Journal: Nature Communications

Article Title: RSPO3 is important for trabecular bone and fracture risk in mice and humans

doi: 10.1038/s41467-021-25124-2

Figure Lengend Snippet: (a) Associations for rs7741021 in the RSPO3 locus (C-allele = effect allele). (b) Associations for rs3734626 in the RSPO3 locus (T-allele = effect allele). (c) Associations for rs2489623 in the RSPO3 locus (C-allele = effect allele).

Article Snippet: BMMs were spot seeded in 48-well plates (20,000 cells in 20 μl complete α-MEM) and allowed to attach for 5 min before addition of complete α-MEM supplemented with 30 ng ml −1 M-CSF, with or without 4 ng ml −1 RANKL (R&D Systems, 462-TEC-010) and 100 ng ml −1 recombinant RSPO3 (R&D Systems, 4120-RS-025) for 3 days to induce osteoclast differentiation.

Techniques:

Journal: Nature Communications

Article Title: RSPO3 is important for trabecular bone and fracture risk in mice and humans

doi: 10.1038/s41467-021-25124-2

Figure Lengend Snippet: Mendelian randomization studies of the estimated causal associations of circulating RSPO3 level on fractures and bone parameters.

Article Snippet: BMMs were spot seeded in 48-well plates (20,000 cells in 20 μl complete α-MEM) and allowed to attach for 5 min before addition of complete α-MEM supplemented with 30 ng ml −1 M-CSF, with or without 4 ng ml −1 RANKL (R&D Systems, 462-TEC-010) and 100 ng ml −1 recombinant RSPO3 (R&D Systems, 4120-RS-025) for 3 days to induce osteoclast differentiation.

Techniques:

Journal: Nature Communications

Article Title: RSPO3 is important for trabecular bone and fracture risk in mice and humans

doi: 10.1038/s41467-021-25124-2

Figure Lengend Snippet: a Rspo3 mRNA expression pattern in various tissues of male, wildtype mice ( n = 6). Mean of individual values are presented as horizontal lines and ±SEM as vertical lines. Data are presented as % of expression in cortical diaphyseal bone. b Rspo3 is expressed in primary calvarial osteoblasts (OBL), but not in bone marrow-derived macrophages (BMM) or RANKL-differentiated osteoclasts (OCL). Rspo3 gene expression increase from day 2 (d2) to day 7 (d7) in primary calvarial osteoblasts cultured in osteogenic media. Presented as % of expression in osteoblasts day 2. ND=not detectable. Mean of individual values are presented as horizontal lines and ±SEM as vertical lines. Difference in expression between day 2 and 7 was analyzed by two-sided Student’s t test, n = 4 wells per group. c–f Representative in situ hybridization images in mice. Transverse sections across lumbar vertebra 5 in mouse showing the mRNA expression pattern (red) of Rspo3 ( c – f , upper) and Runx2 ( c , lower), Col1a1 ( d , lower), Dmp1 ( e , lower), and Acp5/Trap ( f , lower). The lower images are consecutive to the upper images. Rspo3 mRNA could be observed in Runx2- expressing ( c ) , Col1a1- expressing ( d ), and Dmp1- expressing ( e ) osteoblast lineage cells (OBL) on the bone surface. In contrast, Rspo3 was not detectable in osteocytes (Ocy; e ), or osteoclasts (OCL; f ). Scale bar 50 µm. g–i Representative in situ hybridization images in humans. Sections from human proximal femur ( g ) and iliac crest ( h , i ) confirmed moderate RSPO3 mRNA expression. Staining for RSPO3 mRNA (red) were observed in immature osteoblast lineage cells (OBL) on eroded lining surfaces (reversal cells), and on quiescent bone surfaces (bone lining cells), as well as in a subset of cells close to blood vessels (bv; g , h ). No RSPO3 mRNA expression was observed in osteocytes (Ocy) or TRAP-immunostained osteoclasts (OCL, black; i ). Scale bar 20 µm. Experiments were repeated one ( a ), two ( g – i ) or at least three ( b – f ) times. Source data are provided as a Source Data file.

Article Snippet: BMMs were spot seeded in 48-well plates (20,000 cells in 20 μl complete α-MEM) and allowed to attach for 5 min before addition of complete α-MEM supplemented with 30 ng ml −1 M-CSF, with or without 4 ng ml −1 RANKL (R&D Systems, 462-TEC-010) and 100 ng ml −1 recombinant RSPO3 (R&D Systems, 4120-RS-025) for 3 days to induce osteoclast differentiation.

Techniques: Expressing, Derivative Assay, Gene Expression, Cell Culture, In Situ Hybridization, Staining

Journal: Nature Communications

Article Title: RSPO3 is important for trabecular bone and fracture risk in mice and humans

doi: 10.1038/s41467-021-25124-2

Figure Lengend Snippet: Single cell RNA sequencing analysis of Cxcl12 expressing bone marrow cells. a Uniform Manifold Approximation and Projection (UMAP)-based visualization of major cell clusters (Cluster 0–7) and cluster expressed genes (right). Feature plot ( b ) and violin plot ( c ) of Rspo3 expression.

Article Snippet: BMMs were spot seeded in 48-well plates (20,000 cells in 20 μl complete α-MEM) and allowed to attach for 5 min before addition of complete α-MEM supplemented with 30 ng ml −1 M-CSF, with or without 4 ng ml −1 RANKL (R&D Systems, 462-TEC-010) and 100 ng ml −1 recombinant RSPO3 (R&D Systems, 4120-RS-025) for 3 days to induce osteoclast differentiation.

Techniques: RNA Sequencing, Expressing

Journal: Nature Communications

Article Title: RSPO3 is important for trabecular bone and fracture risk in mice and humans

doi: 10.1038/s41467-021-25124-2

Figure Lengend Snippet: a Schematic figure of the conditional osteoblast-lineage specific Rspo3 -inactivated mouse model. b mRNA expression analyses of Rspo3 in vertebral trabecular bone, cortical diaphyseal bone, liver, and brain cortex in male Runx2-creRspo3 flox/flox mice, compared to Rspo3 flox/flox mice. c Body weight in Runx2-creRspo3 flox/flox mice compared to Rspo3 flox/flox mice at 5, 9, and 13 weeks-of-age. d Weight of liver, gonadal fat, kidneys, and spleen, per body weight (BW) in Runx2-creRspo3 flox/flox mice, and Rspo3 flox/flox mice. e Representative 3D µCT images of vertebra L5 in Runx2-creRspo3 flox/flox mouse (left) and Rspo3 flox/flox mouse (right). f–i Trabecular bone volume over total volume (BV/TV; f ), trabecular thickness (Tb.Th; g ), trabecular number (Tb.N; h ), and trabecular separation (Tb.Sp; i ) in vertebra L5 from Runx2-creRspo3 flox/flox mice, compared to Rspo3 flox/flox mice, as measured by µCT. j Correlation between Rspo3 mRNA levels in vertebral body (relative quantity, x -axis) and trabecular BV/TV (%, y -axis) in male Runx2-creRspo3 flox/flox and Rspo3 flox/flox mice. Variance explained ( r 2 ) is given in the figure. k Maximal load at failure ( N ) of vertebra L5 as measured by compression test in 14-week-old male Runx2-creRspo3 flox/flox ( n = 10) mice and Rspo3 flox/flox ( n = 9) mice. l , m Cortical volumetric bone mineral density (Ct.vBMD; l ) and cortical thickness (Ct.Th; m ) in femur from Runx2-creRspo3 flox/flox mice, compared to Rspo3 flox/flox mice. n mRNA expression of sclerostin ( Sost) , Dickkopf-1 ( Dkk1) , and R-spondins 1, -2, and -4 ( Rspo1, Rspo2 , and Rspo4 ) in vertebral body in male Runx2-creRspo3 flox/flox mice, compared to Rspo3 flox/flox mice. o Levels of procollagen type I N-terminal propeptide (P1NP, left) and C-terminal type I collagen (CTX, right) in male Runx2-creRspo3 flox/flox mice, compared to Rspo3 flox/flox mice. Unless otherwise stated, the results refer to 13-week-old Runx2-creRspo3 flox/flox mice, males n = 14; females n = 12, and Rspo3 flox/flox mice, males n = 15; females n = 12. Individual values are presented in all graphs with the mean presented as horizontal lines and ±SEM as vertical lines. Statistical analyses were performed using two-sided Student’s t test. Source data are provided as a Source Data file.

Article Snippet: BMMs were spot seeded in 48-well plates (20,000 cells in 20 μl complete α-MEM) and allowed to attach for 5 min before addition of complete α-MEM supplemented with 30 ng ml −1 M-CSF, with or without 4 ng ml −1 RANKL (R&D Systems, 462-TEC-010) and 100 ng ml −1 recombinant RSPO3 (R&D Systems, 4120-RS-025) for 3 days to induce osteoclast differentiation.

Techniques: Expressing

Journal: Nature Communications

Article Title: RSPO3 is important for trabecular bone and fracture risk in mice and humans

doi: 10.1038/s41467-021-25124-2

Figure Lengend Snippet: a Schematic image of the conditional osteocyte/late osteoblast-specific Rspo3- inactivated mouse model. b mRNA expression analysis of Rspo3 in vertebral trabecular bone in male Dmp1cre-Rspo3 flox/flox mice, compared to Rspo3 flox/flox mice. c Body weight in Dmp1cre-Rspo3 flox/flox mice, and Rspo3 flox/flox mice, at 9 and 15 weeks-of-age. d Weight of liver, gonadal fat, kidneys, and spleen per body weight (BW) in Dmp1-creRspo3 flox/flox mice, and Rspo3 flox/flox mice. e–h Trabecular bone volume over total volume (BV/TV; e ), trabecular thickness (Tb.Th; f ), trabecular number (Tb.N; g ), and trabecular separation (Tb.Sp; h ) in vertebra L5 from Dmp1-creRspo3 flox/flox mice, compared to Rspo3 flox/flox mice. i Representative 3D µCT images of vertebra L5 in Rspo3 flox/flox mouse (left) and Dmp1creRspo3 flox/flox mouse (right). j and k Cortical volumetric bone mineral density (Ct.vBMD, j ) and cortical thickness (Ct.Th, k ) in femur from Dmp1-creRspo3 flox/flox mice, compared to Rspo3 flox/flox mice. l Schematic image of the global, tamoxifen-inducible Rspo3 -inactivated mouse model. m mRNA expression analysis of Rspo3 in vertebral trabecular bone in CAGGCre-ER-Rspo3 flox/flox ( n = 10) mice, compared to Rspo3 flox/flox ( n = 14) mice. ( n ) Body weight in male CAGGCre-ER-Rspo3 flox/flox ( n = 11) mice, and Rspo3 flox/flox ( n = 17) mice, at 8, 9, and 13 weeks-of-age. o Weight of liver, gonadal fat, kidneys, and spleen per body weight in CAGGCre-ER-Rspo3 flox/flox ( n = 10) mice, and Rspo3 flox/flox mice ( n = 14). p–s Trabecular BV/TV ( p ), trabecular thickness (Tb.Th; q ), trabecular number (Tb.N; r ), and trabecular separation (Tb.Sp; s ) in vertebra L5 from CAGGCre-ER-Rspo3 flox/flox ( n = 11) mice, compared to Rspo3 flox/flox ( n = 16) mice. t , u Cortical volumetric BMD (Ct.vBMD; t ) and cortical thickness (Ct.Th; u ) in femur from CAGGCre-ER-Rspo3 flox/flox ( n = 11) mice, compared to Rspo3 flox/flox ( n = 17) mice. v mRNA expression of sclerostin ( Sost ), Dickkopf-1 ( Dkk1 ), and R-spondins 1, -2, and -4 ( Rspo1 , Rspo2 , and Rspo4 ) in vertebral body in CAGGCre-ER-Rspo3 flox/flox ( n = 10) mice, compared to Rspo3 flox/flox ( n = 14) mice. For Dmp1-creRspo3 flox/flox mice, males n =14; females n = 16, and littermate control Rspo3 flox/flox mice, males n = 14; females n = 14. Individual values are presented in all graphs with the mean presented as horizontal lines and ±SEM as vertical lines. Statistical analyses were performed using two-sided Student’s t test. Source data are provided as a Source Data file.

Article Snippet: BMMs were spot seeded in 48-well plates (20,000 cells in 20 μl complete α-MEM) and allowed to attach for 5 min before addition of complete α-MEM supplemented with 30 ng ml −1 M-CSF, with or without 4 ng ml −1 RANKL (R&D Systems, 462-TEC-010) and 100 ng ml −1 recombinant RSPO3 (R&D Systems, 4120-RS-025) for 3 days to induce osteoclast differentiation.

Techniques: Expressing, Control

Journal: Nature Communications

Article Title: RSPO3 is important for trabecular bone and fracture risk in mice and humans

doi: 10.1038/s41467-021-25124-2

Figure Lengend Snippet: a Rspo3 mRNA expression in primary calvarial osteoblasts (cOBL) isolated from CAGGCre-ER-Rspo3 flox/flox mice cultured for 2 and 6 days (d) with or without prior Rspo3 inactivation using tamoxifen (TAM). Veh=vehicle. b Representative photos of alkaline phosphatase staining of CAGGCre-ER-Rspo3 flox/flox and Rspo3 flox/flox cOBL cultured for 6 days after veh treatment or Rspo3 inactivation. c , d Representative photos ( c ) and quantification ( d ) of mineralized noduli in cultures of CAGGCre-ER-Rspo3 flox/flox and Rspo3 flox/flox cOBL cultured for 14 days after veh treatment or Rspo3 inactivation. e , f mRNA expression analysis of Alpl ( e ) and Col1a1 ( f ) in CAGGCre-ER-Rspo3 flox/flox cOBL cultured for 2 and 6 days with or without prior Rspo3 inactivation. g Haematoxylin (Htx) and Dapi staining of CAGGCre-ER-Rspo3 flox/flox cOBL cultured for 3 days with or without prior Rspo3 inactivation. Scale bar 100 μm. h Amount of DNA per well in CAGGCre-ER-Rspo3 flox/flox cOBL at the time of TAM addition (day −1), directly after removal of TAM (day 0) and after culture for 1, 3, 6, and 10 days. i Mki67 mRNA expression in CAGGCre-ER-Rspo3 flox/flox cOBL cultured for 2 days after Rspo3 inactivation. j , k mRNA expression of Tnfsf11 ( j ) and Tnfrsf11b ( k ) in CAGGCre-ER-Rspo3 flox/flox cOBL cultured with or without PTH and PGE2 for 5 days with or without prior Rspo3 inactivation. n = 3–4 wells. l–n TRAP staining ( l ), counting ( m ), and Acp5 mRNA expression ( n ) of bone marrow macrophages cultured in M-CSF (M) or in M-CSF and RANKL (RL) to induce osteoclastogenesis in the absence or presence of recombinant RSPO3 (rRSPO3). Scale bar 200 μm. Individual values are presented in all graphs with the mean presented as horizontal lines and ±SEM as vertical lines. If not otherwise stated, n = 4 wells per group. Experiments were repeated two ( g , j – n ) or at least three ( a – f , h , i ) times. For a , e , f , h , two-way ANOVA was used to determine the overall effect of Rspo3 inactivation by treatment of TAM or veh, time, as well as their interaction. For j , k , two-way ANOVA was used to determine the overall effect of PTH/PGE2 treatment, Rspo3 inactivation by treatment of TAM or veh, as well as their interaction. When only the effect of Rspo3 inactivation was evaluated, two-sided Student’s t was used. Source data are provided as a Source Data file.

Article Snippet: BMMs were spot seeded in 48-well plates (20,000 cells in 20 μl complete α-MEM) and allowed to attach for 5 min before addition of complete α-MEM supplemented with 30 ng ml −1 M-CSF, with or without 4 ng ml −1 RANKL (R&D Systems, 462-TEC-010) and 100 ng ml −1 recombinant RSPO3 (R&D Systems, 4120-RS-025) for 3 days to induce osteoclast differentiation.

Techniques: Expressing, Isolation, Cell Culture, Staining, Recombinant

Journal: Nature Communications

Article Title: RSPO3 is important for trabecular bone and fracture risk in mice and humans

doi: 10.1038/s41467-021-25124-2

Figure Lengend Snippet: a , b LRP6 protein analysis in primary calvarial osteoblasts isolated from CAGGCre-ER-Rspo3 flox/flox mice cultured in osteogenic media for 1 day with or without prior Rspo3 inactivation using tamoxifen (TAM). Lane view from the software in the capillary-based electrophoresis immunodetection system ( a ) and protein levels ( b ) normalized to total protein. c , d pLRP6 protein analysis in primary calvarial osteoblasts isolated from CAGGCre-ER-Rspo3 flox/flox mice cultured in osteogenic media for 1 day with or without prior Rspo3 inactivation using tamoxifen (TAM). Lane view from the software in the capillary-based electrophoresis immunodetection system ( c ) and protein levels ( d ) normalized to total protein. e–h mRNA expression analyses of Znrf3 ( e ), Rnf43 ( f ), Tcf7 ( g ) and Lef1 ( h ) in primary calvarial osteoblasts isolated from CAGGCre-ER-Rspo3 flox/flox mice cultured in osteogenic media for 6 days with or without prior Rspo3 inactivation using TAM. Veh=vehicle. Individual values are presented in all graphs with the mean presented as horizontal lines and ±SEM as vertical lines. n = 4 wells per group. Experiments were repeated one ( e , f ), two ( a , b , g , h ) or at least three ( c , d ) times. Statistical analyses were performed using two-sided Student’s t test. Source data are provided as a Source Data file.

Article Snippet: BMMs were spot seeded in 48-well plates (20,000 cells in 20 μl complete α-MEM) and allowed to attach for 5 min before addition of complete α-MEM supplemented with 30 ng ml −1 M-CSF, with or without 4 ng ml −1 RANKL (R&D Systems, 462-TEC-010) and 100 ng ml −1 recombinant RSPO3 (R&D Systems, 4120-RS-025) for 3 days to induce osteoclast differentiation.

Techniques: Isolation, Cell Culture, Software, Electrophoresis, Immunodetection, Expressing

Journal: iScience

Article Title: RSPO2 promotes progression of ovarian cancer through dual receptor-mediated FAK/Src signaling activation.

doi: 10.1016/j.isci.2022.105184

Figure Lengend Snippet: Figure 1. Expression and pathological significance of RSPO2 in ovarian cancer (A) Relative mRNA expression levels of RSPO2 in ovarian cancer cell lines. RSPO2 mRNA levels were determined using RT-qPCR. mRNA expression levels were normalized to those in normal ovarian epithelial cells (IOSE80). (B) Representative images of immunohistochemical staining for RSPO2 in a tissue microarray containing thirty ovarian tumors (T) and paired normal ovarian (N) tissues. Scale bar, 50 mm. (C) Immunohistochemical staining score for RSPO2 in the tissue microarray (n = 30). **p < 0.01, calculated by two-tailed unpaired t-test. (D) Kaplan-Meier survival analysis of ovarian cancer patients with RSPO2-high vs. RSPO2-low tumors from the TCGA cohort. (E) Kaplan-Meier survival analysis of ovarian cancer patients with RSPO2-high vs. RSPO2-low tumors from the GSE26193 cohort.

Article Snippet: His-tag Cell Signaling Technology Cat# 12698; RRID: AB_2744546 Fibronectin BD transduction laboratories Cat# 610077; RRID: AB_2105706 Normal Rabbit IgG Cell Signaling Technology Cat# 2729; RRID: AB_1031062 Mouse mAb IgG1 Isotype Control Cell Signaling Technology Cat# 5415; RRID: AB_10829607 LAMP1 Santa Cruz Biotechnology Cat# sc-20011; RRID: AB_626853 Frizzled6 (D16E5) Rabbit mAb Cell Signaling Technology Cat# 5158; RRID: AB_10621242 Anti-Frizzled 7 antibody Abcam Cat# ab64636; RRID: AB_1640522 Biological samples Human ovarian cancer and paired nontumor ovarian tissues Shanghai Superbiotek Inc. (China) http://www.superbiotek.com Chemicals, peptides, and recombinant proteins Recombinant human RSPO2 protein R&D Systems (USA) 3266-rs-025/cf Wnt3a R&D Systems (USA) 5036-WN-500 LY294002 Selleck S1105 Cycloheximide (CHX) Sigma C7698 Defactinib RayStar Biosystems (China) 1073154-85-4 Saracatinib Selleck S1006 Niclosamide Sigma-Aldrich 50-65-7 MTT Sangon Biotech TB0799-1G-N vitronectin Sigma-Aldrich 5051 MG132 Sigma-Aldrich M7449

Techniques: Expressing, Quantitative RT-PCR, Immunohistochemical staining, Staining, Microarray, Two Tailed Test

Journal: iScience

Article Title: RSPO2 promotes progression of ovarian cancer through dual receptor-mediated FAK/Src signaling activation.

doi: 10.1016/j.isci.2022.105184

Figure Lengend Snippet: Figure 2. RSPO2 promotes ovarian cancer growth and metastasis (A) Representative images (upper panel) and quantification (lower panel) of the colony formation results of A2780 and OVCAR3 cells after RSPO2 overexpression. Cells stably overexpressing RSPO2 (RS2, pooled) or empty vector (Vec) were used. ***p < 0.001 vs. Vec; two-tailed Student’s t-test. Error bars indicate mean G SD. (B) Colony formation assay in A2780 and OVCAR3 cells after shRNA-mediated RSPO2 knockdown. The lentiviral vector containing shRNA against RSPO2 (shRS2) was designed using the most effective interference sequence, siRS2-2#. **p < 0.01 and ***p < 0.001 vs. control (shNC); two-tailed Student’s t-test. Error bars indicate mean G SD. (C and D) Representative images (upper panel) and quantification (lower panel) of the Transwell assay results show that the overexpression of RSPO2 enhanced the migration (C) and invasion (D) of ovarian cancer cells. (E) Wound healing assay showing increased wound closure in the RSPO2-overexpressing cell group. (F) Representative images of tumor nodules in the peritoneum (upper panel) and ovary (lower panel) in the orthotopic mouse model. A2780 and OVCAR3 cells stably overexpressing RSPO2 (RS2, pooled) or empty vector (Vec) were inoculated into the ovary (n = 5 per group). The white dashed circles indicate the metastatic nodules and the black arrow shows the tumor formed in ovarian in situ.

Article Snippet: His-tag Cell Signaling Technology Cat# 12698; RRID: AB_2744546 Fibronectin BD transduction laboratories Cat# 610077; RRID: AB_2105706 Normal Rabbit IgG Cell Signaling Technology Cat# 2729; RRID: AB_1031062 Mouse mAb IgG1 Isotype Control Cell Signaling Technology Cat# 5415; RRID: AB_10829607 LAMP1 Santa Cruz Biotechnology Cat# sc-20011; RRID: AB_626853 Frizzled6 (D16E5) Rabbit mAb Cell Signaling Technology Cat# 5158; RRID: AB_10621242 Anti-Frizzled 7 antibody Abcam Cat# ab64636; RRID: AB_1640522 Biological samples Human ovarian cancer and paired nontumor ovarian tissues Shanghai Superbiotek Inc. (China) http://www.superbiotek.com Chemicals, peptides, and recombinant proteins Recombinant human RSPO2 protein R&D Systems (USA) 3266-rs-025/cf Wnt3a R&D Systems (USA) 5036-WN-500 LY294002 Selleck S1105 Cycloheximide (CHX) Sigma C7698 Defactinib RayStar Biosystems (China) 1073154-85-4 Saracatinib Selleck S1006 Niclosamide Sigma-Aldrich 50-65-7 MTT Sangon Biotech TB0799-1G-N vitronectin Sigma-Aldrich 5051 MG132 Sigma-Aldrich M7449

Techniques: Over Expression, Stable Transfection, Plasmid Preparation, Two Tailed Test, Colony Assay, shRNA, Knockdown, Sequencing, Control, Transwell Assay, Migration, Wound Healing Assay, In Situ

Journal: iScience

Article Title: RSPO2 promotes progression of ovarian cancer through dual receptor-mediated FAK/Src signaling activation.

doi: 10.1016/j.isci.2022.105184

Figure Lengend Snippet: Figure 3. Effects of RSPO2 on ovarian cancer cell adhesion, EMT, and cell cycle progression (A) GSEA of the TCGA ovarian cancer cohort showing that pathways associated with the cytoskeleton and focal adhesion were enriched in tumors with high RSPO2 expression. TCGA dataset was separated into two groups based on the mRNA expression of RSPO2. The expression cutoff for KEGG analysis is first (RSPO2-High) and last quartile (RSPO2-Low) of mRNA expression of RSPO2. (B) Effect of RSPO2 on cell adhesion. Quantitation of adhesion and cell attachment, as evaluated by cellular binding to vitronectin, was performed using vector control cells (Vec) and cells stably overexpressing RSPO2 (RS2). ***p < 0.001 vs. Vec. (C) Representative light micrographs showing that stable overexpression of RSPO2 altered the morphology of ovarian cancer cells (magnification, 2003). (D) Phalloidin staining showing actin filaments in A2780 and OVCAR3 cells stably overexpressing RSPO2. Scale bar, 20 mm. (E) Western blot analysis of EMT and cell cycle markers in cells stably overexpressing RSPO2. (F) Flow cytometric analysisofthecell cycleinRSPO2-silencedovarian cancercells.Cellswereinfected with lentivirus containingshRS2 ornontargetingshNC for48 h. The data shown are the mean G SD of a representative experiment performed in triplicate (lower panel). **p < 0.01 and p*** <0.001 vs. shNC; two-tailed Student’s t-test. Error bars indicate mean G SD.

Article Snippet: His-tag Cell Signaling Technology Cat# 12698; RRID: AB_2744546 Fibronectin BD transduction laboratories Cat# 610077; RRID: AB_2105706 Normal Rabbit IgG Cell Signaling Technology Cat# 2729; RRID: AB_1031062 Mouse mAb IgG1 Isotype Control Cell Signaling Technology Cat# 5415; RRID: AB_10829607 LAMP1 Santa Cruz Biotechnology Cat# sc-20011; RRID: AB_626853 Frizzled6 (D16E5) Rabbit mAb Cell Signaling Technology Cat# 5158; RRID: AB_10621242 Anti-Frizzled 7 antibody Abcam Cat# ab64636; RRID: AB_1640522 Biological samples Human ovarian cancer and paired nontumor ovarian tissues Shanghai Superbiotek Inc. (China) http://www.superbiotek.com Chemicals, peptides, and recombinant proteins Recombinant human RSPO2 protein R&D Systems (USA) 3266-rs-025/cf Wnt3a R&D Systems (USA) 5036-WN-500 LY294002 Selleck S1105 Cycloheximide (CHX) Sigma C7698 Defactinib RayStar Biosystems (China) 1073154-85-4 Saracatinib Selleck S1006 Niclosamide Sigma-Aldrich 50-65-7 MTT Sangon Biotech TB0799-1G-N vitronectin Sigma-Aldrich 5051 MG132 Sigma-Aldrich M7449

Techniques: Expressing, Quantitation Assay, Cell Attachment Assay, Binding Assay, Plasmid Preparation, Control, Stable Transfection, Over Expression, Staining, Western Blot, Two Tailed Test

Journal: iScience

Article Title: RSPO2 promotes progression of ovarian cancer through dual receptor-mediated FAK/Src signaling activation.

doi: 10.1016/j.isci.2022.105184

Figure Lengend Snippet: Figure 4. RSPO2 promotes ovarian cancer growth and metastasis through FAK/Src signaling cascades (A) KEGG enrichment analysis of the pathways affected by overexpression of RSPO2 in A2780 cells. (B) Effects of RSPO2 overexpression on the phosphorylation of FAK (Tyr397), Src (Tyr416, Y416), Src (Tyr527, Y527), EGFR (Tyr1068), and Akt (Ser473). Proteins were extracted from A2780 and OVCAR3 cells stably overexpressing RSPO2 (RS2, pooled) or empty vector (Vec) and were analyzed by Western blotting. (C) The effects of RSPO2 knockdown on the phosphorylation of FAK, Src, and Akt were analyzed by Western blotting. Cells were infected with lentivirus containing shRS2 or nontargeting shNC for 48 h.

Article Snippet: His-tag Cell Signaling Technology Cat# 12698; RRID: AB_2744546 Fibronectin BD transduction laboratories Cat# 610077; RRID: AB_2105706 Normal Rabbit IgG Cell Signaling Technology Cat# 2729; RRID: AB_1031062 Mouse mAb IgG1 Isotype Control Cell Signaling Technology Cat# 5415; RRID: AB_10829607 LAMP1 Santa Cruz Biotechnology Cat# sc-20011; RRID: AB_626853 Frizzled6 (D16E5) Rabbit mAb Cell Signaling Technology Cat# 5158; RRID: AB_10621242 Anti-Frizzled 7 antibody Abcam Cat# ab64636; RRID: AB_1640522 Biological samples Human ovarian cancer and paired nontumor ovarian tissues Shanghai Superbiotek Inc. (China) http://www.superbiotek.com Chemicals, peptides, and recombinant proteins Recombinant human RSPO2 protein R&D Systems (USA) 3266-rs-025/cf Wnt3a R&D Systems (USA) 5036-WN-500 LY294002 Selleck S1105 Cycloheximide (CHX) Sigma C7698 Defactinib RayStar Biosystems (China) 1073154-85-4 Saracatinib Selleck S1006 Niclosamide Sigma-Aldrich 50-65-7 MTT Sangon Biotech TB0799-1G-N vitronectin Sigma-Aldrich 5051 MG132 Sigma-Aldrich M7449

Techniques: Over Expression, Phospho-proteomics, Stable Transfection, Plasmid Preparation, Western Blot, Knockdown, Infection

Journal: iScience

Article Title: RSPO2 promotes progression of ovarian cancer through dual receptor-mediated FAK/Src signaling activation.

doi: 10.1016/j.isci.2022.105184

Figure Lengend Snippet: Figure 5. RSPO2 promotes the plasma membrane translocation and autophosphorylation of Src via the LGR4 receptor (A) Western blot analysis of LGR4 and LGR5 in ovarian cancer cells stably overexpressing RSPO2 (RS2, pooled) or vector control (Vec). (B) Representative images (left panel) and quantification (right panel) of colony formation assay results showing that knockdown of LGR4 diminished RSPO2- induced cell growth. A2780 and OVCAR3 cells stably overexpressing RSPO2 (RS2, pooled) or empty vector (Vec) were transfected with siRNA against LGR4 (siLGR4) or a scrambled nontargeting sequence (siNC), and a colony formation assay was then performed. n.s., not significant; *p < 0.05 and p*** <0.001; two-tailed Student’s t test. Error bars indicate mean G SD. (C) Cycloheximide (CHX)-chase assay to determine the stability of LGR4 in ovarian cancer cells. The LGR4 protein level was measured by Western blotting. A2780 cells without (Vec) or with RSPO2 expression were treated with CHX (500 nM) for the times indicated. Quantification of the LGR4 protein levels was normalized to the GAPDH and zero-time control data. (D) RSPO2 treatment enhanced the plasma membrane distribution of LGR4 and Src in A2780 and OVCAR3 cells. Colocalization of LGR4 with Src was analyzed by fluorescence confocal microscopy. Cells were treated with PBS or RSPO2 protein (200 ng/mL) at 4C for 2 h. Scale bar, 10 mm. (E) Presilencing of LGR4 attenuated RSPO2-induced Src/Akt phosphorylation. Cells pretransfected with siRNA against LGR4 (siLGR4) or siNC were treated with PBS control or recombinant RSPO2 protein (200 ng/mL) for 12 h.

Article Snippet: His-tag Cell Signaling Technology Cat# 12698; RRID: AB_2744546 Fibronectin BD transduction laboratories Cat# 610077; RRID: AB_2105706 Normal Rabbit IgG Cell Signaling Technology Cat# 2729; RRID: AB_1031062 Mouse mAb IgG1 Isotype Control Cell Signaling Technology Cat# 5415; RRID: AB_10829607 LAMP1 Santa Cruz Biotechnology Cat# sc-20011; RRID: AB_626853 Frizzled6 (D16E5) Rabbit mAb Cell Signaling Technology Cat# 5158; RRID: AB_10621242 Anti-Frizzled 7 antibody Abcam Cat# ab64636; RRID: AB_1640522 Biological samples Human ovarian cancer and paired nontumor ovarian tissues Shanghai Superbiotek Inc. (China) http://www.superbiotek.com Chemicals, peptides, and recombinant proteins Recombinant human RSPO2 protein R&D Systems (USA) 3266-rs-025/cf Wnt3a R&D Systems (USA) 5036-WN-500 LY294002 Selleck S1105 Cycloheximide (CHX) Sigma C7698 Defactinib RayStar Biosystems (China) 1073154-85-4 Saracatinib Selleck S1006 Niclosamide Sigma-Aldrich 50-65-7 MTT Sangon Biotech TB0799-1G-N vitronectin Sigma-Aldrich 5051 MG132 Sigma-Aldrich M7449

Techniques: Clinical Proteomics, Membrane, Translocation Assay, Western Blot, Stable Transfection, Plasmid Preparation, Control, Colony Assay, Knockdown, Transfection, Sequencing, Two Tailed Test, Expressing, Confocal Microscopy, Phospho-proteomics, Recombinant

Journal: iScience

Article Title: RSPO2 promotes progression of ovarian cancer through dual receptor-mediated FAK/Src signaling activation.

doi: 10.1016/j.isci.2022.105184

Figure Lengend Snippet: Figure 6. RSPO2 potentiates FAK phosphorylation by binding to integrin b3 (A) Alterations in integrin expression in A2780 and OVCAR3 cells following RSPO2 depletion were analyzed by Western blotting. Cells were transfected with a scrambled nontargeting sequence (siNC) or siRNA against RSPO2 (siRS2#). (B) Presilencing of integrin av/b3 impaired the enhancing effect of RSPO2 on FAK and Src phosphorylation. Cells pretransfected with siRNAs against both integrin av and b3 (siITGAV/B3) or siNC () were treated with recombinant RSPO2 protein (200 ng/mL) for 12 h. (C) Transwell assay results showing that the silencing of integrin av/b3 blocked RSPO2-induced cell migration. A2780 and OVCAR3 cells stably overexpressing RSPO2 (RS2, pooled) or empty vector (Vec) were transfected with siRNAs against integrin av and b3 (siITGAV/B3) or siNC and were then subjected to a Transwell assay. n.s., not significant; ***p < 0.001; two-tailed Student’s t test. Error bars indicate mean G SD. (D) Effects of RSPO2 knockdown on the ubiquitination levels of integrin av and b3 in A2780 and OVCAR3 cells. (E) Co-IP analysis of the interactions between endogenous RSPO2 and integrin av and b3 in A2780 and OVCAR3 cells. (F) An in vitro pulldown assay was performed to validate the interaction between RSPO2 and integrin subunits. Recombinant His-tagged RSPO2 protein was used to pull down Flag-tagged integrin-av or -b3 from HEK293T cell lysates. (G) Co-IP analysis of the interactions between RSPO2 mutants and LGR4 or ITGB3. OVCAR3 cells were transfected with empty vector (Vec), myc-tagged RSPO2 (WT) or mutants containing only two furin domains (FUs) or a TSP-1 domain (TSP), and cell lysates were immunoprecipitated and probed with anti- Myc, anti-LGR4, or anti-integrin-b3 antibodies. Upper panel, schematic diagram of the RSPO2 domain structure and truncation mutants.

Article Snippet: His-tag Cell Signaling Technology Cat# 12698; RRID: AB_2744546 Fibronectin BD transduction laboratories Cat# 610077; RRID: AB_2105706 Normal Rabbit IgG Cell Signaling Technology Cat# 2729; RRID: AB_1031062 Mouse mAb IgG1 Isotype Control Cell Signaling Technology Cat# 5415; RRID: AB_10829607 LAMP1 Santa Cruz Biotechnology Cat# sc-20011; RRID: AB_626853 Frizzled6 (D16E5) Rabbit mAb Cell Signaling Technology Cat# 5158; RRID: AB_10621242 Anti-Frizzled 7 antibody Abcam Cat# ab64636; RRID: AB_1640522 Biological samples Human ovarian cancer and paired nontumor ovarian tissues Shanghai Superbiotek Inc. (China) http://www.superbiotek.com Chemicals, peptides, and recombinant proteins Recombinant human RSPO2 protein R&D Systems (USA) 3266-rs-025/cf Wnt3a R&D Systems (USA) 5036-WN-500 LY294002 Selleck S1105 Cycloheximide (CHX) Sigma C7698 Defactinib RayStar Biosystems (China) 1073154-85-4 Saracatinib Selleck S1006 Niclosamide Sigma-Aldrich 50-65-7 MTT Sangon Biotech TB0799-1G-N vitronectin Sigma-Aldrich 5051 MG132 Sigma-Aldrich M7449

Techniques: Phospho-proteomics, Binding Assay, Expressing, Western Blot, Transfection, Sequencing, Recombinant, Transwell Assay, Migration, Stable Transfection, Plasmid Preparation, Two Tailed Test, Knockdown, Ubiquitin Proteomics, Co-Immunoprecipitation Assay, In Vitro, Immunoprecipitation

Journal: iScience

Article Title: RSPO2 promotes progression of ovarian cancer through dual receptor-mediated FAK/Src signaling activation.

doi: 10.1016/j.isci.2022.105184

Figure Lengend Snippet: Figure 7. Schematic diagram illustrating the dual mechanism of RSPO2-mediated FAK/Src signaling activation In the presence of RSPO2, endocytic degradation of LGR4 is attenuated, which increases the accumulation of LGR4 on the plasma membrane, leading to plasma membrane translocation and autophosphorylation of Src. RSPO2 can also increase the stability of integrins by directly binding to integrin b3, leading to enhanced FAK phosphorylation. Constitutive activation of FAK/Src signaling promotes the growth and metastasis of ovarian cancer.

Article Snippet: His-tag Cell Signaling Technology Cat# 12698; RRID: AB_2744546 Fibronectin BD transduction laboratories Cat# 610077; RRID: AB_2105706 Normal Rabbit IgG Cell Signaling Technology Cat# 2729; RRID: AB_1031062 Mouse mAb IgG1 Isotype Control Cell Signaling Technology Cat# 5415; RRID: AB_10829607 LAMP1 Santa Cruz Biotechnology Cat# sc-20011; RRID: AB_626853 Frizzled6 (D16E5) Rabbit mAb Cell Signaling Technology Cat# 5158; RRID: AB_10621242 Anti-Frizzled 7 antibody Abcam Cat# ab64636; RRID: AB_1640522 Biological samples Human ovarian cancer and paired nontumor ovarian tissues Shanghai Superbiotek Inc. (China) http://www.superbiotek.com Chemicals, peptides, and recombinant proteins Recombinant human RSPO2 protein R&D Systems (USA) 3266-rs-025/cf Wnt3a R&D Systems (USA) 5036-WN-500 LY294002 Selleck S1105 Cycloheximide (CHX) Sigma C7698 Defactinib RayStar Biosystems (China) 1073154-85-4 Saracatinib Selleck S1006 Niclosamide Sigma-Aldrich 50-65-7 MTT Sangon Biotech TB0799-1G-N vitronectin Sigma-Aldrich 5051 MG132 Sigma-Aldrich M7449

Techniques: Activation Assay, Clinical Proteomics, Membrane, Translocation Assay, Binding Assay, Phospho-proteomics

Journal: STAR Protocols

Article Title: Functional assays with human patient-derived enteroid monolayers to assess the human gut barrier

doi: 10.1016/j.xpro.2021.100680

Figure Lengend Snippet: Preparation of Human monolayer EDMs media (100 mL) (stored at 4°C for 2–4 days)

Article Snippet: Embed the living stem cells into a basement membrane matrix (Matrigel) dome and cultured using 50% stem cell-enriched WRN conditioned media (WNT 3a, R-spondin, and Noggin) (prepared from L-WRN cells, ATCC CRL-3276 TM ) containing specific components such as Y27632 (ROCK inhibitor) and SB431542 (an inhibitor for TGF-β type I receptor), nicotinamide, A83-01 (an ACTIVIN /NODAL/TGF-β pathway inhibitor), Epidermal growth factor, and SB202190 (a p38 MAPK inhibitor) to support the growth and long-term cultures of 3D organoids ( , , , ).

Techniques: Concentration Assay