Journal: bioRxiv

Article Title: Gut Microbiota-derived Acetate Safeguards the Colonic Epithelial Acetyl-CoA Reserve to Avert Colonic Senescence

doi: 10.64898/2026.05.15.725523

Figure Lengend Snippet: (A) Breeding strategy for the production of CEC- Acly −/− / Acss 2 −/− double knockout (DKO) mice; (B) Body weight changes in Acly f/f / Acss2 f/f ;Cdx2-CreERT2 mice following tamoxifen (TAM) treatment with or without acetate (Ac) supplementation; (C) Kaplan-Meier survival curves of Acly f/f / Acss2 f/f ;Cdx2-CreERT2 mice following tamoxifen (TAM) treatment with or without acetate supplementation; (D) Western blot assessment of senescence hallmarks in colonic crypt cells from Acly f/f / Acss2 f/f (Ctrl) and Acly f/f / Acss2 f/f ;Cdx2-CreERT2 (DKO) mice on day 13 following TAM and acetate treatment; (E) RT-qPCR quantitation of SASP cytokines in colonic crypt cells from Acly f/f / Acss2 f/f and Acly f/f / Acss2 f/f ;Cdx2-CreERT2 mice on day 13 following TAM and acetate treatment; (F) Serum cytokine concentrations in Acly f/f / Acss2 f/f and Acly f/f / Acss2 f/f ;Cdx2-CreERT2 mice on day 13 following TAM and acetate treatment; (G) H&E stained lung sections from Acly f/f / Acss2 f/f and Acly f/f / Acss2 f/f ;Cdx2-CreERT2 mice on day 13 following TAM treatment; (H) Quantitation of fecal SCFAs (acetate, propionate, butyrate) in Acly f/f / Acss2 f/f and Acly f/f / Acss2 f/f ;Cdx2-CreERT2 mice on day 13 following TAM and acetate treatment; (I) Alcian blue staining of colon sections from Acly f/f / Acss2 f/f and Acly f/f / Acss2 f/f ;Cdx2-CreERT2 mice on day 13 following TAM and acetate treatment; (J) Confocal microscopic images of ψH2AX immunofluorescence staining in colonic sections from Acly f/f / Acss2 f/f and Acly f/f / Acss2 f/f ;Cdx2-CreERT2 mice on day 13 following TAM and acetate treatment. Arrows indicate ψH2AX loci in the nucleus of crypt epithelial cells; (K) Quantitation of ψH2AX-positive cells in colonic mucosa of Acly f/f / Acss2 f/f and Acly f/f / Acss2 f/f ;Cdx2-CreERT2 mice on day 13 following TAM and acetate treatment; (L) Quantitation of acetyl-CoA concentrations in total cell lysates and nuclear, cytosolic and mitochondrial fractions of colonic crypt cells from Acly f/f / Acss2 f/f and Acly f/f / Acss2 f/f ;Cdx2-CreERT2 mice on day 13 following TAM and acetate treatment. Data are presented as mean ± SD. *p<0.05; **p<0.01; ***p<0.001, ****p<0.0001 by two-way ANOVA.

Article Snippet: CDX2-Cre transgenic mice (B6.Cg-Tg(CDX2-cre)101Erf/J; Stock # 009350), CDX2-CreERT2 transgenic mice (B6.Cg-Tg(CDX2-Cre/ERT2)752Erf/J; Stock # 022390), p16-3MR transgenic mice (B6.Cg-Tg(Cdkn2a/luc/RFP/TK)1Cmps/J, Stock # 037045) and Trp53 −/− mice (B6.129S2- Trp53 tm1Tyj /J; Stock # 002101) were purchased from Jackson Laboratory.

Techniques: Double Knockout, Western Blot, Quantitative RT-PCR, Quantitation Assay, Staining, Immunofluorescence

Journal: bioRxiv

Article Title: Gut Microbiota-derived Acetate Safeguards the Colonic Epithelial Acetyl-CoA Reserve to Avert Colonic Senescence

doi: 10.64898/2026.05.15.725523

Figure Lengend Snippet: (A) Schematic illustration for the derivation of Acly -deleted colonic organoids from Acly f/f ;Cdx2-CreERT2 mice; (B) SA-β-gal expression in Acly f/f ;Cdx2-CreERT2 colonic organoids on days 0, 4 and 6 after 4-hydroxytamoxyfen (4-OHT) treatment; (C) Western blot assessment of canonical senescence hallmarks in Acly f/f ;Cdx2-CreERT2 colonic organoids on days 0, 4 and 6 after 4-OHT treatment; (D) RT-qPCR quantitation of SASP cytokine expression in Acly f/f ;Cdx2-CreERT2 colonic organoids on days 0, 4 and 6 after 4-OHT treatment; *p<0.05; **p<0.01; ***p<0.001, ****p<0.0001 by two-way ANOVA. (E) Western blot analysis of IL-6 and TNF-α expression in Acly f/f ;Cdx2-CreERT2 colonic organoids on days 0, 4 and 6 after 4-OHT treatment; (F) Western blot analysis of senescence-related biomarkers in Acly f/f ;Cdx2-CreERT2 colonic organoids on days 0, 4 and 6 after 4-OHT treatment; (G) Western blot analysis of senescence hallmarks in Acly f/f ;Cdx2-CreERT2 colonic organoids treated with 4-OHT with or without acetate (Ac) supplementation in the culture media; (H) Human colonic organoids transduced with lentivirus carrying control shRNA (shCtrl) or ACLY-specific shRNA (shACLY) and cultured in the presence or absence of acetate (Ac) supplementation. Note the unique senescent morphology of shACLY-transduced organoids; (I) Western blot confirmation of ACLY knockdown, induction of senescence hallmarks, and suppression of these hallmarks by Ac supplementation in human organoids transduced with shACLY-lentivirus.

Article Snippet: CDX2-Cre transgenic mice (B6.Cg-Tg(CDX2-cre)101Erf/J; Stock # 009350), CDX2-CreERT2 transgenic mice (B6.Cg-Tg(CDX2-Cre/ERT2)752Erf/J; Stock # 022390), p16-3MR transgenic mice (B6.Cg-Tg(Cdkn2a/luc/RFP/TK)1Cmps/J, Stock # 037045) and Trp53 −/− mice (B6.129S2- Trp53 tm1Tyj /J; Stock # 002101) were purchased from Jackson Laboratory.

Techniques: Expressing, Western Blot, Quantitative RT-PCR, Quantitation Assay, Transduction, Control, shRNA, Cell Culture, Knockdown

Journal: bioRxiv

Article Title: Gut Microbiota-derived Acetate Safeguards the Colonic Epithelial Acetyl-CoA Reserve to Avert Colonic Senescence

doi: 10.64898/2026.05.15.725523

Figure Lengend Snippet: (A) Acetyl-protein profiles revealed by immunoprecipitation with anti-acetyl-lysine antibody in purified colonic crypt cells from Acly f/f and CEC- Acly −/− mice on day 7 following ABX and acetate (Ac) feeding; (B) Kaplan-Meier survival curves of Acly f/f / Acss2 f/f ;Cdx2-CreERT2 mice following tamoxifen (TAM) and SAHA treatment; (C) Acetyl-protein profiles in colonic crypt cells of Acly f/f / Acss2 f/f ;Cdx2-CreERT2 mice on day 13 following TAM and SAHA treatment; (D) Western blot assessment of senescence hallmarks in purified colonic crypt cells from Acly f/f / Acss2 f/f ;Cdx2-CreERT2 mice on day 13 following TAM and SAHA treatment; (E) Schematic illustration of acetyl-proteomics procedure; (F) Heatmap showing changes in specific lysine acetylation in a cohort of nuclear and mitochondrial proteins identified by acetyl-proteomics in CEC- Acly −/− colonic crypt cells with or without ABX; (G) Western blot analysis of H4 acetylation at K8 and K16 in purified colonic crypt cells from Acly f/f and CEC- Acly −/− mice on day 7 following ABX and Ac feeding; (H) Immunoprecipitation analysis of nuclear protein acetylation using anti-acetyl-lysine antibody in purified colonic crypt cells from Acly f/f and CEC- Acly −/− mice on day 7 following ABX and Ac feeding; (I) Immunoprecipitation analysis of mitochondrial protein acetylation using anti-acetyl-lysine antibody in purified colonic crypt cells from Acly f/f and CEC- Acly −/− mice on day 7 following ABX and Ac feeding.

Article Snippet: CDX2-Cre transgenic mice (B6.Cg-Tg(CDX2-cre)101Erf/J; Stock # 009350), CDX2-CreERT2 transgenic mice (B6.Cg-Tg(CDX2-Cre/ERT2)752Erf/J; Stock # 022390), p16-3MR transgenic mice (B6.Cg-Tg(Cdkn2a/luc/RFP/TK)1Cmps/J, Stock # 037045) and Trp53 −/− mice (B6.129S2- Trp53 tm1Tyj /J; Stock # 002101) were purchased from Jackson Laboratory.

Techniques: Immunoprecipitation, Purification, Western Blot

Journal: bioRxiv

Article Title: Gut Microbiota-derived Acetate Safeguards the Colonic Epithelial Acetyl-CoA Reserve to Avert Colonic Senescence

doi: 10.64898/2026.05.15.725523

Figure Lengend Snippet: (A) Acetyl-protein profiles in Acly f/f ;Cdx2-CreERT2 colonic organoids on day 6 after 4-OHT treatment with or without acetate supplementation in the media; (B) Western blot analysis of senescence markers in Acly f/f ;Cdx2-CreERT2 colonic organoids treated with 4-OHT and Vorinostat (SAHA); (C) Western blot analysis of H4 acetylation in colonic crypt cells from Acly f/f / Acss2 f/f ;Cdx2-CreERT2 mice treated with TAM and SAHA; (D) Immunoprecipitation analysis of nuclear protein acetylation using anti-acetyl-lysine antibody in colonic crypt cells prepared from Acly f/f / Acss2 f/f ;Cdx2-CreERT2 mice treated with TAM and SAHA; (E) Immunoprecipitation analysis of mitochondrial protein acetylation using anti-acetyl-lysine antibody in colonic crypt cells from Acly f/f / Acss2 f/f ;Cdx2-CreERT2 mice treated with TAM and SAHA; (F) FACS analysis of JC-1 stained Acly f/f ;Cdx2-CreERT2 colonic organoids on day 6 after 4-OHT treatment; (G) Mitochondrial membrane potentials in Acly f/f ;Cdx2-CreERT2 colonic organoids on day 6 after 4-OHT treatment, expressed as red (Q2) to green (Q3) ratio based on the FACS data; (H) Cellular ATP concentration in purified colonic crypt cells from Acly f/f / Acss2 f/f and Acly f/f / Acss2 f/f ;Cdx2-CreERT2 mice on day 13 after TAM and acetate treatment; (I) Cellular ROS production in purified colonic crypt cells from Acly f/f / Acss2 f/f and Acly f/f / Acss2 f/f ;Cdx2-CreERT2 mice on day 13 after TAM and acetate treatment; (J) Western blot assessment of senescence markers in Acly f/f ;Cdx2-CreERT2 colonic organoids on day 6 after 4-OHT and NAC treatment. (K) Molecular mechanism whereby acetyl-CoA deficiency triggers colonic senescence. Colonic epithelial Acly ablation combined with intestinal bacterial depletion or Acss2 ablation causes epithelial acetyl-CoA deficiency, which depletes lysine acetylation in a cohort of nuclear and mitochondrial proteins. Particularly, the depletion of ATP5F1A acetylation at K161 disrupts ATP synthesis and the depletion of H4 acetylation at K5 and K8 disrupts its DNA repair activity. These lead to increased oxidative stress and increased DNA damage response, which together trigger robust colonic epithelial senescence. Widespread colonic senescence causes severe systemic inflammation and premature death of the mice.

Article Snippet: CDX2-Cre transgenic mice (B6.Cg-Tg(CDX2-cre)101Erf/J; Stock # 009350), CDX2-CreERT2 transgenic mice (B6.Cg-Tg(CDX2-Cre/ERT2)752Erf/J; Stock # 022390), p16-3MR transgenic mice (B6.Cg-Tg(Cdkn2a/luc/RFP/TK)1Cmps/J, Stock # 037045) and Trp53 −/− mice (B6.129S2- Trp53 tm1Tyj /J; Stock # 002101) were purchased from Jackson Laboratory.

Techniques: Western Blot, Immunoprecipitation, Staining, Membrane, Concentration Assay, Purification, Activity Assay

Journal: bioRxiv

Article Title: Gut Microbiota-derived Acetate Safeguards the Colonic Epithelial Acetyl-CoA Reserve to Avert Colonic Senescence

doi: 10.64898/2026.05.15.725523

Figure Lengend Snippet: (A) Schematic illustration of an organoid rescue strategy using recombinant lentivirus; (B) Microscopic images of Acly f/f ;Cdx2-CreERT2 colonic organoids transduced with recombinant GFP-lentivirus carrying cDNA as indicated. Unless indicated, all organoids were treated with 4-OHT to deplete Acly . GFP fluorescence confirms the success in organoid transduction, and the normal and senescent morphology of the same organoids are identified by regular light microscopy; V, vector. (C,D) Western blot assessment of the induction or suppression of senescence hallmarks in organoids transduced with a recombinant lentivirus indicated on the top. Overexpression of each corresponding protein confirms the successful transduction of the organoids; EV: Empty vector. (E) Schematic illustration of acetylation of the lysine (K) residues identified by acetyl-proteomics in H4 and ATP5F1A, their mutation to glutamine (Q) or arginine (R) in each construct, and the outcome of the rescue experiments; (F) Microscopic images of Acly f/f ;Cdx2-CreERT2 colonic organoids transduced with a recombinant GFP-lentivirus carrying wildtype H4c1 or Atp5f1a cDNA, or their mutants in which all four K residues were converted to Q or R. (G) Microscopic images of Acly f/f ;Cdx2-CreERT2 colonic organoids transduced with a recombinant GFP-lentivirus carrying one of the H4c1 double mutants in which two K residues were mutated to R, or carrying one of the Atp5f1a single mutants in which each K residue was individually converted to R; (H) Western blot assessment of senescence hallmarks in Acly f/f ;Cdx2-CreERT2 organoids transduced with a recombinant lentivirus carrying wildtype H4c1, H4c1(4K>4Q) or H4c1(4K>4R) mutant, or carrying wildtype Atp5f1a, Atp5f1a(4K>4Q) or Atp5f1a(4K>5R) mutant; (I) Western blot assessment of senescence hallmarks in Acly f/f ;Cdx2-CreERT2 organoids transduced with a recombinant lentivirus carrying one of the H4c1 double mutants or one of the Atp5f1a single mutants as indicated; (J) Quantitation of cellular ATP concentration in Acly f/f ;Cdx2-CreERT2 organoids transduced with indicated recombinant GFP-lentivirus constructs; (I) Quantitation of cellular ROS production in Acly f/f ;Cdx2-CreERT2 organoids transduced with indicated recombinant GFP-lentivirus constructs; (L) Kaplan-Meier survival curves of Acly f/f / Acss2 f/f ;Cdx2-CreERT2 mice following tamoxifen (TAM) and N-acetylcysteine (NAC) treatment; (M) Quantitation of ROS production in purified colonic crypt cells from Acly f/f / Acss2 f/f ;Cdx2-CreERT2 mice on day 13 following TAM and NAC treatment; (N) Western blot assessment of senescence hallmarks in purified colonic crypt cells from Acly f/f / Acss2 f/f ;Cdx2-CreERT2 mice on day 13 following TAM and NAC treatment; (O) RT-qPCR quantitation of SASP cytokines in purified colonic crypt cells from Acly f/f / Acss2 f/f ;Cdx2-CreERT2 mice on day 13 following TAM and NAC treatment.

Article Snippet: CDX2-Cre transgenic mice (B6.Cg-Tg(CDX2-cre)101Erf/J; Stock # 009350), CDX2-CreERT2 transgenic mice (B6.Cg-Tg(CDX2-Cre/ERT2)752Erf/J; Stock # 022390), p16-3MR transgenic mice (B6.Cg-Tg(Cdkn2a/luc/RFP/TK)1Cmps/J, Stock # 037045) and Trp53 −/− mice (B6.129S2- Trp53 tm1Tyj /J; Stock # 002101) were purchased from Jackson Laboratory.

Techniques: Recombinant, Transduction, Fluorescence, Light Microscopy, Plasmid Preparation, Western Blot, Over Expression, Mutagenesis, Construct, Residue, Quantitation Assay, Concentration Assay, Purification, Quantitative RT-PCR

Journal: Disease Models & Mechanisms

Article Title: Chiari II brain malformation is secondary to open spina bifida

doi: 10.1242/dmm.052528

Figure Lengend Snippet: Generation of the Cdx2 cre/+ ;Pax3 fl/fl mouse model. (A) Genetic cross used to generate mice with spina bifida (SB). Cdx2 cre drives Pax3 knockout in the body only (light-green highlight), whereas the head remains wild type. See Table 1 for offspring genotypes. (B,C) Cdx2 cre driving mTmG reporter expression in the body at embryonic day (E)10.5 (B; blue) and E15.5 (C; green). Magenta in B indicates a region of no Cdx2 cre -mediated recombination. Dashed line in C indicates outline of the fetus. See also Fig. S1 . (D,E) Representative images of E15.5 control ( Cdx2 +/+ ;Pax3 fl/fl ; D) and mutant ( Cdx2 cre/+ ;Pax3 fl/fl ; E) fetuses. White arrowheads indicate the location of the open SB lesion (in E). See also Fig. S2 . (F) Phenotypic developmental timeline of Cdx2 cre/+ ;Pax3 fl/fl embryos and fetuses, with dashed lines indicating the extent of open SB lesions. Scale bars: 1.0 mm.

Article Snippet: Cdx2 cre/+ male mice ( ) were obtained from The Jackson Laboratory [JAX; strain 009350 : B6.Cg-Tg(CDX2-cre)101Erf/J] and maintained by breeding with C57BL/6J females.

Techniques: Knock-Out, Expressing, Control, Mutagenesis

Journal: Disease Models & Mechanisms

Article Title: Chiari II brain malformation is secondary to open spina bifida

doi: 10.1242/dmm.052528

Figure Lengend Snippet: PAX3 expression in E10.5 control and Cdx2 cre/+ ;Pax3 fl/fl embryos. Sagittal cryosections of control ( n =3) and SB ( n =3) embryos, immunostained for PAX3 (yellow) and nuclear stained with DAPI (magenta). (A,B) Head sections: PAX3 expression is present in the neural tube (hindbrain, outlined by dashed lines) in both control (A) and SB (B) embryos (yellow arrowheads). (C,D) Lower body: PAX3 expression is present in the dorsal neural tube and dermomyotomes of the control spinal region (yellow arrowheads in C), but is not detectable in the SB embryo (D), consistent with knockout of Pax3 in the lower body. Bracket in D indicates spina bifida region. Scale bar: 0.5 mm.

Article Snippet: Cdx2 cre/+ male mice ( ) were obtained from The Jackson Laboratory [JAX; strain 009350 : B6.Cg-Tg(CDX2-cre)101Erf/J] and maintained by breeding with C57BL/6J females.

Techniques: Expressing, Control, Staining, Knock-Out