human colonic epithelial cell line ht29 c1  (ATCC)

Journal: Nature Communications

Article Title: Targeting NAT10 alleviates colonic senescence and elderly-onset colitis by disrupting N4-acetylation of DYRK1A

doi: 10.1038/s41467-026-70220-w

Figure Lengend Snippet: a Flow diagram of mRNA modification screening. m/z, mass-to-charge ratio. Created in BioRender. Chen, J. (2026) https://BioRender.com/h61m193 . b Analysis of SA-Gal activity in proliferation, H-Sen and D-Sen cells of NCM460 and CCD841. The SA-Gal - positive percentages in senescent cell groups were compared to the proliferation cell group. H-Sen, D-Sen: senescent cell induced by H 2 O 2 or DOX. Scale bar for NCM460, 100 µm. Scale bar for CCD841, 50 µm. n = 3 independent experiments. Data were represented as mean ± SD. c , d mRNA modification levels in proliferation cells and senescent cell groups of NCM460 and CCD841. n = 3 independent experiments. Data were represented as mean ± SD and represented relative to the control. e Dot blot of RNA ac 4 C modification level and western blot of Cyclin D1, γ-H2AX, p53, p21, p16 and NAT10 in senescent NCM460 and CCD841 cells. f Cell morphology and SA-Gal staining of P5 and P20 CCD841 cells. Scale bar, 50 µm. n = 3 independent experiments. Data were represented as mean ± SD. g mRNA ac 4 C modification level in P5 and P20 CCD841cells. n = 3 independent experiments. Data were represented as mean ± SD. Comparisons were performed by a Two-tailed unpaired Student’s t test ( b – d , f , g ). *** p < 0.001, ** p < 0.01, ns p > 0.05. Source data and exact p -value are provided as a Source data file.

Article Snippet: The colon epithelial cell line NCM460 (Cellosaurus, #305430, RRID: CVCL_0460) was cultured in RPMI 1640 medium (GIBCO, Waltham, MA, USA), and the finite colon cell line CCD841 (ATCC, CRL-1790, RRID: CVCL_2871) was maintained in Eagle’s minimum essential medium (ATCC, Manassas, VA, USA).

Techniques: Modification, Activity Assay, Control, Dot Blot, Western Blot, Staining, Two Tailed Test

Journal: Nature Communications

Article Title: Targeting NAT10 alleviates colonic senescence and elderly-onset colitis by disrupting N4-acetylation of DYRK1A

doi: 10.1038/s41467-026-70220-w

Figure Lengend Snippet: a Dot blot of RNA ac 4 C modification levels in NAT10-reexpressed (NAT10-WT or NAT10-MUT) NCM460 cells after transfected with NAT10-Si and treated with H 2 O 2 or DOX. n = 3 independent experiments. Data were represented as mean ± SD. b Western blot of NAT10, CyclinD1, γ-H2AX, p21, p53, and p16 in NAT10-reexpressed (NAT10-WT or NAT10-MUT) cells after transfected with NAT10-Si and treated with H 2 O 2 or DOX. c Analysis of SA-Gal activity and positive immunofluorescence staining of γ-H2AX in NAT10-reexpressed NCM460 cells after transfected with NAT10 siRNA and treated with H 2 O 2 . Scale bar for SA-Gal, 100 µm. Scale bar for IF, 20 µm. n = 3 independent experiments. Data were represented as mean ± SD. d Flow diagram for senescent cell viability and caspase3/7 activity detection. Created in BioRender. Chen, J. (2026) https://BioRender.com/w97m115 . e Colony formation assay using H-Sen NCM460 cells after co-transfection of NAT10-Si with either NAT10-WT or NAT10-MUT. f Cell viability analysis in H-Sen NCM460 cells after co-transfection of NAT10-Si with either NAT10-WT or NAT10-MUT. n = 3 independent experiments. Data were represented as mean ± SD. g Caspase3/7 activity analysis in H-Sen NCM460 cells after co-transfection of NAT10-Si with either NAT10-WT or NAT10-MUT. n = 3 independent experiments. Data were represented as mean ± SD. h Apoptosis flow cytometry analysis in H-Sen NCM460 cells after co-transfection of NAT10-Si with either NAT10-WT or NAT10-MUT. n = 3 independent experiments. Data were represented as mean ± SD. Comparisons were performed by Two-tailed unpaired Student’s t test ( a , c , f , g , h ). *** p < 0.001, ** p < 0.01, ns p > 0.05. Source data and exact p -value are provided as a Source data file.

Article Snippet: The colon epithelial cell line NCM460 (Cellosaurus, #305430, RRID: CVCL_0460) was cultured in RPMI 1640 medium (GIBCO, Waltham, MA, USA), and the finite colon cell line CCD841 (ATCC, CRL-1790, RRID: CVCL_2871) was maintained in Eagle’s minimum essential medium (ATCC, Manassas, VA, USA).

Techniques: Dot Blot, Modification, Transfection, Western Blot, Activity Assay, Immunofluorescence, Staining, Colony Assay, Cotransfection, Flow Cytometry, Two Tailed Test

Journal: Nature Communications

Article Title: Targeting NAT10 alleviates colonic senescence and elderly-onset colitis by disrupting N4-acetylation of DYRK1A

doi: 10.1038/s41467-026-70220-w

Figure Lengend Snippet: a Schematic illustration of acRIP-seq in NCM460 cells. Created in BioRender. Chen, J. (2026) https://BioRender.com/j92d013 . b Pie charts showed the distribution of ac 4 C peak in the acetylated transcripts in the control NC-Si, H-Sen NC-Si, and H-Sen NAT10-Si groups. c GO-BP enrichment analysis of hyperacetylated genes in H-Sen NCM460 cells compared to control NCM460 cells (left). GO-BP enrichment analysis of hypoacetylated genes in H-Sen NCM460 cells with NAT10-Si compared to H-Sen NCM460 cells with NC-Si (right). d Overlapping the potential downstream targets of NAT10-meditated RNA ac 4 C modification in hyperacetylated gene (H-Sen NC-Si vs Control NC-Si) and hypoacetylated gene (H-Sen NAT10-Si vs H-Sen NC-Si) by the acRIP-seq analysis. e acRIP-qPCR validation of ac 4 C level alterations in four potential downstream targets across the control NC-Si, H-Sen NC-Si, and H-Sen NAT10-Si groups. n = 3 independent experiments. Data were represented as mean ± SD. f qPCR was used to validate DYRK1A and LMLN RNA expression across the three groups. n = 3 independent experiments. Data were represented as mean ± SD. g Actinomycin D (ActD) chase RNA-seq showing DYRK1A mRNA level change in H-Sen NC-Si and H-Sen NAT10-Si cells. n = 3 independent samples. Data were represented as mean ± SD. h IGV plots showed that ac 4 C peaks changes in the CDS domains of DYRK1A mRNA in the control NC-Si, H-Sen NC-Si, and H-Sen NAT10-Si groups from acRIP-seq data. i RNA ac 4 C modifications and THAP5, DYRK1A and LMLN protein levels were detected by dot blot or western blot across the three groups. Two-way ANOVA analysis with Tukey’s multiple comparison was used for ( g ). Two-tailed unpaired Student’s t test was used for other analyses ( e , f ). *** p < 0.001, ** p < 0.01, ns p > 0.05. Source data and exact p -value are provided as a Source data file.

Article Snippet: The colon epithelial cell line NCM460 (Cellosaurus, #305430, RRID: CVCL_0460) was cultured in RPMI 1640 medium (GIBCO, Waltham, MA, USA), and the finite colon cell line CCD841 (ATCC, CRL-1790, RRID: CVCL_2871) was maintained in Eagle’s minimum essential medium (ATCC, Manassas, VA, USA).

Techniques: Control, Modification, Biomarker Discovery, RNA Expression, RNA Sequencing, Dot Blot, Western Blot, Comparison, Two Tailed Test

Journal: Nature Communications

Article Title: Targeting NAT10 alleviates colonic senescence and elderly-onset colitis by disrupting N4-acetylation of DYRK1A

doi: 10.1038/s41467-026-70220-w

Figure Lengend Snippet: a acRIP-qPCR analysis of ac 4 C levels in the CDS domain of DYRK1A among control, H-Sen, and D-Sen NCM460 cells. n = 3 independent experiments. Data were represented as mean ± SD. b DYRK1A mRNA expression and protein expression in control, H-Sen, and D-Sen NCM460 cells. n = 3 independent experiments. Data were represented as mean ± SD. c Immunofluorescence staining of DAPI, γ-H2AX, and DYRK1A in control, H-Sen, and D-Sen NCM460 cells. Scale bar for immunofluorescence, 20 μm. d Immunoblotting of NAT10 after NAT10-RIP assay with cell lysate among control, H-Sen, and D-Sen NCM460 cells. qPCR analysis of the CDS domain of DYRK1A was performed after NAT10-RIP. n = 3 independent experiments. Data were represented as mean ± SD. e DYRK1A mRNA stability was measured by adding actinomycin D in control, H-Sen, D-Sen NCM460 cells and in H-Sen and D-Sen NCM460 cells with NAT10 knockdown. n = 3 independent experiments. Data were represented as mean ± SD. f Sucrose gradient fractionation of H-Sen NCM460 cells and H-Sen NCM460 cells with NAT10 knockdown. Relative DYRK1A mRNA abundance across the polysome fraction was quantified by RT-qPCR. n = 3 independent experiments. Data were represented as mean ± SD. g Analysis of SA-Gal activity in DYRK1A-reexpressed NCM460 cells after transfected with NAT10 siRNA and treated with H 2 O 2 or DOX. Scale bar for SA-Gal, 100 µm. h qPCR analysis of IL-1β and IL-6 in DYRK1A-reexpressed NCM460 cells after transfected with NAT10 siRNA and treated with H 2 O 2 or DOX. n = 3 independent experiments. Data were represented as mean ± SD. i Western blot of NAT10, DYRK1A, γ-H2AX, and p21 in DYRK1A-reexpressed cells after transfected with NAT10-Si and treated with H 2 O 2 or DOX. j Caspase3/7 activity analysis in DYRK1A-reexpressed H-Sen and D-Sen NCM460 cells after transfected with NAT10-Si. n = 3 independent experiments. Data were represented as mean ± SD. Two-way ANOVA analysis with Tukey’s multiple comparison was used for ( e ). Two-tailed unpaired Student’s t test was used for other analyses ( a , b , d , e , f , g , h , j ). *** p < 0.001, ** p < 0.01, * p < 0.05. Source data and exact p -value are provided as a Source data file.

Article Snippet: The colon epithelial cell line NCM460 (Cellosaurus, #305430, RRID: CVCL_0460) was cultured in RPMI 1640 medium (GIBCO, Waltham, MA, USA), and the finite colon cell line CCD841 (ATCC, CRL-1790, RRID: CVCL_2871) was maintained in Eagle’s minimum essential medium (ATCC, Manassas, VA, USA).

Techniques: Control, Expressing, Immunofluorescence, Staining, Western Blot, Knockdown, Fractionation, Quantitative RT-PCR, Activity Assay, Transfection, Comparison, Two Tailed Test