Fig 1: miR-373-target genes mediate NE-induced effects. (A) The mRNA levels of TIMP2 and APC were analyzed using qRT-PCR in HCT116 and RKO cells treated with NE. (B) The protein levels of TIMP2, APC, and ß-catenin in HCT116 and RKO cells treated with NE were detected by western blotting. ß-actin was used as the loading control. (C) HCT116 and RKO cells were transfected with TIMP2 or vehicle plasmids followed by NE treatment. Representative images and quantification of the wound healing assay are shown. Scale bars represent 100 µm. (D) Representative images and percentage migration in the transwell assay for migration and invasion. Scale bars represent 100 µm. The experiments were independently repeated three times with reproducible results. Error bars are represented as mean ± SD (n = 5). P-values were calculated using Student's t-test. * represents P < 0.05, ** represents P < 0.01, and *** represents P < 0.001.
Fig 2: miR-373 targets TIMP2 and APC. (A) mRNA levels of TIMP2 and APC as analyzed by qRT-PCR in HCT116 and RKO cells transfected with miR-373 or miR-Ctrl. (B) Protein levels of TIMP2, APC, and ß-catenin in HCT116 and RKO cells transfected with miR-373, miR-Ctrl, miR-373 inhibitor, or mNC were detected by western blotting. ß-actin levels were used as the loading control. (C) The luciferase reporter assay was performed in HCT116 cells containing miR-373 or miR-Ctrl cotransfected with pmirGLO-wild-type (TIMP2-WT or APC-WT) or pmirGLO-mutant constructs (TIMP2-MT or APC-MT). (D) HCT116 and RKO cells were transfected with miR-Ctrl, miR-373, or miR-373 and the TIMP2 plasmid. Subsequently, wound healing assays were performed and quantified. Representative images and percentage of wound closure are shown. Scale bars represent 100 µm. The experiments were independently repeated three times with reproducible results. Error bars are represented as mean ± SD (n = 5). P-values were calculated using Student's t-test. * represents P < 0.05, ** represents P < 0.01, and *** represents P < 0.001.
Fig 3: MiR-26b inhibits hepatocellular carcinoma cell proliferation, invasion and migration in vitro by targeting Zinc ribbon domain-containing 1 (ZNRD1). SMMC-7721 or Hep 3B cells were transfected with NC, miR-26b mimics or miR-26b mimics & ZNRD1 overexpression plasmid. A, The expression levels of ZNRD1, Wnt3a, ß-catenin, APC, and cyclinD1 were analyzed by western blot 48 h later. B, Cell proliferation was determined by CCK-8 cell proliferation assays. C, Colonies of SMMC-7721 or Hep 3B in different groups were determined by colony formation assay. D, Cell invasion capability of SMMC-7721 or Hep 3B in different groups was analyzed by transwell assay. E, Cell migration capability of SMMC-7721 or Hep 3B cells in different groups was analyzed by wound-healing assay. *P < .05, **P < .01
Fig 4: The expression of Wnt receptors and DCs in back skin sample of psoriatic mice. (a) Western blot of Frizzled-2, LRP6, and LRP5 protein levels in skin samples of psoriasis mice. (b) Western blot of GSK-3ß, APC, and Axin2 protein levels in skin samples of psoriasis mice. Data are presented as the mean ± SD of three independent experiments performed in triplicate. *P < 0.0001 vs. control group; #P < 0.0001 vs. IMQ group.
Fig 5: Analysis of TH, CREB1, hsa-miR-373-3p, APC, and TIMP2 expression in human CRC using TCGA database and clinical CRC samples. (A) Data on TH, CREB1, hsa-miR-373-3p, APC, and TIMP2 expression in CRC tumor tissues were extracted from the RNAseq Illumina HiSeq dataset in TCGA database. There were 23 cases of TCGA colon cancer (COAD) and 14 cases of TCGA rectal cancer (READ) with simultaneous data on TH, CREB1, hsa-miR-373-3p, APC, and TIMP2. The cases were ranked according to the expression level of TH and divided into two groups; the half with lower TH expression was designated the Low TH RNA level group, and the other half was designated the High TH RNA level group. The expression of CREB1, hsa-miR-373-3p, APC, and TIMP2 in these two groups was compared, and P-values were calculated using unpaired t-test. (B) Protein lysates were harvested from the tumors of each clinical CRC case. The levels of TH, phospho-CREB1, CREB1, APC, and TIMP2 were detected by western blotting. ß-actin was used as the loading control. (C) RNA was extracted from the tumors of clinical CRC patients. The levels of hsa-miR-373-3p were measured by qRT-PCR and compared between the group with Low TH protein level and the group with High TH protein level. Error bars are represented as mean ± SD (n = 12). P-values were calculated using unpaired t-test. * represents P < 0.05 and ** represents P < 0.01.
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