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Additional file 1: of WDR76 degrades RAS and suppresses cancer stem cell activation in colorectal cancer

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posted on 31.07.2019, 03:59 by Eun Ro, Yong-Hee Cho, Woo-Jeong Jeong, Jong-Chan Park, Do Min, Kang-Yell Choi
Figure S1. Ras protein level is increased in Lgr5+ stem cells in the murine small intestine. A-B Immunofluorescence of A Ras (green) and B Lgr5- GFP (green) in Lgr5-EGFP mouse intestinal sections. Lgr5+ ISCs are indicated by arrows. Scale bars represent 20 μm. Figure S2. Loss of Wdr76 affects lineage differentiation in the murine small intestine. A Quantification of the length of small intestinal crypts, based on at least 10 crypts per 5 fields of view. *** p < 0.001. B-C Immunofluorescence analysis of lineage differentiation into goblet cells (mucin2, red) and Paneth cell (lysozyme, red) in intestinal sections of B Wdr76+/+ and Wdr76-/- mice and C Wdr76+/+; ApcMin/+ and Wdr76−/−; ApcMin/+ mice. Boxes indicate the enlarged areas. B Crypts and C tumors are indicated by dotted lines. Scale bars represent 20 μm. Figure S3. RAS protein level is increased in CSC-like cells compared with that in non-CSC-like cells in CRC. A-B Non-CSC-like cells (CD44lowCD133-CD166-) and CSC-like cells (CD44highCD133+CD166+ cells) were sorted from D-MT cells by flow cytometry and were analyzed by A brightfield images of spheroid cultures and B western blots using the indicated antibodies. A Scale bars represent 20 μm. Figure S4. Cytosolic WDR76 destabilizes RAS and suppresses CSC activation in CRC. A-G D-MT cells stably expressing GFP-Control, GFP-WDR76FL, or GFP-WDR76ΔNLS were analyzed. A Western blots using the indicated antibodies. B-C After treatment of ALLN (25 μg/mL, 12 h), extracts were analyzed by B immunoprecipitation and C ubiquitination of K-RAS by immunoblotting against the indicated antibodies. D-G Five-day spheroid cultures were analyzed. D Number and size of spheroids were quantified using Image J. *** p < 0.001. E Cell viability assay was performed. *** p < 0.001. F Immunocytochemistry was performed using the indicated antibodies and counterstaining with DAPI. Scale bars represent 20 μm. G Relative mRNA levels of the indicated genes were quantified by RT-qPCR. * p < 0.05, ** p < 0.01. (DOCX 3962 kb)


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