PSD93 Antibody - #DF3995

Figure 3 miR-6836 induces tumor growth, metastasis and stemness of EOC cells in vivo. a-c For spheroid cells tumorigenicity assay, four-week-old female NOD-SCID mice were injected with different concentration of single cell suspensions from SKOV3 Ctrl or Lv-miR-6836 spheroids. a Schematic diagram presents the experimental procedure of spheroid cells tumorigenicity assay. b The left image presents the tumor formation of SKOV3 ctrl or Lv-miR-6836 stem cells isolated from spheroids. The tumor incidence of concentration gradient is listed in the right table. c Representative IHC staining of Sox2 and CD44 in subcutaneous xenografts. Scale bar, 100 μm. d,e For subcutaneous tumor formation model, five-week-old female BALB/c nude mice were injected subcutaneously with 1×106 CAOV3 cells infected with negative control lentivirus (Ctrl) or lenti-miR-6836 (Lv-miR-6836) in 100 μL per mouse (n=9). d Representative images of tumor tissues generated from Ctrl or Lv-miR-6836 cells are shown on the left and tumor volumes were measured and shown as scatter plot on the right. e Representative IHC staining of PCNA and DLG2 in subcutaneous xenografts. Scale bar, 100 μm. f,g For peritoneal metastatic model, five-week-old female BALB/c nude mice were injected intraperitoneally with 2×106 Ctrl or Lv-miR-6836 CAOV3 cells in 100 μL per mouse (n=8). f Representative images of peritoneal metastatic tumors are exhibited on the left, the number of organ spread with metastatic tumors is counted and quantified using scatter plot (right) and peritoneal metastasis incidence is listed and analyzed in the table (below). g Hematoxylin-eosin (HE) staining of tumor metastatic organs is conducted and representative images of HE staining (including lung, colon, kidney and Liver) from Lv-miR-6836 group are shown. Scale bar, 500 μm. Results are presented as mean ± SEM

Figure 4 miR-6836 regulates Yap1 subcellular localization by targeting DLG2. a Bubble chart revealing the functional enrichment pattern of SKOV3 attached cells and spheroids. b Bubble chart revealing the functional enrichment pattern of CAOV3 transfected with mimic/mi-NC. c Venn diagram of putative miR-6836 candidate target genes predicted by Targetscan, and genes involved in Notch signaling and Hippo signaling pathway. d Spearman correlation analysis between DLG2 mRNA and miR-6836 expression levels in EOC tissues (n=46). The expression level of DLG2 and miR-6836 was measured by RT-qPCR. e RT-qPCR analysis of DLG2 expression level in A2780/CAOV3 treated with mimic/mi-NC or inhibitor/in-NC. f Western blot analysis of DLG2 expression in A2780/CAOV3 treated with mimic/mi-NC or inhibitor/in-NC. g The schematic diagram on the left shows the dual luciferase reporter plasmids construction containing wild type (WT) or mutated (MUT) miR-6836 binding site in DLG2 3' UTR. 293T cells were transfected with WT/MUT recombined luciferase report vector and mimic/mi-NC. Luciferase reporter activity was normalized to Renilla luciferase activity. h Western blot analysis of p-Yap1, Yap1, Bax, Bcl2 and β-actin expression in OVCAR3/A2780 treated with mimic/mi-NC or A2780/CAOV3 with inhibitor/in-NC. i Western blot analysis of Yap1 expression in the nucleus and cytoplasm of CAOV3/SKOV3 Ctrl or Lv-miR-6836. j Immunofluorescence assay for the subcellular localization of Yap1 caused by mimic/mi-NC or inhibitor/in-NC treatment. Scale bar, 10 μm. k RT-qPCR analysis of miR-6836 expression in A2780/CAOV3 treated with si-TEAD1/si-NC. l Schematic diagram shows the possible TEAD1 binding regions of miR-6836 promoter region. ChIP assay is performed using TEAD1 antibodies and IgG as the negative control. m Luciferase reporter assay: 293T cells were transfected with si-TEAD1/si-NC and recombined luciferase report vector containing miR-6836 promoter region. Luciferase reporter activity was normalized to Renilla luciferase activity. n The schematic diagram of miR-6836-DLG2-Yap1-TEAD1 positive feedback loops in regulating EOC cisplatin resistance. In resistant EOC cells, miR-6836 suppressed apoptosis and activated stemness by directly targeting DLG2, and further promoted Yap1 nuclear translocation and interaction with TEAD1. TEAD1 is both the transcriptional regulator and downstream for miR-6836. Results are presented as mean ± SEM