Journal: Biology Direct

Article Title: NCOA4-mediated ferritinophagy modulates colitis-associated ferroptosis in intestinal epithelial cells and mucosal repair

doi: 10.1186/s13062-026-00747-x

Figure Lengend Snippet: NCOA4 participates in regulating ferroptosis in NCM460 cells. LV transfection was applied to overexpress or knock down the NCOA4 expression in vitro (1×10 8 TU/mL, MOI = 40) and DSS solution (20 mg/mL) was added to NCM460 to induce the inflammation model. Knockdown of NCOA4 expression alleviated the ferroptosis in NCM460 cells. ( A ) Experiment flowchart. ( B ) Cell viability (CCK8). ( C ) MDA content. ( D , J ) Fe 2+ content and location (Fluorescent staining, Scale bar = 100 μm, Magnification:200). Blue: nuclei; red: Fe 2+ ; green. ( E , K ) ROS content and location (Fluorescent staining, Scale bar = 100 μm, Magnification:200). Blue: nuclei; green: ROS. ( L ) Cell ultrastructure (TEM, Scale bar = 2 μm, Magnification:6000). Red arrow: mitochondrial structure. ( F , I , N ) Comparison of the expression of GPX4 protein and location (IF, Scale bar = 100 μm, Magnification:200). Blue: nuclei; green: GPX 4. ( G , H , M ) Comparison of the expression of COX2 protein and location (IF, Scale bar = 50 μm, Magnification:400). Blue: nuclei; red: COX2. Data are presented as the mean ± SD ( n = 3). vs. NG * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001; vs. MG # P < 0.05; ## P < 0.01, ### P < 0.001, ### P < 0.001, #### P < 0.0001; n.s.: no significance. BC: blank control group; MG: model group (DSS); DSS + LV NC: LV negative control + DSS group; DSS + LV NCOA4 OE: LV NCOA4 overexpression + DSS group; DSS + LV NCOA4 KD: LV NCOA4 knockdown + DSS group. LV: lentivirus; ROS: reactive oxygen species; MDA: malondialdehyde; GPX4: glutathione peroxidase 4; COX2: cyclooxygenase-2; DAPI: 4’,6-diamidino-2-phenylindole; DSS: dextran sulfate sodium salt; TEM: transmission electron microscopy; IHC: immunohistochemistry; IF: immunofluorescence; CCK8: cell counting kit 8

Article Snippet: NCOA4 knockdown (KD) and OE were achieved using the adeno-associated virus (AAV) transfection system (1×10 11 v.g./200uL) (Shanghai Genechem Co., Ltd., Shanghai, China) via tail vein injection.

Techniques: Transfection, Knockdown, Expressing, In Vitro, Staining, Comparison, Control, Negative Control, Over Expression, Transmission Assay, Electron Microscopy, Immunohistochemistry, Immunofluorescence, Cell Counting

Journal: Biology Direct

Article Title: NCOA4-mediated ferritinophagy modulates colitis-associated ferroptosis in intestinal epithelial cells and mucosal repair

doi: 10.1186/s13062-026-00747-x

Figure Lengend Snippet: NCOA4 is involved in regulating ferroptosis in IECs in vivo. AAV transfection was used to overexpress or knock down the NCOA4 expression in vivo (1×10 11 v.g./200uL, tvi) and a colitis mice model was induced via drinking 3% DSS solution. Suppressing NCOA4 mitigated the ferroptosis of IECs. ( A ) Experiment flowchart. ( B ) Ultrastructure of IECs (TEM, Scale bar = 2 μm, Magnification:6000). Red arrow: mitochondrial structure. ( C ) The ROS content in IECs. ( D ) The Fe 2+ content in IECs. ( E ) The MDA content in IECs. ( F , H ) Comparison of the expression of GPX4 protein. ( G , I ) Comparison of the expression of COX2 protein. Data are presented as the mean ± SD ( n = 8). vs. NG, * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001; vs. MG, # P < 0.05, ## P < 0.01, ### P < 0.001, ### P < 0.001, #### P < 0.0001; n.s.: no significance. NG: normal control group; MG: model group (DSS); AAV-NC-DSS: AAV negative control + DSS group; AAV-NCOA4 OE-DSS: AAV NCOA4 overexpression + DSS group; AAV-NCOA4 KD-DSS: AAV NCOA4 knockdown + DSS group. AAV: adeno-associated virus; ROS: reactive oxygen species; MDA: Malondialdehyde; GPX4: glutathione peroxidase 4; COX2: cyclooxygenase-2; DSS: dextran sulfate sodium salt; IEC: intestinal epithelial cell; TEM: transmission electron microscopy; TVI: tail vein injection

Article Snippet: NCOA4 knockdown (KD) and OE were achieved using the adeno-associated virus (AAV) transfection system (1×10 11 v.g./200uL) (Shanghai Genechem Co., Ltd., Shanghai, China) via tail vein injection.

Techniques: In Vivo, Transfection, Knockdown, Expressing, Comparison, Control, Negative Control, Over Expression, Virus, Transmission Assay, Electron Microscopy, Injection

Journal: Biology Direct

Article Title: NCOA4-mediated ferritinophagy modulates colitis-associated ferroptosis in intestinal epithelial cells and mucosal repair

doi: 10.1186/s13062-026-00747-x

Figure Lengend Snippet: LV transfection was applied to overexpress or knock down the NCOA4 expression in vitro (1×108 TU/mL, MOI=40) and DSS solution (20mg/mL) was added to NCM460 cells to induce the inflammation model. Knockdown of NCOA4 reduced the key markers of ferritinophagy in NCM460 cells. (A, B) Comparison of the expression of NCOA4 protein. (C, D) Comparison of the expression of ATG5 protein. (E, F) Comparison of the expression of LC3 protein. (G, H) Comparison of the expression of FTH1 protein. Data are presented as the mean ± SD (n = 3). vs NG *P<0.05, **P<0.01; vs MG #P<0.05, ##P<0.01, ###P<0.001, #### P<0.0001; n.s.: no significance. BC: blank control group; MG: model group (DSS); DSS+LV NC: LV negative control+DSS group; DSS+LV NCOA4 OE: LV NCOA4 overexpression+DSS group; DSS+LV NCOA4 KD: LV NCOA4 knockdown+DSS group. LV: lentivirus; NCOA4: nuclear receptor coactivator 4; FTH1: ferritin heavy chain 1; LC3: microtubule-associated protein light chain 3; ATG5: autophagy-related protein 5; DSS: dextran sulfate sodium salt

Article Snippet: NCOA4 knockdown (KD) and OE were achieved using the adeno-associated virus (AAV) transfection system (1×10 11 v.g./200uL) (Shanghai Genechem Co., Ltd., Shanghai, China) via tail vein injection.

Techniques: Transfection, Knockdown, Expressing, In Vitro, Comparison, Control, Negative Control, Over Expression

Journal: Biology Direct

Article Title: NCOA4-mediated ferritinophagy modulates colitis-associated ferroptosis in intestinal epithelial cells and mucosal repair

doi: 10.1186/s13062-026-00747-x

Figure Lengend Snippet: NCOA4 alters the ferritinophagy in IECs in vivo. AAV transfection was used to overexpress and knock down the NCOA4 expression in vivo (1×10 11 v.g./200uL, tvi) and a colitis mice model was established by drinking 3% DSS solution. Suppression of NCOA4 weakened the ferritinophagy in IECs. ( A , B ) Comparison of the expression of NCOA4 protein. ( C , D ) Comparison of the expression of ATG5 protein. ( E , F ) Comparison of the expression of LC3 protein. ( G , H ) Comparison of the expression of FTH1 protein. Data are presented as the mean ± SD ( n = 8). vs. NG, * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001; vs. MG, # P < 0.05, ## P < 0.01, ### P < 0.001, ### P < 0.001, #### P < 0.0001; n.s.: no significance. NG: normal control group; MG: model group (DSS); AAV-NC-DSS: AAV negative control + DSS group; AAV-NCOA4 OE-DSS: AAV NCOA4 overexpression + DSS group; AAV-NCOA4 KD-DSS: AAV NCOA4 knockdown + DSS group. AAV: adeno-associated virus; NCOA4: nuclear receptor coactivator 4; FTH1: ferritin heavy chain 1; LC3: microtubule-associated protein light chain 3; ATG5: autophagy-related protein 5; DSS: dextran sulfate sodium salt; IEC: intestinal epithelial cell; TVI: tail vein injection

Article Snippet: NCOA4 knockdown (KD) and OE were achieved using the adeno-associated virus (AAV) transfection system (1×10 11 v.g./200uL) (Shanghai Genechem Co., Ltd., Shanghai, China) via tail vein injection.

Techniques: In Vivo, Transfection, Knockdown, Expressing, Comparison, Control, Negative Control, Over Expression, Virus, Injection

Journal: Biology Direct

Article Title: NCOA4-mediated ferritinophagy modulates colitis-associated ferroptosis in intestinal epithelial cells and mucosal repair

doi: 10.1186/s13062-026-00747-x

Figure Lengend Snippet: NCOA4 inflences colonic inflammatory injuries in colitis mice. AAV transfection was applied to overexpress and knock down the NCOA4 expression in vivo (1×10 11 v.g./200uL, tvi) and a colitis mice model was established via drinking 3% DSS solution. Inhibiting NCOA4 expression improved the colonic inflammatory damage. ( A ) Morphology and structure of colonic tissues (HE staining, Scale bar=100 μm, Magnification:200). ( B ) Pathological score of colonic injuries. ( C , D ) Measurement and comparison of colon length. ( E ) CMDI. ( F ) DAI. ( G ) Serum DAO. ( H ) Serum D-LA. Data in a normal distribution are expressed as the mean ± SD ( n = 8). Data in a skewed distribution are expressed as the median ( P 25, P 75) ( n = 8). vs. NG, * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001; vs. MG, # P < 0.05, ## P < 0.01, ### P < 0.001, ### P < 0.001, #### P < 0.0001; n.s.: no significance. NG: normal control group; MG: model group (DSS); AAV-NC-DSS: AAV negative control + DSS group; AAV-NCOA4 OE-DSS: AAV NCOA4 overexpression + DSS group; AAV-NCOA4 KD-DSS: AAV NCOA4 knockdown + DSS group. AAV: adeno-associated virus. DAI: disease activity index; CMDI: colon macroscopic damage index; DAO: diamine oxidase; D-LA: D-lactate; HE: hematoxylin-eosin; TVI: tail vein injection

Article Snippet: NCOA4 knockdown (KD) and OE were achieved using the adeno-associated virus (AAV) transfection system (1×10 11 v.g./200uL) (Shanghai Genechem Co., Ltd., Shanghai, China) via tail vein injection.

Techniques: Transfection, Knockdown, Expressing, In Vivo, Staining, Comparison, Control, Negative Control, Over Expression, Virus, Activity Assay, Injection