Journal: Cell death & disease

Article Title: CKMT1 deficiency contributes to mitochondrial dysfunction and promotes intestinal epithelial cell apoptosis via reverse electron transfer-derived ROS in colitis.

doi: 10.1038/s41419-025-07504-4

Figure Lengend Snippet: Fig. 1 CKMT1 protein was down-regulated in UC and experimental colitis. A Representative images of immunohistochemical staining of colon sections from UC and healthy. B Western blotting analysis of CKMT1 in colonic biopsy tissues from UC (n = 6) and healthy (n = 3), and C in colonic tissues from DSS-induced colitis mice and controls (n = 6). D Representative images of immunofluorescence staining of CKMT1 in colon sections from DSS-induced colitis mice and controls. E Immunofluorescence staining analysis of the co-localization of CKMT1 and Occludin/EpCAM. *P < 0.05; **P < 0.01; ***P < 0.001.

Article Snippet: The generation of intestinal epithelial-specific Ckmt1 conditional knockout (Ckmt1flox/flox, Vil-Cre) mice (C57BL/6J) was assisted by Cyagen Biosciences (Suzhou, China) through CRISPR/Cas-mediated genome engineering.

Techniques: Immunohistochemical staining, Staining, Western Blot

Journal: Cell death & disease

Article Title: CKMT1 deficiency contributes to mitochondrial dysfunction and promotes intestinal epithelial cell apoptosis via reverse electron transfer-derived ROS in colitis.

doi: 10.1038/s41419-025-07504-4

Figure Lengend Snippet: Fig. 2 Epithelial CKMT1 deficiency aggravated DSS-induced colitis in mice. A Targeting strategy for mouse conditional Ckmt1 knockout. (B- C) The knockout was verified by Western blotting in colon tissues (B) and isolated primary IECs (C) from mice. D Weight curves showing similar growth of male and female CKMT1 KOIEC and WT mice (n = 5 for each group). E Weight loss following DSS treatment was aggravated after epithelial CKMT1 deletion (n = 6–8). F Representative megascopic viewing of colon, and colon length. G DAI scores at day 8. H Evaluation of intestinal barrier function (E-Cadherin, Occludin, and Claudin-1) via Western blotting (n = 6). (I) Immunofluorescent staining of Ly6G and F4/80 in the colon tissues. *P < 0.05; **P < 0.01; ***P < 0.001.

Article Snippet: The generation of intestinal epithelial-specific Ckmt1 conditional knockout (Ckmt1flox/flox, Vil-Cre) mice (C57BL/6J) was assisted by Cyagen Biosciences (Suzhou, China) through CRISPR/Cas-mediated genome engineering.

Techniques: Knock-Out, Western Blot, Isolation, Staining

Journal: Cell death & disease

Article Title: CKMT1 deficiency contributes to mitochondrial dysfunction and promotes intestinal epithelial cell apoptosis via reverse electron transfer-derived ROS in colitis.

doi: 10.1038/s41419-025-07504-4

Figure Lengend Snippet: Fig. 3 Epithelial CKMT1 deficiency aggravated mitochondrial dysfunction and apoptosis in colitis. A Representative images by transmission electron microscopy showing mitochondrial structural changes in colonic IECs of mice. Quantitative real-time PCR analysis of the expression of key genes involved in B mitochondrial biogenesis (n = 6–10), C mitochondrial dynamics and mitophagy (Pink1) (n = 7–10), and D mitochondrial antioxidant system (n = 7–10). E Western blotting analysis for the protein expressions of mitochondrial antioxidant genes in colon tissues. F Dihydroethidium (DHE) staining showing ROS levels in colon tissues. G Mitochondrial morphology of NCM460 cells with CKMT1 overexpression (CKOE) or control (NC) after TNF-α (50 ng/ml) and CHX (20 μg/mL) treatment for 1 h, was observed via confocal microscopy and analyzed via Image-J software. H TUNEL staining showing the apoptosis levels in colon tissues. *P < 0.05; **P < 0.01; ***P < 0.001. One-way ANOVA with a post hoc LSD test was used for pairwise comparisons.

Article Snippet: The generation of intestinal epithelial-specific Ckmt1 conditional knockout (Ckmt1flox/flox, Vil-Cre) mice (C57BL/6J) was assisted by Cyagen Biosciences (Suzhou, China) through CRISPR/Cas-mediated genome engineering.

Techniques: Transmission Assay, Electron Microscopy, Real-time Polymerase Chain Reaction, Expressing, Western Blot, Staining, Over Expression, Control, Confocal Microscopy, Software, TUNEL Assay

Journal: Cell death & disease

Article Title: CKMT1 deficiency contributes to mitochondrial dysfunction and promotes intestinal epithelial cell apoptosis via reverse electron transfer-derived ROS in colitis.

doi: 10.1038/s41419-025-07504-4

Figure Lengend Snippet: Fig. 5 CKMT1 deficiency increased mitochondrial ROS via RET. After treated with TNF-α for 24 h (50 ng/ml), DCFH-DA staining showing cellular ROS levels in (A) Lovo cell, or (B) NCM460 cells, via flow cytometry. After treated with TNF-α for 24 h, MitoSOX staining of mitochondrial ROS (mtROS) was assessed via C flow cytometry in Lovo cells, or D via fluorescence microscopy in NCM460 cells. E The effects of FCCP (100 nM) or diazoxide (200 nM) pretreatment on TNF-α-induced mtROS in siCK Lovo cells were assessed via flow cytometry. F The mtROS levels were measured by flow cytometry in Lovo cells pretreated with rotenone (10 nM). G Illustration of electron transport chain-derived ROS production at complex I via RET. H AOX expression markedly reduced the TNF-α-induced mtROS levels in siCK cell. I NADH/NAD+ ratio was measured in cell lysis via microplate reader (n = 3 or 4), and J MMP (stained with TMRE) was measured via flow cytometry. All flow cytometry experiments were repeated three times. *P < 0.05; **P < 0.01; ***P < 0.001.

Article Snippet: The generation of intestinal epithelial-specific Ckmt1 conditional knockout (Ckmt1flox/flox, Vil-Cre) mice (C57BL/6J) was assisted by Cyagen Biosciences (Suzhou, China) through CRISPR/Cas-mediated genome engineering.

Techniques: Staining, Cytometry, Microscopy, Derivative Assay, Expressing, Lysis

Journal: Cell death & disease

Article Title: CKMT1 deficiency contributes to mitochondrial dysfunction and promotes intestinal epithelial cell apoptosis via reverse electron transfer-derived ROS in colitis.

doi: 10.1038/s41419-025-07504-4

Figure Lengend Snippet: Fig. 6 ROS and mPTP were involved in CKMT1-mediated IECs apoptosis. A Flow cytometry analysis of the opening of mPTP (Calcein fluorescence) in Lovo cells. B Flow cytometry analysis of the effect of NAC pretreatment (5 mM for 3 h) on the opening of mPTP induced by TNF-α in siCK cells. C Flow cytometry analysis of the effect of CsA pretreatment (1 μM for 3 h) on TNF-α-induced mtROS generation in siCK cells. D Assessment of cytochrome c, ATP5A, and SPG7 in cytosolic or mitochondrial fraction of siCK cells after apoptosis induction via Western blotting. E Assessment of AIF and BAX expressions after apoptosis induction via Western blotting. F Assessment of the levels of apoptosis- related proteins in siCK cells upon NAC and CsA pretreatment via western blotting (n = 4). All flow cytometry experiments were repeated three times. *P < 0.05; **P < 0.01; ***P < 0.001.

Article Snippet: The generation of intestinal epithelial-specific Ckmt1 conditional knockout (Ckmt1flox/flox, Vil-Cre) mice (C57BL/6J) was assisted by Cyagen Biosciences (Suzhou, China) through CRISPR/Cas-mediated genome engineering.

Techniques: Flow Cytometry, Western Blot, Cytometry