original dld1 cells  (ATCC)

Journal: EMBO Reports

Article Title: Paneth cell SIRT1 deficiency increases intestinal stress resistance by modulating the gut microbiota

doi: 10.1038/s44319-026-00726-3

Figure Lengend Snippet: ( A ) Pseudotime analysis of IECs in the small intestine of young and aged Flox and SIRT1 PKO mice. ( B ) The expression of Wnt3 is increased in cluster 16 Paneth cells of aged SIRT1 KO mice. The expression of Wnt3 is projected onto tSNE (upper) or shown in violin plots (bottom, two-sided Wilcoxon test). ( C ) Deletion of Paneth cell SIRT1 reduces age-induced depletion of cluster 16 Paneth cells and Lgr5 + ISCs. ( D – F ) Aged SIRT1 PKO mice have increased nuclear β-catenin accumulation in the crypts than Flox mice. The nuclear β-catenin intensity in total ileal epithelium was quantified by AI as described in Methods. Bars in ( D ), 20 μm. ( E ) The fraction of nuclei with high β-catenin intensity scores (>9, nuclei in the crypts; n = 6 Flox-Young, 12 Flox-Aged, 7 PKO-Young, and 6 PKO-Aged mice, Student’s t-test). ( F ) The average nuclear β-catenin intensity scores in all epithelial tissue ( n = 6 Flox-Young, 12 Flox-Aged, 7 PKO-Young, and 6 PKO-Aged mice, two-way ANOVA). ( G ) The protein level of β-catenin is increased in the ileum of SIRT1 PKO mice ( n = 6 Flox and 6 PKO, Student’s t-test). ( H ) SIRT1 KO colon epithelial cells have increase mRNA levels of WNT3 but not CTNNB1 genes. WT and SIRT1 KO DLD1 cells were cultured in regular RMPI medium ( n = 3 biological replicates, Student’s t-test). ( I ) SIRT1 KO colon epithelial cells have increased β-catenin protein. WT and SIRT1 KO DLD1 cells were cultured in regular RMPI medium. ( J ) β-catenin is hyper-acetylated and hypo-ubiquitinated in SIRT1 KO colon epithelial cells. WT and SIRT1 KO DLD1 cells cultured in regular RMPI medium were treated with 5 μM TSA and 10 μM MG-132 for 3 h before harvesting. Total cell lysates were immunoprecipitated with anti-β-catenin antibodies (IP-β-catenin), then immunoblotted with anti-β-catenin, acetyl-lysine (Ac-K), or anti-ubiquitin antibodies. Data information: in ( E , F , and G ), values are expressed as mean ± SEM; * p < 0.05, ** p < 0.01; no marks, not significant. .

Article Snippet: WT and SIRT1 KO DLD1 human colorectal cell lines , (Ren et al, ) , Original DLD1 cells were from ATCC (cat# CCL-221), routinely tested in the lab for mycoplasma contamination.

Techniques: Expressing, Cell Culture, Immunoprecipitation, Ubiquitin Proteomics

Journal: EMBO Reports

Article Title: Paneth cell SIRT1 deficiency increases intestinal stress resistance by modulating the gut microbiota

doi: 10.1038/s44319-026-00726-3

Figure Lengend Snippet: ( A ) The transcription activity of ATF4 is increased in the SIRT1 KO Paneth cells. All the combined promoters of the genes expressed in a cell were used to infer the activity of a transcription factor by pySCENIC, and the activity of ATF4 in Paneth cells was projected onto tSNE space with its activity color coded. ( B ) ER stress response pathways are enriched in ATF4 target genes that are significantly increased in SIRT1 KO Paneth cells (Permutation testing with the Benjamini–Hochberg adjusted p-values). Enrichment score represents −log 10 -transformed adjusted p -values. ( C ) SIRT1 KO colon epithelial cells have increased response to ER stresses. WT and SIRT1 KO DLD1 cells were treated with indicated stress conditions for 24 h. The expression of indicated genes was analyzed by qPCR ( n = 3 biological replicates, two-way ANOVA). ( D ) SIRT1 KO colon epithelial cells have reduced cell death in response to ER stresses. WT and SIRT1 KO DLD1 cells were cultured in glucose free RPMI medium for 3 days ( n = 3 biological replicates, Student’s t-test). Bar, 100 μm. ( E ) SIRT1 KO DLD1 cells have increased induction of ATF4 protein during glucose free medium induced ER stress. ( F ) ATF4 is hypo-ubiquitinated in SIRT1 KO colon epithelial cells. WT and SIRT1 KO DLD1 cells cultured in regular RMPI medium were treated with 5 μM TSA and 10 μM MG-132 for 3 h before harvesting. Total cell lysates were immunoprecipitated with anti-ATF4 antibodies (IP-ATF4), then immunoblotted with anti-ATF4, acetyl-lysine (Ac-K), or anti-ubiquitin antibodies. ( G ) SIRT1 KO DLD1 cells have increased expression of defensin genes ( n = 3 biological replicates, two-way ANOVA). ( H ) Small intestinal organoids from PKO mice have increased induction of ATF4 targets and reduced suppression of anti-microbial peptide genes in response to ER stress. Small intestinal organoids from Flox and PKO mice were cultured in regular medium or glucose free medium overnight. The expression of indicated genes was analyzed by qPCR ( n = 3 biological replicates/group, two-way ANOVA). Data information: in ( C , D , G , and H ), values are expressed as mean ± SEM; * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001; no marks, not significant. .

Article Snippet: WT and SIRT1 KO DLD1 human colorectal cell lines , (Ren et al, ) , Original DLD1 cells were from ATCC (cat# CCL-221), routinely tested in the lab for mycoplasma contamination.

Techniques: Activity Assay, Transformation Assay, Expressing, Cell Culture, Immunoprecipitation, Ubiquitin Proteomics