Fig 1: Abnormal differentiation of IECs in the DM state is associated with overexpression of Rspo3. a The knockdown efficiencies after transfection of siRNAs in vitro. b The mRNA expression of SI, Tff3, Lyz1, and ChgA after si-Rspo3 transfection into the IECs of the db/db mice. c, d Western blot analysis showed the protein expression of SI, Tff3, Lyz1, and ChgA in the si-Rspo3-treated db/db mice. e, f The numbers of SI-, Tff3-, Lyz1-, and ChgA-positive cells in the si-Rspo3-administered db/db mice. Scale bar, 50 μm; mean ± SD, n = 6; *P < 0.05 vs db/+-NS
Fig 2: RSPO3 expands Lgr5+ stem cells in the DM state. a RT-qPCR revealed that Lgr5 in the db/db-NS mice showed higher expression than that in the db/+-NS mice, and the high Lgr5 expression was decreased after si-Rspo3 administration. b, c Western blot analysis showed that the Lgr5 protein levels were upregulated and downregulated in the si-Rspo3-treated db/db mice. d, e Immunohistochemistry showed that Lgr5 expression was predominantly localized in the crypts of IECs and that Lgr5+ stem cell zones were expanded in the db/db-NS mice. After si-Rspo3 administration in the db/db-NS mice, high Lgr5 expression was inhibited, and the expansion of the Lgr5+ stem cell zone was reduced. Scale bar, 50 µm; mean ± SD, n = 6; *P < 0.05 vs db/+-NS; #P < 0.05 vs db/db-NS
Fig 3: Functional enrichment analysis based on transcriptome sequencing data showing correlation between RSPO3 and PI3K/AKT pathway as well as EMT process, while Wnt signaling is not involved. (A,B,C) The gene set enrichment analysis (GSEA) based on RNA-seq data from TCGA’s ovarian cancer cohort (379 samples). (D,E) The Gene Ontology (GO, D) and Kyoto Encyclopedia of Genes and Genomes (KEGG, E) functional analysis based on RNA-seq data of our experimental RSPO3-overexpression cells vs. RSPO3 wild-type cells of OVCAR3. P<0.05 defined as statistically significant. ns, not significant.
Fig 4: RSPO3 regulates the cell growth and colony formation abilities of ovarian cancer cells. (A) Western blotting verifying the construction of RSPO3-knockdown and RSPO3-overexpression ovarian cancer cell models of SKOV3 and OVCAR3 cells. (B,C,D,E) Cell proliferation rates evaluated by Cell Counting Kit-8 assay in RSPO3-knockdown (B,C) and RSPO3-overexpression (D,E) cells vs. RSPO3 wild-type cells. (F,G) Colony formation abilities evaluated by colony formation assay in RSPO3-knockdown (F) and RSPO3-overexpression (G) cells vs. RSPO3 wild-type cells. The clones were stained with crystal violet and photographed under camera (×1). Data are shown as mean ± SD. *, P<0.05; **, P<0.01.
Fig 5: miRNA expression profiles were evaluated in the IECs from mice with DM. a The red point in the plot represents the differentially expressed miRNAs with statistical significance in the volcano plot. b Microarray analysis was used to evaluate the miRNA expression profiles in the db/db mice. C, control mice; D, diabetic mice. c Rspo3 targets Rspo3, as shown by an analysis of publicly available algorithms. d RT-qPCR revealed that miR-380-5p expression was downregulated in the IECs of the db/db mice. e In situ hybridization with a DIG-labeled LNA-miR-380-5p probe showed that miR-380-5p was predominantly localized near the crypt base. Scale bar, 50 µm; mean ± SD, n = 6; *P < 0.05
Supplier Page from Abcam for Anti-RSPO3 antibody