p53 wt or mut fkbp12f36v inducible cell line plenti cmv rtta3 blast w756 1  (Addgene inc)

Journal: HemaSphere

Article Title: Casein kinase 1δ/ε inhibition suppresses CLL proliferation through cell‐intrinsic and microenvironmental mechanisms

doi: 10.1002/hem3.70343

Figure Lengend Snippet: Validation of the cell cycle and proliferation effects of casein kinase 1δ/ε (CK1δ/ε) inhibition in vivo and in vitro. (A) Percentages of EdU‐Alexa Fluor 647+ leukemic B cells within the spleen (SPL) of treated and control TCL1 adoptive transfer (AT) recipient mice ( N (AT CTRL) = 3; N (AT + PF‐670462) = 4), tested by the t ‐test. (B) Relative cell counts (% of CTRL) originating from in vitro treated MEC‐1 wild‐type (WT) cells after a 72 h treatment with PF‐670462 or MU1742 (performed on the following biological replicates: N (CTRL) = 6; N (3µM PF‐670462) = 3; N (10µM PF‐670462) = 6; N (3µM MU1742) = 3; and N (10µM MU1742) = 3), tested by the Kruskal–Wallis test with post hoc pairwise Wilcoxon rank sum tests with Benjamini–Hochberg correction. (C) Relative cell counts (% of CTRL) originating from in vitro treated HG‐3 WT cells after 72 h treatment with PF‐670462 or MU1742 (performed on the following biological replicates: N (CTRL) = 4; N (3µM PF‐670462) = 4; N (10µM PF‐670462) = 4; N (3µM MU1742) = 4; and N (10µM MU1742) = 4), tested by the Kruskal–Wallis test with post hoc pairwise Wilcoxon rank sum tests with Benjamini–Hochberg correction. (D) Cell cycle assay setup with initial CK1 inhibitor treatment and mitotic arrest with nocodazole and the representative example of cell cycle alterations between analyzed conditions in MEC‐1 and HG‐3 cell lines. (E) Cell cycle phase distribution in MEC‐1 WT cells upon 9 h pre‐treatment with 3 µM PF‐670462 and 10 µM PF‐670462 and subsequent mitotic arrest with nocodazole (performed on the following biological replicates: N (CTRL) = 11; N (NOCODAZOLE) = 11; N (3µM PF‐670462) = 7; and N (10µM PF‐670462) = 11); for all cases together, the generalized linear mixed‐effects model, followed by estimated marginal means calculation (P‐value < 0.05), was used separately for comparison of CTRL versus NOCODAZOLE and NOCODAZOLE versus PF‐670462 and corrected due to usage of 2 models. (F, G) Cell cycle phase distribution in MEC‐1 WT cells upon 9 h pre‐treatment with 3 and 10 µM concentrations of MU1742 and AH078 (respectively) and subsequent mitotic arrest with nocodazole (performed on the following biological replicates: N (CTRL) = 11; N (NOCODAZOLE) = 11; N (3µM) = 3; and N (10µM) = 3); for all cases together, the generalized linear mixed‐effects model followed by estimated marginal means calculation (P‐value < 0.05), was used separately for comparison of CTRL versus NOCODAZOLE and NOCODAZOLE versus MU1742/AH078 and corrected due to usage of 2 models. (H–J) Cell cycle phase distribution in HG‐3 WT cells upon 9 h pre‐treatment with 3 and 10 µM concentrations of PF‐670462, MU1742, and AH078 (respectively) and subsequent mitotic arrest with nocodazole (performed on the following biological replicates: N (CTRL) = 4; N (NOCODAZOLE) = 4; N (3µM) = 4; and N (10µM) = 4); for all cases together, the generalized linear mixed‐effects model, followed by estimated marginal means calculation (P‐value < 0.05), was used separately for comparison of CTRL versus NOCODAZOLE and NOCODAZOLE versus PF‐670462/MU1742/AH078 and corrected due to usage of 2 models. DMSO, dimethyl sulfoxide; PI, propidium iodide.

Article Snippet: CLL cell lines MEC‐1 WT (DSMZ, #ACC497) and HG‐3 WT (DSMZ, #ACC765) were treated with PF‐670462 (DC Chemicals, #DC2086), an in‐house CK1δ/ε inhibitor MU1742 or CK1δ/ε degrader AH078, and subjected to cell proliferation tracking and cell cycle tracking via PI staining, EdU Click‐iT assays, and/or western blotting, as described in more detail in the Supporting Information S1: .

Techniques: Biomarker Discovery, Inhibition, In Vivo, In Vitro, Control, Adoptive Transfer Assay, Cell Cycle Assay, Comparison

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 ) Defa6Cre drives specific expression of TdTomato in Paneth cells. The expression of TdTomato and lysozyme in the ileum of Defa6-Cre/TdTomato mice were analyzed by IF. Bar, 50 μm. ( B ) Sirt1 is efficiently deleted in Paneth cells in SIRT1 PKO mice. Paneth cells in Defa6-Cre control and PKO mice on a TdTomato+ background were sorted out and the levels of Sirt1 mRNA in sorted Paneth cells were analyzed by qPCR ( n = 3 pairs of mice, paired t-test). ( C ) H&E staining of intestinal sections of young Flox and SIRT1 PKO mice. Bars, 250 μm. ( D ) Young Flox and SIRT1 PKO mice have comparable cell proliferation under normal feeding condition. Intestinal sections from 4-month-old Flox and SIRT1 PKO mice were stained with an anti-Ki67 antibody. ( E ) Young Flox and SIRT1 PKO mice have comparable expression of inflammatory genes in the gut under normal feeding condition. The expression of indicated genes was analyzed by qPCR ( n = 6 Flox and 7 PKO, Student’s t-test). ( F ) Immune cells from the ileum and colon of young Flox and SIRT1 PKO mice. The fraction of indicated immune cells in total live cells isolated from Flox and PKO mice was analyzed by FACS as described in Methods (Ileum: n = 7 Flox and 5 PKO mice; Colon: n = 6 Flox and 5 PKO mice, Student’s t-test). Data information: in ( B , E , and F ), 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, Control, Staining, Isolation

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 ) Annotation of total live cells from the small intestine of Flox and SIRT1 PKO mice. Total live cells isolated from the small intestine of young (2–3 months) and aged (12–14 months) mice were analyzed by scRNA-seq. ( B ) SIRT1 PKO mice have increased Paneth cell abundance in both young and aged mice. Total intestinal live cells were analyzed by scRNA-seq as in ( A ). ( C ) SIRT1 PKO mice have increased abundance of Paneth cells in the small intestine. The abundance of Paneth cells in Flox and PKO mice on the Lgr5-GFP background was analyzed by FACS ( n = 15 pairs, paired t-test). ( D ) The small intestinal organoids from SIRT1 PKO mice have a higher density of Paneth cells compared to those from SIRT1 PHet mice. The freshly isolated small intestinal epithelial cells (mainly crypts) from SIRT1 PHet/TdTomato control and SIRT1 PKO/TdTomato mice were cultured in the Intesticult medium for 6 days. The area of organoids and Paneth cell number in organoids were quantified in Fiji ( n = 13 PHet and 14 PKO organoids, Student’s t-test). Bar, 200 μm. ( E ) Aged SIRT1 PKO mice (10–11 months) have reduced CD45 + immune cells in the small intestine. The fraction of CD45 + immune cells in total live cells isolated from Flox and PKO mice was analyzed by FACS as described in Methods ( n = 6 Flox and 6 PKO, Student’s t-test). ( F ) Two-year-old SIRT1 PKO mice have reduced mucosal immune cells (arrows) in the ileum ( n = 5 out of 12 Flox mice and 1 out of 6 PKO mice display patches of mucosal lymphoid follicles; right panel, % of tissue area with mucosal immune cells was quantified by Fiji and categorized as indicated). Bars, 500 μm. Data information: in ( C , D , and E ), values are expressed as mean ± SEM; * p < 0.05; 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: Isolation, Control, Cell Culture

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 ) Dot plot showing the expression of marker genes in different function groups of total live cells isolated from the small intestine. ( B ) Relative composition of different cell types in the small intestine of young and aged mice. ( C ) Gating strategy for FACS analysis of CD24 hi SSC hi Paneth cells and GFP + intestinal stem cells (ISCs). Small intestinal epithelial cells from 2- to 3-month-old Flox and SIRT1 PKO mice on the Lgr5-EGFP background were isolated and analyzed as described in Methods. The FACS plots from the same Flox mouse in Fig. are used here to demonstrate the gating strategy. ( D ) SIRT1 PKO mice have normal abundance of ISCs in the small intestine. The abundance of ISCs in Flox and PKO mice on the Lgr5-GFP background was analyzed by FACS ( n = 15 pairs, paired t-test). ( E ) Age induces immune cell expansion in the colon. The fraction of CD45 + immune cells in total live cells isolated from Flox and PKO mice at indicated ages was analyzed by FACS as described in Methods ( n = 6, 5, 7, 6, 6, 6 mice per indicated group, two-way ANOVA). Data information: ( D and E ), values are expressed as mean ± SEM; * p < 0.05, ** p < 0.01, **** 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: Expressing, Marker, Isolation

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 ) SIRT1 KO Paneth cells have increased expression of gene sets involved in ER stress response but reduced expression of gene sets mediating mitochondrial activities. Transcriptomes of Flox and SIRT1 KO Paneth cells from scRNA-seq dataset were analyzed. Top enriched Gene set enrichment (GSEA) pathways in SIRT1 KO vs Flox Paneth cells in young mice are shown. ( B ) SIRT1 KO Paneth cells have increased expression of genes involved in transport and host defense compared to Flox Paneth cells. Differentially Expressed Genes (DEGs) in SIRT1 KO vs Flox Paneth cells from scRNA-seq dataset were analyzed by GO biological process and top enriched pathways are shown (Permutation testing with the Benjamini–Hochberg adjusted p -values). Enrichment score represents −log 10 -transformed adjusted p -values. ( C ) Annotation of intestinal epithelial cells (IECs) in the small intestine of Flox and SIRT1 PKO mice. ( D ) Two clusters of Paneth cells. ( E ) SIRT1 KO Paneth cells have increased expression of anti-microbial peptide genes. The color-coded violin plots display the expression distribution of anti-microbial peptide genes involved in host defense in two clusters of Paneth cells from indicated scRNA-seq samples. Each dot represents a single cell. Gene expression changes between samples were compared and the significance of change was labeled (two-sided Wilcoxon test). ( F ) SIRT1 PKO mice have increased expression of several anti-microbial peptide genes in the ileum. The mRNA levels of indicated anti-microbial peptide genes in the whole ileum tissue were analyzed by qPCR ( n = 12 Flox and 10 PKO, Student’s t-test). Data information: in ( F ), 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, Transformation Assay, Single Cell, Gene Expression, Labeling

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

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 – D ) SIRT1 PKO female mice have altered small intestinal microbiota. Total small intestinal DNA from Flox and SIRT1 PKO mice were analyzed for mucosal adherent microbiota using 16S rRNA gene amplicon sequencing as described in Methods ( n = 10 Flox and 8 PKO females and 10 Flox and 10 PKO males). ( A ) Alpha diversity of small intestinal bacteria (Student’s t-test). ( B ) Beta diversity of small intestinal bacteria (Pair-wise permanova test). ( C ) SIRT1 PKO females have altered levels of several major classes of small intestinal microbiota. ( D ) SIRT1 PKO females have increased abundance of Lactobacillus in the small intestine ( n = 10 Flox and 8 PKO females, Student’s t-test). ( E ) Depletion of microbiota reduces the expression of anti-microbial peptides in the ileum in both Flox and PKO mice. The expression of indicated genes in the ileum of regular and germ-free (GF) Flox and PKO mice were analyzed by qPCR ( n = 18 regular Flox, 16 regular PKO, 6 GF Flox, and 6 GF PKO mice, two-way ANOVA). ( F ) The effect of microbiota on the expression of other intestinal epithelial cell markers. The expression of indicated genes in the ileum of regular and germ-free (GF) Flox and PKO mice were analyzed by qPCR ( n = 12 regular Flox, 10 regular PKO, 6 GF Flox, and 6 GF PKO mice, two-way ANOVA). Data information: in ( A and B ), box-and-whisker plot with the box representing the interquartile range (Q1 to Q3), a line inside indicating the median, and whiskers representing the 2.5–97.5 percentile; in ( D , E , and F ), values are expressed as mean ± SEM; * p < 0.05, ** p < 0.01, *** p < 0.001; 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: Amplification, Sequencing, Bacteria, Expressing, Whisker Assay

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 – C ) SIRT1 PKO mice have altered colonic microbiota. Total colonic DNA from Flox and SIRT1 PKO mice were analyzed for mucosal adherent microbiota using 16S rRNA gene amplicon sequencing as described in Methods ( n = 10 Flox and 9 PKO females and 10 Flox and 10 PKO males). ( A ) Alpha diversity of colonic bacteria (Student’s t-test). ( B ) Beta diversity of colonic bacteria (Pair-wise permanova test). ( C ) The abundance of major genera in the colon ( n = 10 Flox and 9 PKO females and 10 Flox and 10 PKO males, Student’s t-test). ( D – G ) SIRT1 PKO female mice have altered fecal microbiota. Total fecal DNA from Flox and SIRT1 PKO females were analyzed using 16S rRNA gene amplicon sequencing. ( D ) Alpha diversity of fecal bacteria ( n = 6 mice/genotype, Student’s t-test). ( E ) Beta diversity of fecal bacteria ( n = 6 mice/genotype, Pair-wise permanova test). ( F ) Altered abundance of several major classes of fecal microbiota in SIRT1 PKO females. ( G ) SIRT1 PKO females have altered levels of several major genera in feces ( n = 6/genotype, Student’s t-test). Data information: in ( A , B , D , and E ), box-and-whisker plot with the box representing the interquartile range (Q1 to Q3), a line inside indicating the median, and whiskers representing the 2.5–97.5 percentile; in ( C 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: Amplification, Sequencing, Bacteria, Whisker Assay

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 – C ) Young SIRT1 PKO mice are more protected against DSS-induced colitis compared to Flox controls. Three-month-old Flox and SIRT1 PKO mice were treated with 2.5% DSS in drinking water for 5–7 days. Their body weight ( A ), rectal bleeding ( B ), and colon length ( C ) were analyzed ( n = 43/group, Student’s t-test). ( D , E ) Young SIRT1 PKO mice have reduced pathological severity in the colon after DSS treatment. Three-month-old Flox and SIRT1 PKO mice were treated as in ( A ). The histopathological severity was evaluated by a professional pathologist as described in Methods ( n = 6 Flox and 8 PKO, Student’s t-test). Red arrowheads, segmental mucosal ulceration and consequent replacement with granulation tissue; orange arrowhead, submucosal edema; blue arrowhead, sloughed off necrotic and cellular debris within the lumen. Bars, 200 μm. ( F ) Altered gene expression in the colon of mice after 2 cycles of DSS treatment. Nine-month-old Flox and SIRT1 PKO mice were treated with 2 cycles of DSS as described in Methods and the expression of indicated genes were analyzed by qPCR ( n = 6 Flox and 12 PKO mice, Student’s t-test). ( G ) The ileum of DSS-treated 9-month-old SIRT1 PKO mice have increased expression of genes involved in cell cycle, cell proliferation, as well as increased levels of stem cell and Paneth cell genes. The expression of indicated genes was analyzed by qPCR ( n = 6 Flox and 12 PKO mice, Student’s t-test). ( H , I ) The ileum of 9-month-old SIRT1 PKO mice treated with 2 cycles of DSS water have increased β-catenin signaling. ( H ) The expression of β-catenin was analyzed by IHC. Arrowheads, nuclear β-catenin in Paneth cells. Bar, 20 μm. ( I ) The nuclear β-catenin intensity in the epithelial tissue was quantified by AI as described in Methods ( n = 6 Flox and 12 PKO mice, Student’s t-test). Data information: in ( A , B , C , F , G , and I ), values are expressed as mean ± SEM; in ( E ), box-and-whisker plot with the box representing the interquartile range (Q1 to Q3), a line inside indicating the median, and whiskers representing the 2.5–97.5 percentile; * p < 0.05, ** p < 0.01, *** p < 0.001; 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: Gene Expression, Expressing, Whisker Assay

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 – C ) Nine-month-old SIRT1 PKO mice are more resistant to DSS-induced colitis than Flox mice. Nine-month-old Flox and SIRT1 PKO mice were treated with 2 cycles of 2.5% DSS for 7 days with 14 day of regular water break in between. Their body weight ( A ), rectal bleeding ( B ), and colon length ( C ) were analyzed ( n = 6 Flox and 12 PKO mice, Student’s t-test). ( D , E ) Colon histology of 9-month-old Flox and SIRT1 PKO mice after 2 cycles of DSS treatment. ( D ) Representative H&E staining of Swiss swirl from 2 Flox and 2 PKO mice are shown. Bars, 500 μm. ( E ) The histopathological severity was evaluated by a professional pathologist as described in Methods ( n = 6 Flox and 13 PKO, Mann-Whitney test). ( F ) Ileum histology of 9-month-old Flox and SIRT1 PKO mice after 2 cycles of DSS treatment. Representative H&E staining of Swiss swirl from 2 Flox and 2 PKO mice are shown. Bar, 1 mm. ( G ) The top enriched functional pathways in the colon or ileum of 9-month-old Flox and SIRT1 PKO mice after 2 cycles of DSS treatment. Total RNA from the indicated mice were analyzed by RNA-seq. DEGs between SIRT1 KO vs Flox were analyzed by GO biological process and top enriched pathways are shown (Permutation testing with the Benjamini–Hochberg adjusted p -values). Enrichment score represents −log 10 -transformed p -values after adjusted for FDR false discovery rate. ( H ) The small intestine of DSS-treated SIRT1 PKO mice have increased expression of immune cell genes. The expression of indicated genes was analyzed by qPCR ( n = 6 Flox and 12 PKO mice, Student’s t-test). ( I ) The expression of β-catenin was analyzed by IHC in the ileum of DSS-treated Flox and SIRT1 PKO mice. Bar, 50 μm. Data information: in ( A , B , C , and H ), values are expressed as mean ± SEM; in ( E ), box-and-whisker plot with the box representing the interquartile range (Q1 to Q3), a line inside indicating the median, and whiskers representing the 2.5–97.5 percentile; * 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: Staining, MANN-WHITNEY, Functional Assay, RNA Sequencing, Transformation Assay, Expressing, Whisker Assay

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 – D ) Antibiotics (Abx)-mediated depletion of gut microbiota ameliorates DSS-induced colitis in Flox but not PKO mice. Flox and PKO mice were treated with an antibiotic cocktail (Abx) for 4 weeks then with 2.5% DSS for 8 days (Regular: n = 9 Flox and 8 PKO; Abx: n = 5 Flox and 8 PKO; Student’s t-test). Bar in ( D ), 100 μm. ( E ) Gene expression in the colon and ileum of regular and Abx mice after the 7-day DSS-treatment. Flox and SIRT1 PKO mice were treated as in Fig. (Regular, n = 6 Flox and 8 PKO mice) or 6A (Abx, n = 5 Flox and 8 PKO mice). Student’s t-test. ( F ) Fecal transplantation switches the abundance of Muribaculaceae uc . Fecal transplantation was performed as described in Methods and fecal microbiota were analyzed by 16S rRNA amplicon sequencing ( n = 6, 6, 11, and 8 mice; two-way ANOVA). ( G – I ) Mice transplanted with fecal microbes from PKO mice are protected from DSS-induced rectal bleeding and colonic tissue damage. GF Flox and PKO mice were transplanted with fecal microbes from either regular Flox mice or PKO mice, then treated with 2.5% DSS ( n = 6 GF Flox FT Flox, 7 GF PKO FT PKO, 16 GF Flox FT PKO, and 11 GF PKO FT Flox; two-way ANOVA). Bar in ( I ), 200 μm. Data information: in ( A , B , C , E , F , G , and H ), values are expressed as mean ± SEM; * p < 0.05, ** p < 0.01, **** 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: Gene Expression, Transplantation Assay, Amplification, Sequencing

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: scRNA-seq datasets (Accession DUOS-000146 CD_Atlas_2021_GIDER; DUOS-000145 CD_Atlas_2021_PRISM) of intestinal epithelial cells from healthy donors or CD patients was analyzed. ( A ) The expression levels of SIRT1 in the terminal ileum were compared between Healthy controls, inflamed, and non-inflamed CD patients (two-sided Wilcoxon test, and adjusted p-values were obtained using the Holm–Bonferroni correction). ( B ) The expression of SIRT1 in different cell types in the terminal ileum (two-sided Wilcoxon test, and adjusted p -values were obtained using the Holm–Bonferroni correction). ( C ) pySCENIC analysis of transcription activity of TFs was performed using above scRNA-seq datasets and top 100 TFs altered in indicated Paneth cells from the terminal ileum. ( D ) The top enriched functional pathways in the Paneth cells isolated from Healthy and Noninflamed terminal ileum of CD patients. Paneth cell DEGs between Non-inflamed vs Healthy were analyzed by g:Prolifer and top enriched pathways are shown (Permutation testing with the Benjamini–Hochberg adjusted p -values). Enrichment score represents −log 10 -transformed p -values after adjusted for FDR false discovery rate.

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, Activity Assay, Functional Assay, Isolation, Transformation Assay

Journal: bioRxiv

Article Title: FGL-1 binding to LAG-3 inhibits T cell activation via disruption of CD28 and TCR signaling

doi: 10.1101/2025.08.05.668721

Figure Lengend Snippet: (A) Flow cytometry analysis of tetrameric FGL-1 FD::streptavidin Phycoerythrin (SA-PE) binding to LAG-3+ Jurkat cells across a concentration range (1 – 16 µM). (B) Comparison of FGL-1 FD tetramer binding to LAG-3+ Jurkat cells versus wild-type (WT) Jurkat cells (LAG-3 low) (n = 3). Data are presented as median fluorescent intensity (MFI), normalized to LAG-3+ Jurkat cells MFI at each concentration. Error bars represent standard deviation (SD). (C) NF-κB::eGFP induction and (D) IL-2 production in LAG-3+ Jurkat T cells stimulated with anti-CD3/CD28-coated beads +/− FGL-1 FD or mouse IgG1 isotype control (mIgG1) for 24 hours (n = 4, n = 3, respectively). NF-κB::eGFP induction data is shown as percent MFI normalized to anti-CD3/CD28 stimulation (set to 100%). Error bars represent standard deviation (SD). (E) Expression of T cell activation markers: PD-1, CD69, CD25, ICOS, 4-1BB and OX40 in LAG-3+ Jurkat T cells, either unstimulated or stimulated with anti-CD3/CD28 +/− FGL-1 FD (n = 4). Data are shown as MFI ± SEM, normalized to the anti-CD3/CD28 condition. (F) Control expression of activation markers in WT (LAG-3 low) Jurkat T cells under identical conditions (n = 4). Data represent MFI ± SEM from triplicate wells across two independent experiments. Statistical significance was performed using one or two-way ANOVA, followed by Tukey or Šídák’s post hoc tests, respectively. **** p < 0.0001, *** p < 0.001, ** p < 0.01, * p < 0.05, ns = not significant p > 0.05.

Article Snippet: WT Jurkat (Clone E6-1) cell line was obtained from the American Type Culture Collection (ATCC).

Techniques: Flow Cytometry, Binding Assay, Concentration Assay, Comparison, Standard Deviation, Control, Expressing, Activation Assay

Journal: bioRxiv

Article Title: FGL-1 binding to LAG-3 inhibits T cell activation via disruption of CD28 and TCR signaling

doi: 10.1101/2025.08.05.668721

Figure Lengend Snippet: (A, B) Label-free LC–MS/MS analysis of phosphotyrosine (pY) peptides in LAG-3+ Jurkat cells, either unstimulated or stimulated for 5 minutes with anti-CD3/CD28 +/− FGL-1 FD (n = 4), (A) number of unique pY peptides identified per condition (B) Log 2 intensity distribution of detected pY peptides. Statistical analysis performed using two-way ANOVA with Tukey’s post hoc tests. Solid black line = median; dashed lines = interquartile range. **** p < 0.0001, ns = not significant (p > 0.05) (C) Venn diagram showing overlap of pY peptides detected in cells stimulated with anti-CD3/anti-CD28 versus anti-CD3/anti-CD28 + FGL-1 FD from one representative experiment. (D – F) Volcano plots illustrating differential log 2 fold changes (log 2 FC) in pY intensity differences, across the three conditions: (D) anti-CD3/CD28 vs. unstimulated; (E) anti-CD3/CD28 + FGL-1 FD vs. unstimulated; (F) anti-CD3/CD28 + FGL-1 FD vs. anti-CD3/CD28. Red = significantly upregulated phosphosites (log 2 FC ≥ 1.5); blue = significantly downregulated phosphosites, (log 2 FC ≤ 1.5); grey = unchanged phosphosites (log 2 FC) < 1.5).

Article Snippet: WT Jurkat (Clone E6-1) cell line was obtained from the American Type Culture Collection (ATCC).

Techniques: Liquid Chromatography with Mass Spectroscopy

Journal: bioRxiv

Article Title: FGL-1 binding to LAG-3 inhibits T cell activation via disruption of CD28 and TCR signaling

doi: 10.1101/2025.08.05.668721

Figure Lengend Snippet: ( A ) Functional enrichment analysis of proteins with upregulated pY sites (log 2 FC ≥ 1.5, red), and downregulated pY (log 2 FC ≤ 1.5; blue) in response to anti-CD3/CD28 and anti-CD3/CD28 + FGL-1 FD stimulation, relative to unstimulated cells and stimulation without FGL-1 FD. Color intensity indicates –Log 10 (p-value) of pathway enrichment; X denotes non-enriched pathways. ( B ) Log 2 fold change (log 2 FC) analysis of TCR signaling-associated pY peptides following anti-CD3/CD28 and anti-CD3/CD28 + FGL-1 FD stimulation compared to unstimulated cells. ( C ) Heatmap of log 2 FC phosphorylation differences in TCR signaling-related pY peptides across three conditions: anti-CD3/CD28 vs. unstimulated; anti-CD3/CD28 + FGL-1 FD vs. unstimulated; anti-CD3/CD28 + FGL-1 FD vs. anti-CD3/CD28. ( D ) Bar graphs showing log 2 FC values of TCR signaling pY sites in CD3 ITAMS, ZAP-70, LAT, ERK1/2, TEC and distal molecules upon FGL-1 FD stimulation relative to stimulation without FGL-1 FD. ( E ) Time-course western blot analysis of phosphorylation at CD3ζ Y142, ZAP70 Y319, and ERK 1/2 Y204/Y187 in LAG-3+ Jurkat cells unstimulated, stimulated with anti-CD3/CD28 +/− FGL-1 FD. Quantification represents phospho/total protein signal intensity, normalized to unstimulated (US) values (n = 3). ( F ) Bar graphs showing log 2 FC values of CD28 signaling pY sites: CD28 pY191, pY209, and PI3K P85A pY467, in response to FGL-1 FD stimulation relative to stimulation without FGL-1 FD. ( G ) Western blot analysis of CD28 Y191 phosphorylation in LAG-3+ cell lysates: unstimulated, stimulated with anti-CD3/CD28 +/− FGL-1 FD. Band intensities were quantified and normalized to total CD28 and unstimulated control (n = 5). Statistical significance was determined using a two-way ANOVA with Šídák’s post hoc tests. **** p < 0.0001, *** p < 0.001, ** p < 0.01, * p < 0.05, ns = not significant (p > 0.05).

Article Snippet: WT Jurkat (Clone E6-1) cell line was obtained from the American Type Culture Collection (ATCC).

Techniques: Functional Assay, Phospho-proteomics, Western Blot, Control

Journal: bioRxiv

Article Title: FGL-1 binding to LAG-3 inhibits T cell activation via disruption of CD28 and TCR signaling

doi: 10.1101/2025.08.05.668721

Figure Lengend Snippet: ( A ) Representative fixed-cell confocal microscopy images showing colocalization of CD3 (green) and LAG-3 (red) in unstimulated and anti-CD3/CD28-stimulated LAG-3+ Jurkat cells. Scale bar: 5 µm. (B) Representative confocal images depicting colocalization of CD28 (green) and LAG-3 (red) under the same conditions: unstimulated and anti-CD3/CD28. Scale bar: 5 µm. (C) Violin plots of Manders’ Overlap Coefficient (MOC) quantifying colocalization of CD3 and CD28 with LAG-3 in unstimulated and stimulated cells (n = 66). Median values are indicated by solid black lines; interquartile ranges by dashed black lines. Statistical significance was assessed using Kruskal-Wallis ANOVA, followed by Dunnett’s post hoc tests. **** p < 0.0001, *** p < 0.001, ** p < 0.01, * p < 0.05, ns = not significant (p > 0.05).

Article Snippet: WT Jurkat (Clone E6-1) cell line was obtained from the American Type Culture Collection (ATCC).

Techniques: Confocal Microscopy

Journal: bioRxiv

Article Title: FGL-1 binding to LAG-3 inhibits T cell activation via disruption of CD28 and TCR signaling

doi: 10.1101/2025.08.05.668721

Figure Lengend Snippet: ( A ) Representative fixed-cell confocal microscopy images showing colocalization of CD28 (red) and Lck (pY394, cyan) in unstimulated, anti-CD3/CD28-stimulated, and anti-CD3/CD28 +/− FGL-FD stimulated LAG-3+ Jurkat cells. Scale bar: 5 µm. (B) Violin plots quantifying CD28 and Lck pY394 colocalization using Manders’ overlap coefficient (MOC) and Pearson’s correlation coefficient (PCC) (n ≥ 50). (C) Representative confocal images showing colocalization of CD28 (green) and total Lck (red) in unstimulated, anti-CD3/CD28 +/− FGL-FD stimulated LAG-3+ Jurkat cells. Scale bar: 5 µm. (D) Colocalization analysis of CD28 and total Lck, measured by MOC and PCC (n ≥ 60). (E) Representative confocal images of CD28 (red) and Lck pY394 (cyan) in unstimulated, anti-CD3/CD28 +/− FGL-FD stimulated Jurkat cells. Scale bar: 5 µm. (F) Colocalization analysis of CD28 and Lck pY394, determined by MOC and PCC (n ≥ 75). (G) Comparison of MOC and PCC values for CD28 and Lck pY394 colocalization between unstimulated and stimulated LAG-3+ Jurkat cells compared to WT (LAG-3 low) Jurkat cells (n ≥ 75). Median values are shown in solid black lines; interquartile ranges as dashed black lines. Statistical analysis was performed using Kruskal-Wallis ANOVA test, followed by Dunnett’s post hoc tests. **** p < 0.0001, *** p < 0.001, ** p < 0.01, * p < 0.05, ns = not significant (p > 0.05).

Article Snippet: WT Jurkat (Clone E6-1) cell line was obtained from the American Type Culture Collection (ATCC).

Techniques: Confocal Microscopy, Comparison