogenicity, migration, invasion and stemness. (A) Subcellular localization by immunofluorescence showed that RSPO4 protein co-localized with the endoplasmic reticulum (ER). Original magnification, ×400. Scale bar, 200μm. (B) RSPO4 protein can be detected in conditioned medium after 24 hrs posttransfection. CM, serum-free conditioned medium; TCL, total cell lysates. (C) Monolayer CFA in KYSE150, A549 and HCT116 cells. 5,000 cells were seeded in each well and colonies were counted after 2 weeks. (D) Anchorage-independent soft agar assay on KYSE150 and HCT116 cells. 5,000 cells were seeded in each well and colonies were counted after 4 weeks. (E) Flow cytometry analysis of apoptosis by Annexin V-FITC/PI staining of A549 and KYSE150 cells. Both early and late apoptotic cells (Annexin V-positive) were counted. (F) Western blot detected the protein level of cleaved caspase 3, 7, 9 and PARP in cancer cells transfected with vector- and RSPO4 . (G) In vivo tumor formation ability of LoVo cells transduced with lentivirus encoding RSPO4 or empty vector, then injected subcutaneously into BALB/c nude mice. (H) Transwell migration and invasion assay of HONE1 cells transfected with empty vector and RSPO4 plasmid. (I) Morphological changes in RSPO4 -transfected cancer cells compared with vector control after genecitin selection for 2 weeks. Original magnification, ×400. Scale bar, 200μm. (J) Indirect immunofluorescence staining of E-cadherin and vimentin in empty vector- and RSPO4 -transfected KYSE150 and A549 cells, respectively. Original magnification, ×400. Scale bar, 200μm. (K) Western blot detected the expression levels of E-cadherin, vimentin and fibronectin in RSPO4 -stably ( Left ) and transiently expressed cancer cells ( Right ). (L) Western blot detected the protein level of vimentin, N-cadherin and fibronectin in H1299 cells with knockdown of RSPO4 by siRNAs. (M) Effect of ectopic RSPO4 expression on cytoskeletal structures of A549 cells. Red, Rhodamine-labeled phalloidin; Blue, DAPI. Original magnification, ×400. Scale bar, 20μm. (N) Sphere-forming assays evaluated the stemness of cancer cells transfected with empty vector and RSPO4 plasmid. Scale bar, 100 μm. For C, D, E, F, H and N, n = 3 biologically independent replicates were examined over three independent experiments with similar results. Data are presented as mean values ± SD. For C, D, E, H and N, Student's test was performed to obtain the P values. For G, n=5 mice were used for RSPO4 and vector control, and one-way ANOVA was performed to obtain the P values.

Techniques Used: Migration, Immunofluorescence, Soft Agar Assay, Flow Cytometry, Staining, Western Blot, Transfection, Plasmid Preparation, In Vivo, Transduction, Injection, Invasion Assay, Control, Selection, Expressing, Stable Transfection, Knockdown, Labeling

RSPO4 antagonizes Wnt/β-catenin signaling in cancer cells. (A) Gene set enrichment analysis of pathways in datasets of TCGA CRC patients ( Left ) and enrichment score of epithelial mesenchymal transition ( Right ). (B) Kyoto Encyclopedia of Genes and Genomes (KEGG) datasets of TCGA CRC patients ( Left ) and enrichment score of ECM receptor interaction ( Right ). (C) Immunofluorescent staining of nuclear β-catenin in cancer cells after 48 hrs transfection of RSPO4 plasmid. Red, active β-catenin (unphosphorylated at Ser33/Ser37/Thr41); Green, RSPO4 protein stained by V5 antibody; Scale bar, 200 μm. (D) TOPflash/FOPflash luciferase reporter assay evaluated β-catenin/TCF activities in vector- and RSPO4 -transfected cancer cells. (E) Transcriptional activities of CCND1 , c-MYC and MMP7 promoter were determined by luciferase reporter assay in vector- and RSPO4 -transfected tumor cells (of either LGR4+/LGR5- or LGR4-/LGR5+ phenotype). (F) Transcriptional activities of AP-1 and SRE responsive element reporters were determined by luciferase reporter assay in vector- and RSPO4 -transfected HCT116 and KYSE150 cells. (G) Western blot detection of the signaling alterations in canonical and non-canonical Wnt signaling in tumor cells transfected with RSPO4 and vector. (H) Western blot detection of the signaling alterations in canonical and non-canonical Wnt signaling in HNE1 cells stably expressing RSPO4 and vector. (I) Luciferase reporter assay detected β-catenin/TCF activities and transcriptional activities of Wnt target genes as well as AP-1 and SRE activities in H1299 cells with RSPO4 knockdown by siRNAs. (J) Western blot examined the levels of the components of canonical and non-canonical Wnt signaling in H1299 cells with RSPO4 knockdown by siRNAs. For D, E, F and I, n = 3 biologically independent replicates were examined over three independent experiments with similar results. Data are presented as mean values ± SD. Student's test was performed to obtain the P values.

Figure Legend Snippet: RSPO4 antagonizes Wnt/β-catenin signaling in cancer cells. (A) Gene set enrichment analysis of pathways in datasets of TCGA CRC patients ( Left ) and enrichment score of epithelial mesenchymal transition ( Right ). (B) Kyoto Encyclopedia of Genes and Genomes (KEGG) datasets of TCGA CRC patients ( Left ) and enrichment score of ECM receptor interaction ( Right ). (C) Immunofluorescent staining of nuclear β-catenin in cancer cells after 48 hrs transfection of RSPO4 plasmid. Red, active β-catenin (unphosphorylated at Ser33/Ser37/Thr41); Green, RSPO4 protein stained by V5 antibody; Scale bar, 200 μm. (D) TOPflash/FOPflash luciferase reporter assay evaluated β-catenin/TCF activities in vector- and RSPO4 -transfected cancer cells. (E) Transcriptional activities of CCND1 , c-MYC and MMP7 promoter were determined by luciferase reporter assay in vector- and RSPO4 -transfected tumor cells (of either LGR4+/LGR5- or LGR4-/LGR5+ phenotype). (F) Transcriptional activities of AP-1 and SRE responsive element reporters were determined by luciferase reporter assay in vector- and RSPO4 -transfected HCT116 and KYSE150 cells. (G) Western blot detection of the signaling alterations in canonical and non-canonical Wnt signaling in tumor cells transfected with RSPO4 and vector. (H) Western blot detection of the signaling alterations in canonical and non-canonical Wnt signaling in HNE1 cells stably expressing RSPO4 and vector. (I) Luciferase reporter assay detected β-catenin/TCF activities and transcriptional activities of Wnt target genes as well as AP-1 and SRE activities in H1299 cells with RSPO4 knockdown by siRNAs. (J) Western blot examined the levels of the components of canonical and non-canonical Wnt signaling in H1299 cells with RSPO4 knockdown by siRNAs. For D, E, F and I, n = 3 biologically independent replicates were examined over three independent experiments with similar results. Data are presented as mean values ± SD. Student's test was performed to obtain the P values.

Techniques Used: Staining, Transfection, Plasmid Preparation, Luciferase, Reporter Assay, Western Blot, Stable Transfection, Expressing, Knockdown

RSPO4 suppresses of Wnt/β-catenin signaling in an LGR4/5 dependent manner. (A) Schematic structure of RSPO4 protein and its mutants. (B) Western blot detected the β-catenin and active β-catenin level in HCT116 and KYSE150 cells transfected with vector-, RSPO4 -WT, FUm1, FUm2, FUm1/2, ΔTSP and ΔTSP/BR. After 48 hrs transfection, cells were harvested for Western blot. (C) Colony formation assay in cancer cells transfected with vector, RSPO4 and FUm1/2. (D) TOPflash/FOPflash luciferase reporter assay in vector-, RSPO4 - and FUm1/2-transfected tumor cells ( left ). Transcriptional activities of CCND1 , c-MYC and MMP7 promoter reporter in vector-, RSPO4 - and FUm1/2-transfected cancer cells ( right ). (E) Western blot detected the signaling alterations in canonical and non-canonical Wnt signaling in cancer cells transfected with vector, RSPO4 and FUm1/2. (F) TOPflash/FOPflash luciferase reporter assay detected the transcriptional activity of β-catenin in cancer cells with knockdown of LGR4 or LGR5 by siRNAs. (G) Western blot detected the signaling alterations in canonical and non-canonical Wnt signaling in cancer cells with vector and LGR4 or LGR5 knockdown by siRNAs. For C, D and F, n = 3 biologically independent replicates were examined over three independent experiments with similar results. Data are presented as mean values ± SD. Student's test was performed to obtain the P values.

Figure Legend Snippet: RSPO4 suppresses of Wnt/β-catenin signaling in an LGR4/5 dependent manner. (A) Schematic structure of RSPO4 protein and its mutants. (B) Western blot detected the β-catenin and active β-catenin level in HCT116 and KYSE150 cells transfected with vector-, RSPO4 -WT, FUm1, FUm2, FUm1/2, ΔTSP and ΔTSP/BR. After 48 hrs transfection, cells were harvested for Western blot. (C) Colony formation assay in cancer cells transfected with vector, RSPO4 and FUm1/2. (D) TOPflash/FOPflash luciferase reporter assay in vector-, RSPO4 - and FUm1/2-transfected tumor cells ( left ). Transcriptional activities of CCND1 , c-MYC and MMP7 promoter reporter in vector-, RSPO4 - and FUm1/2-transfected cancer cells ( right ). (E) Western blot detected the signaling alterations in canonical and non-canonical Wnt signaling in cancer cells transfected with vector, RSPO4 and FUm1/2. (F) TOPflash/FOPflash luciferase reporter assay detected the transcriptional activity of β-catenin in cancer cells with knockdown of LGR4 or LGR5 by siRNAs. (G) Western blot detected the signaling alterations in canonical and non-canonical Wnt signaling in cancer cells with vector and LGR4 or LGR5 knockdown by siRNAs. For C, D and F, n = 3 biologically independent replicates were examined over three independent experiments with similar results. Data are presented as mean values ± SD. Student's test was performed to obtain the P values.

Techniques Used: Western Blot, Transfection, Plasmid Preparation, Colony Assay, Luciferase, Reporter Assay, Activity Assay, Knockdown

RSPO4 recruits LGR4/5 to prevent the ubiquitin-proteasome mediated degradation of ZNRF3. (A) Western blot detection of membrane ZNRF3 in LGR4+/LGR5- and LGR4-/LGR5+ cancer cells transfected with vector, RSPO4 and FUm1/2. Membrane ZNRF3 was isolated after 48 hrs transfection of vector, RSPO4 and FUm1/2. (B) V5-epitope-tagged RSPO4 , FUm1 and FUm2 were co-transfected with HA-epitope-tagged LGR4 in HEK293 cells ( left panel ). V5-epitope-tagged RSPO4 , FUm1 and FUm2 were co-transfected with myc-epitope-tagged LGR5 in HEK293 cells ( middle panel ). V5-epitope-tagged RSPO4 , FUm1 and FUm2 were co-transfected with HA-epitope-tagged ZNRF3 in HEK293 cells ( right panel ). After 48 h transfection, membrane proteins were prepared and immunoprecipitated by using V5 antibody. Immunoblotting was probed by V5, myc and HA antibody. (C) V5-epitope-tagged RSPO4 and FUm1/2 were co-transfected with HA-epitope-tagged ZNRF3 and myc-epitope-tagged LGR4 in HEK293 cells. After 48 h transfection, membrane proteins were prepared and immunoprecipitated by using myc ( left panel ), HA ( middle panel ) and V5 ( right panel ) antibody, respectively. Immunoblotting was probed by V5, myc and HA antibody. The input control was shown at the most right. (D) V5-epitope-tagged RSPO4 and FUm1/2 were co-transfected with HA-epitope-tagged ZNRF3 and myc-epitope-tagged LGR5 in HEK293 cells. After 48 h transfection, membrane proteins were prepared and immunoprecipitated by using myc ( left panel ), HA ( middle panel ) and V5 ( right panel ) antibody, respectively. Immunoblotting was probed by V5, myc and HA antibody. The input control was shown at the most right. (E) Cycloheximide (CHX)-chase assay for the half-life of ZNRF3 in HCT116 and LoVo cells. HCT116 ( left panel ) and LoVo ( right panel ) cells with RSPO4 or vector expression are treated with CHX (20 μg/ml) for the indicated time points, and Western blot with indicated antibodies. (F) RSPO4 decreases ZNRF3 ubiquitination. RSPO4 and FUm1/2 were co-transfected with His-Ub plasmid into HCT116 and LoVo cells followed by treatment of 10 μM MG132 for 6 h. Membrane proteins were prepared and immunoprecipitated by using ZNRF3 antibody. Immunoblotting was probed by His antibody.

Figure Legend Snippet: RSPO4 recruits LGR4/5 to prevent the ubiquitin-proteasome mediated degradation of ZNRF3. (A) Western blot detection of membrane ZNRF3 in LGR4+/LGR5- and LGR4-/LGR5+ cancer cells transfected with vector, RSPO4 and FUm1/2. Membrane ZNRF3 was isolated after 48 hrs transfection of vector, RSPO4 and FUm1/2. (B) V5-epitope-tagged RSPO4 , FUm1 and FUm2 were co-transfected with HA-epitope-tagged LGR4 in HEK293 cells ( left panel ). V5-epitope-tagged RSPO4 , FUm1 and FUm2 were co-transfected with myc-epitope-tagged LGR5 in HEK293 cells ( middle panel ). V5-epitope-tagged RSPO4 , FUm1 and FUm2 were co-transfected with HA-epitope-tagged ZNRF3 in HEK293 cells ( right panel ). After 48 h transfection, membrane proteins were prepared and immunoprecipitated by using V5 antibody. Immunoblotting was probed by V5, myc and HA antibody. (C) V5-epitope-tagged RSPO4 and FUm1/2 were co-transfected with HA-epitope-tagged ZNRF3 and myc-epitope-tagged LGR4 in HEK293 cells. After 48 h transfection, membrane proteins were prepared and immunoprecipitated by using myc ( left panel ), HA ( middle panel ) and V5 ( right panel ) antibody, respectively. Immunoblotting was probed by V5, myc and HA antibody. The input control was shown at the most right. (D) V5-epitope-tagged RSPO4 and FUm1/2 were co-transfected with HA-epitope-tagged ZNRF3 and myc-epitope-tagged LGR5 in HEK293 cells. After 48 h transfection, membrane proteins were prepared and immunoprecipitated by using myc ( left panel ), HA ( middle panel ) and V5 ( right panel ) antibody, respectively. Immunoblotting was probed by V5, myc and HA antibody. The input control was shown at the most right. (E) Cycloheximide (CHX)-chase assay for the half-life of ZNRF3 in HCT116 and LoVo cells. HCT116 ( left panel ) and LoVo ( right panel ) cells with RSPO4 or vector expression are treated with CHX (20 μg/ml) for the indicated time points, and Western blot with indicated antibodies. (F) RSPO4 decreases ZNRF3 ubiquitination. RSPO4 and FUm1/2 were co-transfected with His-Ub plasmid into HCT116 and LoVo cells followed by treatment of 10 μM MG132 for 6 h. Membrane proteins were prepared and immunoprecipitated by using ZNRF3 antibody. Immunoblotting was probed by His antibody.

Techniques Used: Ubiquitin Proteomics, Western Blot, Membrane, Transfection, Plasmid Preparation, Isolation, Immunoprecipitation, Control, Expressing

RSPO4 mitigate cancer cell migration, invasion, stemness through Wnt/β-catenin signaling. (A) Transwell migration and invasion assay of HCT116 cells transfected with empty vector, RSPO4 and FUm1/2 plasmid. (B) Sphere-forming assays evaluated the stemness of cancer cells transfected with empty vector, RSPO4 and FUm1/2 plasmid. Scale bar, 100μm. (C) Western blot detected the expression levels of EMT and stem cell markers in HCT116 and LoVo cells transfected with empty vector, RSPO4 and FUm1/2 plasmid. (D) Indirect immunofluorescence staining of E-caherin in empty vector-, RSPO4 - and FUm1/2 transfected KYSE150 cells. Original magnification, ×400. Scale bar, 200μm. (E) Schematic diagram illustrates the role of RSPO4, functioning as a tumor suppressor through antagonizing Wnt/β-catenin signaling dependent on LGR4/5 and ZNRF3 by forming a negative feedback loop. Diagram was created with BioRender. For A and B, n = 3 biologically independent replicates were examined over three independent experiments with similar results. Data are presented as mean values ± SD. Student's test was performed to obtain the P values.

Figure Legend Snippet: RSPO4 mitigate cancer cell migration, invasion, stemness through Wnt/β-catenin signaling. (A) Transwell migration and invasion assay of HCT116 cells transfected with empty vector, RSPO4 and FUm1/2 plasmid. (B) Sphere-forming assays evaluated the stemness of cancer cells transfected with empty vector, RSPO4 and FUm1/2 plasmid. Scale bar, 100μm. (C) Western blot detected the expression levels of EMT and stem cell markers in HCT116 and LoVo cells transfected with empty vector, RSPO4 and FUm1/2 plasmid. (D) Indirect immunofluorescence staining of E-caherin in empty vector-, RSPO4 - and FUm1/2 transfected KYSE150 cells. Original magnification, ×400. Scale bar, 200μm. (E) Schematic diagram illustrates the role of RSPO4, functioning as a tumor suppressor through antagonizing Wnt/β-catenin signaling dependent on LGR4/5 and ZNRF3 by forming a negative feedback loop. Diagram was created with BioRender. For A and B, n = 3 biologically independent replicates were examined over three independent experiments with similar results. Data are presented as mean values ± SD. Student's test was performed to obtain the P values.

Techniques Used: Migration, Invasion Assay, Transfection, Plasmid Preparation, Western Blot, Expressing, Immunofluorescence, Staining

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Journal: International Journal of Biological Sciences

Article Title: RSPO4 exerts tumor suppression through antagonizing canonical and non-canonical Wnt signaling

doi: 10.7150/ijbs.124734

Figure Lengend Snippet: Identification of RSPO4 as a methylated target gene with clinical significance. (A) MeDIP-chip study identified RSPO4 as a methylated target in CRC and NPC cell lines and primary tumors (C18, OCT83 and NH18). RSPO4 gene structure, promoter and exon 1 (UCSC Genome Browser NCBI36/hg18) are shown on the top panel. e1: exon 1. Positive methylation signal peak (blue) in HCT116 was identified by methylated DNA immunoprecipitation (MeDIP)-chip. Promoter CpG methylation of RSPO4 was also identified by double-enzyme reduced representation bisulfite sequencing (dRRBS) in HCT116 and its double knock-out of DNMT1 and DNMT3A (DKO) cells (bottom panel). (B) β-value as the indicator of methylation level of RSPO4 in cancer tissues and the normal control in TCGA datasets, as retrieved from DNMIVD. HNSC, head and neck squamous carcinoma; LUAD, lung adenocarcinoma; ESCA, esophageal carcinoma; COAD, colon adenocarcinoma; N, normal control; T, tumor. (C) RSPO4 mRNA expression levels in different cancer types in TCGA datasets, as retrieved from SangerBox. LUSC, lung squamous cell carcinoma; STES, stomach and esophageal carcinoma; READ, rectum adenocarcinoma. (D) Analyses of TCGA datasets reveal an inverse correlation between mRNA expression level and promoter methylation level of RSPO4 in ESCA and LUAD, as retrieved from DNMIVD. Each green circle represents a single clinical sample. Pearson correlation coefficient analysis is used. (E) Kaplan-Meier curve analyses show the association between RSPO4 mRNA expression and overall survival of patients with LUAD in TCGA datasets, as retrieved from KM-plotter, and CRC as retrieved from PrognoScan. CRC, colorectal carcinoma. (F) RT-PCR detected RSPO4 mRNA expression in a panel of human normal adult and fetal tissues. (G) Western blot detected RSPO4 protein level in a panel of human normal adult and fetal tissues. 293T cell line ectopically expressing RSPO4 was used as a positive control. (H) Schematic structure of RSPO4 promoter. The primers for RT-PCR and multiplex DNA PCR are indicated with arrows. Exon 1, CpG sites (short vertical lines), MSP sites and BGS region analyzed are shown. RT-PCR and MSP detected RSPO4 mRNA expression and promoter CpG methylation in cancer cell lines and non-transformed epithelial cell lines (NP69, Het-1A, NE1 and NE3), respectively. M, methylated; U, unmethylated. Ca, carcinoma; NPC, nasopharyngeal carcinoma; ESCC, esophageal squamous cell carcinoma. (I) BGS analysis of RSPO4 promoter in non-transformed epithelial cell line (NP69) and cancer cell line (C666-1). Each row of circles represented an individual promoter allele. Filled circle, methylated CpG site; open circle, unmethylated CpG site; open triangle, SNP rs6077512 (C/G). (J) RSPO4 mRNA expression in methylated/silenced cancer cell lines was detected by RT-PCR after pharmacologic demethylation treatment with Aza combined with TSA (A+T). (K) MSP analysis of RSPO4 methylation in different types of primary tumor tissues. Representative samples are shown. (L) BGS analysis of RSPO4 methylation pattern in representative primary tumor tissues and normal tissues. (M) Kaplan Meier analysis shows the association between RSPO4 promoter methylation at specific CpG sites and overall survival of patient with READ from TCGA datasets, as retrieved from MethSurv. The methylation patient groups are dichotomized by higher (β > cut-off) and lower (β < cut-off), according to a best cut-off point in MethSurv.

Article Snippet: Human RSPO4 recombinant protein was commercially purchased (R&D Systems #4575-RS/CF).

Techniques: Methylation, Methylated DNA Immunoprecipitation, Immunoprecipitation, CpG Methylation Assay, Methylation Sequencing, Knock-Out, Control, Expressing, Reverse Transcription Polymerase Chain Reaction, Western Blot, Positive Control, Multiplex Assay, Transformation Assay

Journal: International Journal of Biological Sciences

Article Title: RSPO4 exerts tumor suppression through antagonizing canonical and non-canonical Wnt signaling

doi: 10.7150/ijbs.124734

Figure Lengend Snippet: RSPO4 encodes a secreted protein which inhibits tumor cell clonogenicity, migration, invasion and stemness. (A) Subcellular localization by immunofluorescence showed that RSPO4 protein co-localized with the endoplasmic reticulum (ER). Original magnification, ×400. Scale bar, 200μm. (B) RSPO4 protein can be detected in conditioned medium after 24 hrs posttransfection. CM, serum-free conditioned medium; TCL, total cell lysates. (C) Monolayer CFA in KYSE150, A549 and HCT116 cells. 5,000 cells were seeded in each well and colonies were counted after 2 weeks. (D) Anchorage-independent soft agar assay on KYSE150 and HCT116 cells. 5,000 cells were seeded in each well and colonies were counted after 4 weeks. (E) Flow cytometry analysis of apoptosis by Annexin V-FITC/PI staining of A549 and KYSE150 cells. Both early and late apoptotic cells (Annexin V-positive) were counted. (F) Western blot detected the protein level of cleaved caspase 3, 7, 9 and PARP in cancer cells transfected with vector- and RSPO4 . (G) In vivo tumor formation ability of LoVo cells transduced with lentivirus encoding RSPO4 or empty vector, then injected subcutaneously into BALB/c nude mice. (H) Transwell migration and invasion assay of HONE1 cells transfected with empty vector and RSPO4 plasmid. (I) Morphological changes in RSPO4 -transfected cancer cells compared with vector control after genecitin selection for 2 weeks. Original magnification, ×400. Scale bar, 200μm. (J) Indirect immunofluorescence staining of E-cadherin and vimentin in empty vector- and RSPO4 -transfected KYSE150 and A549 cells, respectively. Original magnification, ×400. Scale bar, 200μm. (K) Western blot detected the expression levels of E-cadherin, vimentin and fibronectin in RSPO4 -stably ( Left ) and transiently expressed cancer cells ( Right ). (L) Western blot detected the protein level of vimentin, N-cadherin and fibronectin in H1299 cells with knockdown of RSPO4 by siRNAs. (M) Effect of ectopic RSPO4 expression on cytoskeletal structures of A549 cells. Red, Rhodamine-labeled phalloidin; Blue, DAPI. Original magnification, ×400. Scale bar, 20μm. (N) Sphere-forming assays evaluated the stemness of cancer cells transfected with empty vector and RSPO4 plasmid. Scale bar, 100 μm. For C, D, E, F, H and N, n = 3 biologically independent replicates were examined over three independent experiments with similar results. Data are presented as mean values ± SD. For C, D, E, H and N, Student's test was performed to obtain the P values. For G, n=5 mice were used for RSPO4 and vector control, and one-way ANOVA was performed to obtain the P values.

Article Snippet: Human RSPO4 recombinant protein was commercially purchased (R&D Systems #4575-RS/CF).

Techniques: Migration, Immunofluorescence, Soft Agar Assay, Flow Cytometry, Staining, Western Blot, Transfection, Plasmid Preparation, In Vivo, Transduction, Injection, Invasion Assay, Control, Selection, Expressing, Stable Transfection, Knockdown, Labeling

Journal: International Journal of Biological Sciences

Article Title: RSPO4 exerts tumor suppression through antagonizing canonical and non-canonical Wnt signaling

doi: 10.7150/ijbs.124734

Figure Lengend Snippet: RSPO4 antagonizes Wnt/β-catenin signaling in cancer cells. (A) Gene set enrichment analysis of pathways in datasets of TCGA CRC patients ( Left ) and enrichment score of epithelial mesenchymal transition ( Right ). (B) Kyoto Encyclopedia of Genes and Genomes (KEGG) datasets of TCGA CRC patients ( Left ) and enrichment score of ECM receptor interaction ( Right ). (C) Immunofluorescent staining of nuclear β-catenin in cancer cells after 48 hrs transfection of RSPO4 plasmid. Red, active β-catenin (unphosphorylated at Ser33/Ser37/Thr41); Green, RSPO4 protein stained by V5 antibody; Scale bar, 200 μm. (D) TOPflash/FOPflash luciferase reporter assay evaluated β-catenin/TCF activities in vector- and RSPO4 -transfected cancer cells. (E) Transcriptional activities of CCND1 , c-MYC and MMP7 promoter were determined by luciferase reporter assay in vector- and RSPO4 -transfected tumor cells (of either LGR4+/LGR5- or LGR4-/LGR5+ phenotype). (F) Transcriptional activities of AP-1 and SRE responsive element reporters were determined by luciferase reporter assay in vector- and RSPO4 -transfected HCT116 and KYSE150 cells. (G) Western blot detection of the signaling alterations in canonical and non-canonical Wnt signaling in tumor cells transfected with RSPO4 and vector. (H) Western blot detection of the signaling alterations in canonical and non-canonical Wnt signaling in HNE1 cells stably expressing RSPO4 and vector. (I) Luciferase reporter assay detected β-catenin/TCF activities and transcriptional activities of Wnt target genes as well as AP-1 and SRE activities in H1299 cells with RSPO4 knockdown by siRNAs. (J) Western blot examined the levels of the components of canonical and non-canonical Wnt signaling in H1299 cells with RSPO4 knockdown by siRNAs. For D, E, F and I, n = 3 biologically independent replicates were examined over three independent experiments with similar results. Data are presented as mean values ± SD. Student's test was performed to obtain the P values.

Article Snippet: Human RSPO4 recombinant protein was commercially purchased (R&D Systems #4575-RS/CF).

Techniques: Staining, Transfection, Plasmid Preparation, Luciferase, Reporter Assay, Western Blot, Stable Transfection, Expressing, Knockdown

Journal: International Journal of Biological Sciences

Article Title: RSPO4 exerts tumor suppression through antagonizing canonical and non-canonical Wnt signaling

doi: 10.7150/ijbs.124734

Figure Lengend Snippet: RSPO4 suppresses of Wnt/β-catenin signaling in an LGR4/5 dependent manner. (A) Schematic structure of RSPO4 protein and its mutants. (B) Western blot detected the β-catenin and active β-catenin level in HCT116 and KYSE150 cells transfected with vector-, RSPO4 -WT, FUm1, FUm2, FUm1/2, ΔTSP and ΔTSP/BR. After 48 hrs transfection, cells were harvested for Western blot. (C) Colony formation assay in cancer cells transfected with vector, RSPO4 and FUm1/2. (D) TOPflash/FOPflash luciferase reporter assay in vector-, RSPO4 - and FUm1/2-transfected tumor cells ( left ). Transcriptional activities of CCND1 , c-MYC and MMP7 promoter reporter in vector-, RSPO4 - and FUm1/2-transfected cancer cells ( right ). (E) Western blot detected the signaling alterations in canonical and non-canonical Wnt signaling in cancer cells transfected with vector, RSPO4 and FUm1/2. (F) TOPflash/FOPflash luciferase reporter assay detected the transcriptional activity of β-catenin in cancer cells with knockdown of LGR4 or LGR5 by siRNAs. (G) Western blot detected the signaling alterations in canonical and non-canonical Wnt signaling in cancer cells with vector and LGR4 or LGR5 knockdown by siRNAs. For C, D and F, n = 3 biologically independent replicates were examined over three independent experiments with similar results. Data are presented as mean values ± SD. Student's test was performed to obtain the P values.

Article Snippet: Human RSPO4 recombinant protein was commercially purchased (R&D Systems #4575-RS/CF).

Techniques: Western Blot, Transfection, Plasmid Preparation, Colony Assay, Luciferase, Reporter Assay, Activity Assay, Knockdown

Journal: International Journal of Biological Sciences

Article Title: RSPO4 exerts tumor suppression through antagonizing canonical and non-canonical Wnt signaling

doi: 10.7150/ijbs.124734

Figure Lengend Snippet: RSPO4 recruits LGR4/5 to prevent the ubiquitin-proteasome mediated degradation of ZNRF3. (A) Western blot detection of membrane ZNRF3 in LGR4+/LGR5- and LGR4-/LGR5+ cancer cells transfected with vector, RSPO4 and FUm1/2. Membrane ZNRF3 was isolated after 48 hrs transfection of vector, RSPO4 and FUm1/2. (B) V5-epitope-tagged RSPO4 , FUm1 and FUm2 were co-transfected with HA-epitope-tagged LGR4 in HEK293 cells ( left panel ). V5-epitope-tagged RSPO4 , FUm1 and FUm2 were co-transfected with myc-epitope-tagged LGR5 in HEK293 cells ( middle panel ). V5-epitope-tagged RSPO4 , FUm1 and FUm2 were co-transfected with HA-epitope-tagged ZNRF3 in HEK293 cells ( right panel ). After 48 h transfection, membrane proteins were prepared and immunoprecipitated by using V5 antibody. Immunoblotting was probed by V5, myc and HA antibody. (C) V5-epitope-tagged RSPO4 and FUm1/2 were co-transfected with HA-epitope-tagged ZNRF3 and myc-epitope-tagged LGR4 in HEK293 cells. After 48 h transfection, membrane proteins were prepared and immunoprecipitated by using myc ( left panel ), HA ( middle panel ) and V5 ( right panel ) antibody, respectively. Immunoblotting was probed by V5, myc and HA antibody. The input control was shown at the most right. (D) V5-epitope-tagged RSPO4 and FUm1/2 were co-transfected with HA-epitope-tagged ZNRF3 and myc-epitope-tagged LGR5 in HEK293 cells. After 48 h transfection, membrane proteins were prepared and immunoprecipitated by using myc ( left panel ), HA ( middle panel ) and V5 ( right panel ) antibody, respectively. Immunoblotting was probed by V5, myc and HA antibody. The input control was shown at the most right. (E) Cycloheximide (CHX)-chase assay for the half-life of ZNRF3 in HCT116 and LoVo cells. HCT116 ( left panel ) and LoVo ( right panel ) cells with RSPO4 or vector expression are treated with CHX (20 μg/ml) for the indicated time points, and Western blot with indicated antibodies. (F) RSPO4 decreases ZNRF3 ubiquitination. RSPO4 and FUm1/2 were co-transfected with His-Ub plasmid into HCT116 and LoVo cells followed by treatment of 10 μM MG132 for 6 h. Membrane proteins were prepared and immunoprecipitated by using ZNRF3 antibody. Immunoblotting was probed by His antibody.

Article Snippet: Human RSPO4 recombinant protein was commercially purchased (R&D Systems #4575-RS/CF).

Techniques: Ubiquitin Proteomics, Western Blot, Membrane, Transfection, Plasmid Preparation, Isolation, Immunoprecipitation, Control, Expressing

Journal: International Journal of Biological Sciences

Article Title: RSPO4 exerts tumor suppression through antagonizing canonical and non-canonical Wnt signaling

doi: 10.7150/ijbs.124734

Figure Lengend Snippet: RSPO4 mitigate cancer cell migration, invasion, stemness through Wnt/β-catenin signaling. (A) Transwell migration and invasion assay of HCT116 cells transfected with empty vector, RSPO4 and FUm1/2 plasmid. (B) Sphere-forming assays evaluated the stemness of cancer cells transfected with empty vector, RSPO4 and FUm1/2 plasmid. Scale bar, 100μm. (C) Western blot detected the expression levels of EMT and stem cell markers in HCT116 and LoVo cells transfected with empty vector, RSPO4 and FUm1/2 plasmid. (D) Indirect immunofluorescence staining of E-caherin in empty vector-, RSPO4 - and FUm1/2 transfected KYSE150 cells. Original magnification, ×400. Scale bar, 200μm. (E) Schematic diagram illustrates the role of RSPO4, functioning as a tumor suppressor through antagonizing Wnt/β-catenin signaling dependent on LGR4/5 and ZNRF3 by forming a negative feedback loop. Diagram was created with BioRender. For A and B, n = 3 biologically independent replicates were examined over three independent experiments with similar results. Data are presented as mean values ± SD. Student's test was performed to obtain the P values.

Article Snippet: Human RSPO4 recombinant protein was commercially purchased (R&D Systems #4575-RS/CF).

Techniques: Migration, Invasion Assay, Transfection, Plasmid Preparation, Western Blot, Expressing, Immunofluorescence, Staining

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