Project description:M-NM-2-catenin activity of dermal cells is crucial for hair follicle neogenesis and strongly suggest that transcriptional target genes of M-NM-2-catenin pathway are essential for maintaining trichogenicity of dermal cells. We used microarrays to identify genes targeted by the M-NM-2-catenin pathway that are essential for maintaining the trichogenicity of dermal cells. Freshly isolated dermal cells from the dorsal skins of C57BL/6 neonates (P0) were cultured for 24 hours and transfected with either control siRNA or M-NM-2-catenin specific siRNAs (M-NM-2-catenin siRNA) at a final concentration of 10 nM for 48 hours followed by RNA extraction and hybridization on Affymetrix microarrays.
Project description:In colorectal cancer, p53 is commonly inactivated, associated with chemo-resistance, and marks the transition from non-invasive to invasive disease. Cancers, including colorectal cancer, are thought to be diseases of aberrant stem cell populations, as stem cells are able to self-renew, making them long-lived enough to acquire mutations necessary to manifest the disease. We have shown that extracts from sweet sorghum stalk components eliminate colon cancer stem cells (CCSC) in a partial p53-dependent fashion. However, the underlying mechanisms are unknown. In the present study, CCSC were transfected with short hairpin-RNA against p53 (CCSC p53 shRNA) and treated with sweet sorghum phenolics extracted from different plant components (dermal layer, leaf, seed head and whole plant). While all components demonstrated anti-proliferative and pro-apoptotic effects in CCSC, phenolics extracted from the dermal layer and seed head were more potent in eliminating CCSC by elevating caspases 3/7 activity, PARP cleavage, and DNA fragmentation in a p53-dependent and p53-independent fashion, respectively. Further investigations revealed that the anti-proliferative and pro-apoptotic effects were associated with decreases in beta-catenin protein levels, and beta-catenin targets cyclin D1, cMyc, and survivin. These results suggest that the anti-proliferative and pro-apoptotic effects of sweet sorghum extracts against human colon cancer stem cells are via suppression of Wnt/beta-catenin pro-survival signaling in a p53-dependent (dermal layer) and partial p53-independent (seed head) fashion. LCMS used to identify phenolic compounds associated with extract activity
Project description:Human colon carcinoma cells SKCO1 were deprived of either beta-catenin alone or beta-catenin together with TNFR1 and changes in mRNA levels were examined before and after TNFR1 ablation SKCO1 cells were transfected with either beta-catenin-specific siRNA alone or beta-catenin-specific siRNA together with a TNFR1-specific siRNA and changes in mRNA levels were examined before and after TNFR1 ablation
Project description:Forced expression of activated beta-catenin in mouse dermal fibroblasts is sufficient to cause spontaneous, progressive skin fibrosis in vivo. We generated triple-transgenic HoxB6CreERT/+; R26-YFP/+; CatnbΔex3/+ "activated beta-catenin" mice and double-transgenic HoxB6CreERT/+; R26-YFP/+ littermate control mice. We induced Cre activity (resulting in expression of activated beta-catenin in triple-transgenic mutant fetuses) by administering tamoxifen to the pregnant dam at embryonic day 16.5. The activated beta-catenin mice developed fibrotic skin, characterized by elevated collagen deposition and increased fibroblast proliferation. We performed RNA-sequencing to profile gene expression in the dermis of control and activated beta-catenin mutant mice with established skin fibrosis at 3 weeks of age.
Project description:To identify novel transcriptional targets of beta-catenin and FoxO1 and FoxO3 in renal epithelial cells, we used conditionally immortalized murine proximal tubule (PT) cells (these cells, from Tgfbr2floxed mice, are described in detail in manuscript PMID: 23160515 in which Leslie Gewin is first author, JASN 2012). PT cells were either treated with Wnt3a (to activate beta-catenin) or the control diluting buffer, H2O2 to induce oxidative stress, and some were transfected with FoxO1, FoxO3, FoxO1 and 3, or scramble siRNA prior to Wnt and H2O2 treatment.
