Fig 1: CRIP1 stimulates the ubiquitination and degradation of Fas. a. Real-time PCR shows the effects of CRIP1 on the expression of Fas at transcriptional level in both SW620 and HCT116 cells. b. Co-Ip shows the interaction between CRIP1 and Fas in both SW620 and HCT116 cells. c. The immunofluorescence staining demonstrated the co-localization of CRIP1 and Fas in both SW620 and HCT116 cells. d. Left panel: western blot assay shows that MG-132 recovered the inhibitory role of CRIP1 on Fas Protein. Right panel: the effects of MG-132 alone on the expression of Fas protein. e. CRIP1 stimulates the ubiquitination of Fas, while both siCRIP1 and MG-132 could recover the ubiquitin-mediated degradation of Fas
Fig 2: CRIP1 is overexpressed in both CRC tissues and cell lines. a. Left panel: Western blot assay for CRIP1 expression in ten CRC tissues (T) and matched normal adjacent normal tissues (N) from the same patient. Right panel: Quantification of CRIP1 expression in CRC tissues and adjacent normal tissues of ten patients. b. Left panel: IHC staining indicated that CRIP1 protein expression was increased in human CRC compared with normal intestinal epithelium (four representative slides was shown). Right panel: Graphical illustration of statistical CRIP1 distribution in CRC tissues. CRIP1 is significantly highly expressed in CRC tissues compared with that in adjacent non-tumorous tissue. c. Real-time PCR analyzed the expression of CRIP1 in NCM460 and six different CRC cell lines. The values represented relative CRIP1 level after normalization to the expression of ß-tubulin (means ±SD, N = 3) d. Western blot analysis for the expression of CRIP1 in NCM460 and CRC cell lines. Immunosignals were quantified by densitometric scanning
Fig 3: Overexpression of CRIP1 recovers 5-FU suppressed CRC cell proliferation both in vitro and in vivo. a. Western blot assay was used to detect the overexpression of CRIP1 in SW620 and RKO cells. b. CCK8 assay was used to study the effects of CRIP1 on 5-FU inhibited CRC cell proliferation. c. Annexin V-FITC/PI flow cytometry assay showed the effects of CRIP1 on 5-FU induced apoptosis. Bars on the right panel represent percentage of cells in Q2 + Q4. d. TUNEL assay showed the effects of CRIP1 on 5-FU induced apoptosis of CRC cells. Bars on the right panel represent the percentage of TUNEL positive cells. e. IHC staining showed the expression of CRIP1 in subcutaneous tumors formed by CRIP1 overexpressed CRC cells. Right panel represents the growth pattern of subcutaneous tumors
Fig 4: CRIP1 inhibits Fas induced apoptosis of CRC cells. a. Western blot analysis of apoptotic related proteins in CRIP1 overexpressed or silencing indicated cells. b. Western blot analysis of the effects of CRIP1 on Fas-mediated apoptosis. c. CCK8 assay shows the effects of CRIP1 on Fas-mediated apoptosis. d. Annexin V-FITC/PI flow cytometry assay showed the effects of CRIP1 on Fas-mediated apoptosis. Bars on the upper panel represent percentage of cells in Q2 + Q4. e. TUNEL assay showed the effects of CRIP1 on Fas-mediated apoptosis of CRC cells. Bars on the right panel represent the percentage of TUNEL positive cells
Fig 5: Knockdown of CRIP1 further suppresses 5-FU suppressed CRC cell proliferation both in vitro and in vivo. a. Western blot assay was used to detect the silencing of CRIP1 in HCT116 and SW480 cells. b. CCK8 assay was used to study the effects of siCRIP1 on 5-FU inhibited CRC cell proliferation. c. Annexin V-FITC/PI flow cytometry assay showed the effects of siCRIP1 on 5-FU induced apoptosis. Bars on the right panel represent percentage of cells in Q2 + Q4. d. TUNEL assay showed the effects of siCRIP1 on 5-FU induced apoptosis of CRC cells. Bars on the right panel represent the percentage of TUNEL positive cells. e. IHC staining showed the expression of CRIP1 in subcutaneous tumors formed by CRIP1 stable silencing CRC cells. Right panel represents the growth pattern of subcutaneous tumors
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