Figure 1. RSPO3 expression is upregulated in bladder cancer

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Figure 1. RSPO3 expression is upregulated in bladder cancer Figure 1. RSPO3 expression is upregulated in bladder cancer. (a and b) RSPO3 mRNA and protein expression in 10 pairs of ... Figure 1. RSPO3 expression is upregulated in bladder cancer. (a and b) RSPO3 mRNA and protein expression in 10 pairs of bladder cancer (T) and adjacent non-tumor tissues (ANT). (c) Representative IHC staining images of RSPO3 expression levels in bladder cancer tissues (left, 200×; right, 400×). (d) RSPO3 mRNA expression in normal bladder urothelial cells (NBUCs), human uroepithelial cells (SV-HUC-1) and bladder cancer cell lines (BIU-87, T24, 5637, RT4, EJ, TCCSUP and UM-UC-3). (e) Recurrent-free survival curves in relation to the RSPO3 status in 196 bladder cancer patients, in 130 patients with Ta-T1 tumor and in 66 patients with T2-T4 tumor. Unless provided in the caption above, the following copyright applies to the content of this slide: © The Author(s) 2018. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.This article is published and distributed under the terms of the Oxford University Press, Standard Journals Publication Model (https://academic.oup.com/journals/pages/open_access/funder_policies/chorus/standard_publication_model) Carcinogenesis, Volume 40, Issue 2, 17 October 2018, Pages 360–369, https://doi.org/10.1093/carcin/bgy140 The content of this slide may be subject to copyright: please see the slide notes for details.

Figure 2. Upregulation of RSPO3 promotes bladder cancer cell aggressiveness in vitro. (a) Western blotting analysis of ... Figure 2. Upregulation of RSPO3 promotes bladder cancer cell aggressiveness in vitro. (a) Western blotting analysis of RSPO3 expression in EJ and T24 cells stably expressing RSPO3; α-tubulin was used as a loading control. (b) Representative micrographs (left panel) and quantification (right panel) of EdU incorporation in the indicated bladder cancer cells. DAPI was used as a DNA/nuclear stain. (c) The mobility of cells was measured by wound-healing assay (left panels); the uncovered areas in the wound-healing assays were quantified as a percentage of the original wound area (right panels). (d) Representative pictures (left panel) and quantification (right panel) of invaded cells were analyzed using a transwell Matrigel assay. (e) Representative images (left panel) and quantification (right panel) of HUVECs cultured on Matrigel-coated plates with conditioned medium from RSPO3-overexpressing cells. Bar graphs show the statistical analysis of three independent experiments (*P < 0.05). (f) Representative micrographs (left) and quantification (right) of colonies formed by the indicated cells, determined by anchorage-independent growth ability assays. Unless provided in the caption above, the following copyright applies to the content of this slide: © The Author(s) 2018. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.This article is published and distributed under the terms of the Oxford University Press, Standard Journals Publication Model (https://academic.oup.com/journals/pages/open_access/funder_policies/chorus/standard_publication_model) Carcinogenesis, Volume 40, Issue 2, 17 October 2018, Pages 360–369, https://doi.org/10.1093/carcin/bgy140 The content of this slide may be subject to copyright: please see the slide notes for details.

Figure 3. Downregulation of RSPO3 inhibits the aggressiveness of bladder cancer cells. (a) Western blotting analysis of ... Figure 3. Downregulation of RSPO3 inhibits the aggressiveness of bladder cancer cells. (a) Western blotting analysis of RSPO3 expression in EJ and T24 cells stably silencing RSPO3; α-tubulin was used as a loading control. (b) Representative micrographs (left panel) and quantification (right panel) of EdU incorporation in the indicated bladder cancer cells. DAPI was used as a DNA/nuclear stain. (c) Representative pictures (left panel) and percentage of the original wound area (right panel) of RSPO3-knockdown cells were analyzed using a wound-healing assay. (d) Representative pictures (left panel) and quantification (right panel) of invaded cells were analyzed using a transwell Matrigel assay. (e) Representative images (left panel) and quantification (right panel) of HUVECs cultured on Matrigel-coated plates with conditioned medium from RSPO3-knockdown cells. Bar graphs show the statistical analysis of three independent experiments (*P < 0.05). (f) Representative micrographs (left) and quantification (right) of colonies formed by the indicated cells, determined by anchorage-independent growth ability assays. Unless provided in the caption above, the following copyright applies to the content of this slide: © The Author(s) 2018. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.This article is published and distributed under the terms of the Oxford University Press, Standard Journals Publication Model (https://academic.oup.com/journals/pages/open_access/funder_policies/chorus/standard_publication_model) Carcinogenesis, Volume 40, Issue 2, 17 October 2018, Pages 360–369, https://doi.org/10.1093/carcin/bgy140 The content of this slide may be subject to copyright: please see the slide notes for details.

Figure 4. RSPO3 overexpression suppresses the tumorigenicity of bladder cancer in vitro. (a) Representative images of ... Figure 4. RSPO3 overexpression suppresses the tumorigenicity of bladder cancer in vitro. (a) Representative images of the tumors from xenograft model in nude mice. (b) Tumor volumes were measured on the indicated days. (c) Tumor weights of all mice in each group. (d) Immunohistochemical staining demonstrated the expression of Ki67, and CD31 in the indicated tissues. Data are mean ± SD of three independent experiments. *P < 0.05. Unless provided in the caption above, the following copyright applies to the content of this slide: © The Author(s) 2018. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.This article is published and distributed under the terms of the Oxford University Press, Standard Journals Publication Model (https://academic.oup.com/journals/pages/open_access/funder_policies/chorus/standard_publication_model) Carcinogenesis, Volume 40, Issue 2, 17 October 2018, Pages 360–369, https://doi.org/10.1093/carcin/bgy140 The content of this slide may be subject to copyright: please see the slide notes for details.

Figure 5. RSPO3 activates the Wnt/β-catenin signaling pathway Figure 5. RSPO3 activates the Wnt/β-catenin signaling pathway. (a) GSEA plot showing that RSPO3 expression positively ... Figure 5. RSPO3 activates the Wnt/β-catenin signaling pathway. (a) GSEA plot showing that RSPO3 expression positively correlated with Wnt/β-catenin-activated gene signatures (KEGG_WNT_SIGNALING_PATHWAY, ST_WNT_BETA_CATENIN_ PATHWAY) in published bladder cancer gene expression profiles. (b) Western blotting analysis of β-catenin expression in the cytoplasm and nucleus of the indicated cells. α-Tubulin and p84 were used as loading controls for the cytoplasmic and nuclear fractions, respectively. (c) TOP/FOP luciferase assay of TCF/LEF transcriptional activity in the indicated cells. (d) Real-time PCR analysis of Wnt/β-catenin signaling pathway downstream genes expression, including c-myc, c-Jun, CTLA4, LEF1, TCF1 and Axin2 in the indicated cells. Bar graphs show the statistical analysis of three independent experiments (*P < 0.05). Unless provided in the caption above, the following copyright applies to the content of this slide: © The Author(s) 2018. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.This article is published and distributed under the terms of the Oxford University Press, Standard Journals Publication Model (https://academic.oup.com/journals/pages/open_access/funder_policies/chorus/standard_publication_model) Carcinogenesis, Volume 40, Issue 2, 17 October 2018, Pages 360–369, https://doi.org/10.1093/carcin/bgy140 The content of this slide may be subject to copyright: please see the slide notes for details.

Figure 6. RSPO3 activates the Hedgehog signaling pathway Figure 6. RSPO3 activates the Hedgehog signaling pathway. (a) GSEA plot showing that RSPO3 expression positively ... Figure 6. RSPO3 activates the Hedgehog signaling pathway. (a) GSEA plot showing that RSPO3 expression positively correlated with Hedgehog-activated gene signatures (PID_Hedgehog_SIGNALING_2PATHWAY, LEE_TARGETS_0F_PTCH1_AND_SUFU_UP) in published bladder cancer gene expression profiles. (b) Western blotting analysis of GLI1 expression in the cytoplasm and nucleus of the indicated cells. α-Tubulin and p84 were used as loading controls for the cytoplasmic and nuclear fractions, respectively. (c) Relative activity of reporter luciferase linked to RSPO3 and GLI1 in the indicated cells. (d) Real-time PCR analysis of Hedgehog signaling pathway downstream genes expression, including PTCH1, HIP1, CCND1, CCNE1 and HDAC1, in the indicated cells. Bar graphs show the statistical analysis of three independent experiments (*P < 0.05). (e) Quantification of the indicated cells in the EdU incorporation assay. (f) Quantification of invaded cells was analyzed using a transwell Matrigel assay. (g) Quantification of tubule formation by HUVECs cultured in Matrigel-coated plates with conditioned media from indicated cells. Each bar represents the mean ± SD of three independent experiments. *P < 0.05. Unless provided in the caption above, the following copyright applies to the content of this slide: © The Author(s) 2018. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.This article is published and distributed under the terms of the Oxford University Press, Standard Journals Publication Model (https://academic.oup.com/journals/pages/open_access/funder_policies/chorus/standard_publication_model) Carcinogenesis, Volume 40, Issue 2, 17 October 2018, Pages 360–369, https://doi.org/10.1093/carcin/bgy140 The content of this slide may be subject to copyright: please see the slide notes for details.

Figure 7. Clinical relevance of RSPO3-induced Wnt/β-catenin and Hedgehog activation in bladder cancer. (a) Western ... Figure 7. Clinical relevance of RSPO3-induced Wnt/β-catenin and Hedgehog activation in bladder cancer. (a) Western blotting assay showing that the protein expression of RSPO3, nuclear β-catenin and nuclear GLI1 expression in 10 bladder cancer samples. α-Tubulin and p84 were used as the loading controls. (b) Correlation analysis of RSPO3 and nuclear β-catenin and nuclear GLI1 expression, respectively. Unless provided in the caption above, the following copyright applies to the content of this slide: © The Author(s) 2018. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.This article is published and distributed under the terms of the Oxford University Press, Standard Journals Publication Model (https://academic.oup.com/journals/pages/open_access/funder_policies/chorus/standard_publication_model) Carcinogenesis, Volume 40, Issue 2, 17 October 2018, Pages 360–369, https://doi.org/10.1093/carcin/bgy140 The content of this slide may be subject to copyright: please see the slide notes for details.