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Alcohol exposure induces anastasis in esophageal epithelial cells. A . An equal number of HEEC cells was seeded on coverslips and incubated with complete medium (control) or medium containing 5.0% ethanol for 2–12 h. Cells were fixed in 4% paraformaldehyde and double-stained for <t>BAX</t> and endoG. A Texas Red- or FITC-conjugated secondary antibody was used to develop the signal for BAX (red) and endoG (green). Cell nuclei were counterstained with DAPI. Scale bar = 10 μm. B . An equal number of HEEC cells was seeded on coverslips and incubated with complete medium (control) or medium containing 5.0% ethanol for 5 min, 30 min, or 2, 6, and 12 h. Cells were fixed in 4% paraformaldehyde and stained for BAX and endoG. Quantitative analyses showing percentages of BAX-endoG co-localized cells in the total number of cells. C . An equal number of HEEC or Het1A cells was seeded on coverslips and incubated with complete medium (control) or medium containing 5.0% ethanol for 2–12 h. Cells were fixed in 4% paraformaldehyde and double-stained for BAX and endoG. Quantitative analyses showing the ratio of endoG to BAX per cell. * indicates a significant change compared to the control. D . An equal number of HEEC or Het1A cells was seeded in 6-well plates and incubated with the complete medium (control) or the medium containing 5.0% ethanol (EtOH) for 2–12 h. Protein was extracted and analyzed by Western blotting for <t>BAX.</t> <t>GAPDH</t> was used as a loading control. E . Quantitative analyses of BAX expression against GAPDH
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Alcohol exposure induces anastasis in esophageal epithelial cells. A . An equal number of HEEC cells was seeded on coverslips and incubated with complete medium (control) or medium containing 5.0% ethanol for 2–12 h. Cells were fixed in 4% paraformaldehyde and double-stained for <t>BAX</t> and endoG. A Texas Red- or FITC-conjugated secondary antibody was used to develop the signal for BAX (red) and endoG (green). Cell nuclei were counterstained with DAPI. Scale bar = 10 μm. B . An equal number of HEEC cells was seeded on coverslips and incubated with complete medium (control) or medium containing 5.0% ethanol for 5 min, 30 min, or 2, 6, and 12 h. Cells were fixed in 4% paraformaldehyde and stained for BAX and endoG. Quantitative analyses showing percentages of BAX-endoG co-localized cells in the total number of cells. C . An equal number of HEEC or Het1A cells was seeded on coverslips and incubated with complete medium (control) or medium containing 5.0% ethanol for 2–12 h. Cells were fixed in 4% paraformaldehyde and double-stained for BAX and endoG. Quantitative analyses showing the ratio of endoG to BAX per cell. * indicates a significant change compared to the control. D . An equal number of HEEC or Het1A cells was seeded in 6-well plates and incubated with the complete medium (control) or the medium containing 5.0% ethanol (EtOH) for 2–12 h. Protein was extracted and analyzed by Western blotting for <t>BAX.</t> <t>GAPDH</t> was used as a loading control. E . Quantitative analyses of BAX expression against GAPDH
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Alcohol exposure induces anastasis in esophageal epithelial cells. A . An equal number of HEEC cells was seeded on coverslips and incubated with complete medium (control) or medium containing 5.0% ethanol for 2–12 h. Cells were fixed in 4% paraformaldehyde and double-stained for <t>BAX</t> and endoG. A Texas Red- or FITC-conjugated secondary antibody was used to develop the signal for BAX (red) and endoG (green). Cell nuclei were counterstained with DAPI. Scale bar = 10 μm. B . An equal number of HEEC cells was seeded on coverslips and incubated with complete medium (control) or medium containing 5.0% ethanol for 5 min, 30 min, or 2, 6, and 12 h. Cells were fixed in 4% paraformaldehyde and stained for BAX and endoG. Quantitative analyses showing percentages of BAX-endoG co-localized cells in the total number of cells. C . An equal number of HEEC or Het1A cells was seeded on coverslips and incubated with complete medium (control) or medium containing 5.0% ethanol for 2–12 h. Cells were fixed in 4% paraformaldehyde and double-stained for BAX and endoG. Quantitative analyses showing the ratio of endoG to BAX per cell. * indicates a significant change compared to the control. D . An equal number of HEEC or Het1A cells was seeded in 6-well plates and incubated with the complete medium (control) or the medium containing 5.0% ethanol (EtOH) for 2–12 h. Protein was extracted and analyzed by Western blotting for <t>BAX.</t> <t>GAPDH</t> was used as a loading control. E . Quantitative analyses of BAX expression against GAPDH
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Alcohol exposure induces anastasis in esophageal epithelial cells. A . An equal number of HEEC cells was seeded on coverslips and incubated with complete medium (control) or medium containing 5.0% ethanol for 2–12 h. Cells were fixed in 4% paraformaldehyde and double-stained for <t>BAX</t> and endoG. A Texas Red- or FITC-conjugated secondary antibody was used to develop the signal for BAX (red) and endoG (green). Cell nuclei were counterstained with DAPI. Scale bar = 10 μm. B . An equal number of HEEC cells was seeded on coverslips and incubated with complete medium (control) or medium containing 5.0% ethanol for 5 min, 30 min, or 2, 6, and 12 h. Cells were fixed in 4% paraformaldehyde and stained for BAX and endoG. Quantitative analyses showing percentages of BAX-endoG co-localized cells in the total number of cells. C . An equal number of HEEC or Het1A cells was seeded on coverslips and incubated with complete medium (control) or medium containing 5.0% ethanol for 2–12 h. Cells were fixed in 4% paraformaldehyde and double-stained for BAX and endoG. Quantitative analyses showing the ratio of endoG to BAX per cell. * indicates a significant change compared to the control. D . An equal number of HEEC or Het1A cells was seeded in 6-well plates and incubated with the complete medium (control) or the medium containing 5.0% ethanol (EtOH) for 2–12 h. Protein was extracted and analyzed by Western blotting for <t>BAX.</t> <t>GAPDH</t> was used as a loading control. E . Quantitative analyses of BAX expression against GAPDH
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Alcohol exposure induces anastasis in esophageal epithelial cells. A . An equal number of HEEC cells was seeded on coverslips and incubated with complete medium (control) or medium containing 5.0% ethanol for 2–12 h. Cells were fixed in 4% paraformaldehyde and double-stained for <t>BAX</t> and endoG. A Texas Red- or FITC-conjugated secondary antibody was used to develop the signal for BAX (red) and endoG (green). Cell nuclei were counterstained with DAPI. Scale bar = 10 μm. B . An equal number of HEEC cells was seeded on coverslips and incubated with complete medium (control) or medium containing 5.0% ethanol for 5 min, 30 min, or 2, 6, and 12 h. Cells were fixed in 4% paraformaldehyde and stained for BAX and endoG. Quantitative analyses showing percentages of BAX-endoG co-localized cells in the total number of cells. C . An equal number of HEEC or Het1A cells was seeded on coverslips and incubated with complete medium (control) or medium containing 5.0% ethanol for 2–12 h. Cells were fixed in 4% paraformaldehyde and double-stained for BAX and endoG. Quantitative analyses showing the ratio of endoG to BAX per cell. * indicates a significant change compared to the control. D . An equal number of HEEC or Het1A cells was seeded in 6-well plates and incubated with the complete medium (control) or the medium containing 5.0% ethanol (EtOH) for 2–12 h. Protein was extracted and analyzed by Western blotting for <t>BAX.</t> <t>GAPDH</t> was used as a loading control. E . Quantitative analyses of BAX expression against GAPDH
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monoclonal mouse anti pcna  (Santa Cruz Biotechnology)
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Alcohol exposure induces anastasis in esophageal epithelial cells. A . An equal number of HEEC cells was seeded on coverslips and incubated with complete medium (control) or medium containing 5.0% ethanol for 2–12 h. Cells were fixed in 4% paraformaldehyde and double-stained for <t>BAX</t> and endoG. A Texas Red- or FITC-conjugated secondary antibody was used to develop the signal for BAX (red) and endoG (green). Cell nuclei were counterstained with DAPI. Scale bar = 10 μm. B . An equal number of HEEC cells was seeded on coverslips and incubated with complete medium (control) or medium containing 5.0% ethanol for 5 min, 30 min, or 2, 6, and 12 h. Cells were fixed in 4% paraformaldehyde and stained for BAX and endoG. Quantitative analyses showing percentages of BAX-endoG co-localized cells in the total number of cells. C . An equal number of HEEC or Het1A cells was seeded on coverslips and incubated with complete medium (control) or medium containing 5.0% ethanol for 2–12 h. Cells were fixed in 4% paraformaldehyde and double-stained for BAX and endoG. Quantitative analyses showing the ratio of endoG to BAX per cell. * indicates a significant change compared to the control. D . An equal number of HEEC or Het1A cells was seeded in 6-well plates and incubated with the complete medium (control) or the medium containing 5.0% ethanol (EtOH) for 2–12 h. Protein was extracted and analyzed by Western blotting for <t>BAX.</t> <t>GAPDH</t> was used as a loading control. E . Quantitative analyses of BAX expression against GAPDH
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Alcohol exposure induces anastasis in esophageal epithelial cells. A . An equal number of HEEC cells was seeded on coverslips and incubated with complete medium (control) or medium containing 5.0% ethanol for 2–12 h. Cells were fixed in 4% paraformaldehyde and double-stained for BAX and endoG. A Texas Red- or FITC-conjugated secondary antibody was used to develop the signal for BAX (red) and endoG (green). Cell nuclei were counterstained with DAPI. Scale bar = 10 μm. B . An equal number of HEEC cells was seeded on coverslips and incubated with complete medium (control) or medium containing 5.0% ethanol for 5 min, 30 min, or 2, 6, and 12 h. Cells were fixed in 4% paraformaldehyde and stained for BAX and endoG. Quantitative analyses showing percentages of BAX-endoG co-localized cells in the total number of cells. C . An equal number of HEEC or Het1A cells was seeded on coverslips and incubated with complete medium (control) or medium containing 5.0% ethanol for 2–12 h. Cells were fixed in 4% paraformaldehyde and double-stained for BAX and endoG. Quantitative analyses showing the ratio of endoG to BAX per cell. * indicates a significant change compared to the control. D . An equal number of HEEC or Het1A cells was seeded in 6-well plates and incubated with the complete medium (control) or the medium containing 5.0% ethanol (EtOH) for 2–12 h. Protein was extracted and analyzed by Western blotting for BAX. GAPDH was used as a loading control. E . Quantitative analyses of BAX expression against GAPDH

Journal: BMC Molecular and Cell Biology

Article Title: Alcohol exposure induces ferroptosis-dominated programmed cell death in esophageal epithelial cells

doi: 10.1186/s12860-026-00589-5

Figure Lengend Snippet: Alcohol exposure induces anastasis in esophageal epithelial cells. A . An equal number of HEEC cells was seeded on coverslips and incubated with complete medium (control) or medium containing 5.0% ethanol for 2–12 h. Cells were fixed in 4% paraformaldehyde and double-stained for BAX and endoG. A Texas Red- or FITC-conjugated secondary antibody was used to develop the signal for BAX (red) and endoG (green). Cell nuclei were counterstained with DAPI. Scale bar = 10 μm. B . An equal number of HEEC cells was seeded on coverslips and incubated with complete medium (control) or medium containing 5.0% ethanol for 5 min, 30 min, or 2, 6, and 12 h. Cells were fixed in 4% paraformaldehyde and stained for BAX and endoG. Quantitative analyses showing percentages of BAX-endoG co-localized cells in the total number of cells. C . An equal number of HEEC or Het1A cells was seeded on coverslips and incubated with complete medium (control) or medium containing 5.0% ethanol for 2–12 h. Cells were fixed in 4% paraformaldehyde and double-stained for BAX and endoG. Quantitative analyses showing the ratio of endoG to BAX per cell. * indicates a significant change compared to the control. D . An equal number of HEEC or Het1A cells was seeded in 6-well plates and incubated with the complete medium (control) or the medium containing 5.0% ethanol (EtOH) for 2–12 h. Protein was extracted and analyzed by Western blotting for BAX. GAPDH was used as a loading control. E . Quantitative analyses of BAX expression against GAPDH

Article Snippet: The following primary antibodies were used: Beclin-1 (Origene, #TA502643), PCNA, CASP1 (Abcam, #ab179515), CASP3 (Santa Cruz Biotechnology, #sc-271759), CASP8 (Origene, #TA374288), CASP9 (Origene, #TA4227045), endoG, GPX4 (Origene, #TA423164M), GSDMD, IL-1β (Santa Cruz Biotechnology, #sc-12742), MLKL, phosphor-MLKL (Abcam, #ab196436), SLC7A11 (Origene, #TA423232), LC3B, BAX, GAPDH (OriGene, #TA800894), and β-actin (Santa Cruz Biotechnology, Inc. #sc-69879).

Techniques: Incubation, Control, Staining, Western Blot, Expressing