Fig 1: Identification of an FRMD5-interacting region and its transcriptional activity. (a) Genomic structure of FRMD5 and the candidate FRMD5-interacting region in intron1. (b) Association of the candidate region with TCF7L2 was analyzed by ChIP-qPCR with anti-TCF7L2 antibody. A TCF7L2-binding region in RNF43 was used as a positive control. Quantitative PCR was performed in triplicate (mean ± SD). (c) Transcriptional activity of the candidate region. Reporter assay was performed using reporter plasmids containing the region with/without ß-catenin siRNA in HCT116 cells. (d) Luciferase activities of the wild type and mutant reporter plasmids. Luciferase activities were measured in triplicate (mean ± SD). An asterisk indicates statistical significance (Student's t-test or Dunnett's test, P < 0.05).
Fig 2: Genes with altered expression by the knockdown of FRMD5. (a) Decreased expression of FRMD5 in response to FRMD5 siRNA #2 and #3. (b) Top five downregulated genes (left) and top five upregulated genes (right) by FRMD5 siRNA in HCT116. Quantitative PCR was performed in triplicate using RNA from the cells treated with FRMD5 siRNA #2 (gray) or #3 (black), or control siRNA (white). Relative expression levels of the ten genes are shown in the histogram (mean ± SD) and result was normalized by GAPDH. An asterisk indicates statistical significance (Dunnett's test, P < 0.05) between FRMD5 siRNA and control siRNA.
Fig 3: FRMD5 regulates cell cycle in a cell context-dependent manner. HCT116, DLD-1, LS174T, and HCT-15 cells were treated with FRMD5 siRNA #2, #3 or control siRNA. G1 (white), S (black) and G2 (grey) phases are measured by FACS. Percentage (%) of each phase was showed in the histogram (mean ± SD). An asterisk indicates statistical significance (Dunnett's test, P < 0.05) between FRMD5 siRNA and control siRNA.
Fig 4: Expression of FRMD5 in CRC cell lines. (a) Reduced expression of FRMD5 protein by the knockdown of ß-catenin in HCT116 and DLD-1 cells. (b) FRMD5 expression in eight CRC cell lines. Expression of ß-actin served as an internal control.
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