Journal: Regenerative Therapy

Article Title: WTAP stabilizes MMP12 expression to promote the malignant phenotypes of esophageal cancer cells

doi: 10.1016/j.reth.2026.101101

Figure Lengend Snippet: Screening of genes in EC. (A) The Venn diagram shows the intersection between the DEGs in EC and normal esophageal tissues and the genes related to apoptosis, proliferation, and glycolysis. (B-D) The Lasso regression, SVM, and RF algorithms were used to further screen the 11 genes and identify key signature genes. (E) The Venn diagram shows the key genes identified by the Lasso regression, SVM, and RF algorithms.

Article Snippet: Apoptosis was evaluated using a Double Staining Apoptosis Kit (Vazyme Biotech, Nanjing, China) following the protocol for Annexin V-FITC and propidium iodide dual staining.

Techniques:

Journal: Regenerative Therapy

Article Title: WTAP stabilizes MMP12 expression to promote the malignant phenotypes of esophageal cancer cells

doi: 10.1016/j.reth.2026.101101

Figure Lengend Snippet: MMP12 knockdown inhibited the migration, invasion, proliferation, and glucose metabolism and induced cell apoptosis. KYSE150 cells were transfected with si-MMP12 or si-NC. (A and B) Cell migration and invasion were analyzed by transwell assays. (C) Cell proliferation was analyzed by EdU assay. (D) Cell apoptosis was assessed by flow cytometry. (E) HK1 and LDHA protein expression were detected by Western blotting. (F–H) Glucose consumption, lactate production, and ATP levels were analyzed by commercial kits. ∗∗ P < 0.01 and ∗∗∗ P < 0.001.

Article Snippet: Apoptosis was evaluated using a Double Staining Apoptosis Kit (Vazyme Biotech, Nanjing, China) following the protocol for Annexin V-FITC and propidium iodide dual staining.

Techniques: Knockdown, Migration, Transfection, EdU Assay, Flow Cytometry, Expressing, Western Blot

Journal: Regenerative Therapy

Article Title: WTAP stabilizes MMP12 expression to promote the malignant phenotypes of esophageal cancer cells

doi: 10.1016/j.reth.2026.101101

Figure Lengend Snippet: WTAP knockdown inhibited the migration, invasion, proliferation, and glucose metabolism and induced cell apoptosis by regulating MMP12 expression. KYSE150 cells were transfected with si-WTAP, MMP12 overexpression plasmid, or the matched control (si-NC and oe-NC). (A) MMP12 protein expression was detected by Western blotting. (B and C) Cell migration and invasion were analyzed by transwell assays. (D and E) Cell proliferation was analyzed by EdU assay. (F) Cell apoptosis was assessed by flow cytometry. (G and H) HK1 and LDHA protein expression were detected by Western blotting. (I–K) Glucose consumption, lactate production, and ATP levels were analyzed by commercial kits. ns: not significant, ∗ P < 0.05, ∗∗ P < 0.01 and ∗∗∗ P < 0.001.

Article Snippet: Apoptosis was evaluated using a Double Staining Apoptosis Kit (Vazyme Biotech, Nanjing, China) following the protocol for Annexin V-FITC and propidium iodide dual staining.

Techniques: Knockdown, Migration, Expressing, Transfection, Over Expression, Plasmid Preparation, Control, Western Blot, EdU Assay, Flow Cytometry

Journal: Bioactive Materials

Article Title: Screening of a quinonoid compounds library identifies decylubiquinone as an antioxidant and anti-apoptotic agent against glucocorticoid-induced osteoporosis via CD39/CD73/adenosine axis

doi: 10.1016/j.bioactmat.2026.03.062

Figure Lengend Snippet: Roles of the CD39/CD73 axis on DUB's multidirectional protection effects in Dex-treated primary BMSCs. ( A ) ELISA for ROS clearance-related enzyme T-SOD and ROS damage biomarkers 8-OHdG, AOPP, and MDA in primary BMSCs of different groups. ( B ) Western blot and quantification for the expression of ROS clearance-related proteins in primary BMSCs of different groups. ( C ) Representative images and quantitative analysis of immunofluorescence staining for MitoSox (red) in primary BMSCs of different groups, and nuclei were stained with Hoechst (blue). ( D ) Western blot and quantification for the expression of apoptosis-related proteins in primary BMSCs of different groups. ( E ) Cellular apoptosis detection in primary BMSCs of different groups by Annexin V-FITC and PI dual-staining assessment via flow cytometry. The proportion of cells in each quadrant was indicated in the plot. ( F ) Tunel (red) staining and quantification of apoptotic cells in primary BMSCs of different groups, and nuclei were stained with DAPI (blue). ( G ) Representative images and quantitative analysis of Alizarin Red S staining for mineralization in primary BMSCs of different groups under osteogenic conditions. ( H ) Western blot and quantification for the expression of osteogenesis-related proteins in primary BMSCs of different groups. n = 4 independent repeats by using different biological samples in each group for in vitro experiments. Data were means ± s.e.m. ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001 by one-way ANOVA. Scale bars: 50 μm (C), 25 μm (F), and 200 μm (G).

Article Snippet: The proportion of early and late apoptotic primary BMSCs under different treatment conditions was determined using an Annexin V-FITC/PI Apoptosis Detection Kit (E-CK-A211, Elabscience, Wuhan, China).

Techniques: Enzyme-linked Immunosorbent Assay, Western Blot, Expressing, Immunofluorescence, Staining, Flow Cytometry, TUNEL Assay, In Vitro

Journal: Bioactive Materials

Article Title: Screening of a quinonoid compounds library identifies decylubiquinone as an antioxidant and anti-apoptotic agent against glucocorticoid-induced osteoporosis via CD39/CD73/adenosine axis

doi: 10.1016/j.bioactmat.2026.03.062

Figure Lengend Snippet: The effect of ADO supplements on primary BMSCs. ( A ) MTT assay for the proliferation of BMSCs treated with different doses of ADO for 2 days and 10 days under osteogenic induction conditions with or without 10 μM Dex. ( B-C ) Representative images and quantitative analysis of mineralized nodule areas by Alizarin Red S staining in primary BMSCs treated with gradient doses of ADO under osteogenic induction with or without 10 μM Dex. ( D ) Western blot and quantification for the expression of osteogenesis-related proteins in primary BMSCs of different groups. ( E ) ELISA for ROS clearance-related enzyme T-SOD and ROS damage biomarkers 8-OHdG, AOPP, and MDA in primary BMSCs of different groups. ( F ) Western blot and quantification for the expression of ROS clearance-related proteins in primary BMSCs of different groups. ( G ) Representative images and quantitative analysis of immunofluorescence staining for MitoSox (red) in primary BMSCs of different groups, and nuclei were stained with Hoechst (blue). ( H ) Western blot and quantification for the expression of apoptosis-related proteins in primary BMSCs of different groups. ( I ) Cellular apoptosis detection in primary BMSCs of different groups by Annexin V-FITC and PI dual-staining assessment via flow cytometry. The proportion of cells in each quadrant was indicated in the plot. ( J ) Tunel (red) staining and quantification of apoptotic cells in primary BMSCs of different groups, and nuclei were stained with DAPI (blue). n = 4 independent repeats by using different biological samples in each group for in vitro experiments. Data were means ± s.e.m. ∗∗ p < 0.01, ∗∗∗ p < 0.001 by one-way ANOVA. Scale bars: 200 μm (B), 50 μm (G), and 25 μm (J).

Article Snippet: The proportion of early and late apoptotic primary BMSCs under different treatment conditions was determined using an Annexin V-FITC/PI Apoptosis Detection Kit (E-CK-A211, Elabscience, Wuhan, China).

Techniques: MTT Assay, Staining, Western Blot, Expressing, Enzyme-linked Immunosorbent Assay, Immunofluorescence, Flow Cytometry, TUNEL Assay, In Vitro

Journal: Bioactive Materials

Article Title: Screening of a quinonoid compounds library identifies decylubiquinone as an antioxidant and anti-apoptotic agent against glucocorticoid-induced osteoporosis via CD39/CD73/adenosine axis

doi: 10.1016/j.bioactmat.2026.03.062

Figure Lengend Snippet: Summary of the study. The schematic diagram illustrates that DUB alleviates GIOP by suppressing oxidative stress and apoptosis via the CD39/CD73/ADO axis and promotes osteogenesis via ADO/A 2b R-mediated activation of the PKA/CREB pathway. The schematic diagram was created by using BioRender.com.

Article Snippet: The proportion of early and late apoptotic primary BMSCs under different treatment conditions was determined using an Annexin V-FITC/PI Apoptosis Detection Kit (E-CK-A211, Elabscience, Wuhan, China).

Techniques: Activation Assay

Journal: Poultry Science

Article Title: NRF2 deficiency impairs proliferation and survival of chicken primordial germ cells via oxidative stress, mitochondrial dysfunction and apoptosis

doi: 10.1016/j.psj.2026.106765

Figure Lengend Snippet: Inhibition of NRF2 results in reduced proliferation and increased apoptosis in chicken PGCs. (A) Morphological observation of PGCs treated with varying concentrations of ML385. Scale bar: 50 μm. (B) Comparison of cell numbers after 3 d of culture in media containing different concentrations of ML385. Different lowercase letters indicate significant differences among groups ( p < 0.05). Based on these dose-dependent effects, the 24 μM concentration was selected for all subsequent experiments. (C and D) Analysis of cell proliferation capacity via EdU assay in ML385-PGCs and Ctrl-PGCs. Scale bar: 50 μm. (E-G) Evaluation of apoptosis levels using Annexin V and PI staining in ML385-PGCs and Ctrl-PGCs. (H and I) Detection of NRF2 protein expression in ML385-PGCs and Ctrl-PGCs by Western blotting. All data are presented as mean ± SEM from three independent biological replicates ( n = 3 per group). Statistical significance is indicated by * ( p < 0.05) and *** ( p < 0.001).

Article Snippet: Apoptosis was assessed using the Annexin V‐FITC/PI Apoptosis Detection Kit (40302ES50, Yeasen, Shanghai, China) according to the manufacturer's instructions.

Techniques: Inhibition, Comparison, Concentration Assay, EdU Assay, Staining, Expressing, Western Blot

Journal: Poultry Science

Article Title: NRF2 deficiency impairs proliferation and survival of chicken primordial germ cells via oxidative stress, mitochondrial dysfunction and apoptosis

doi: 10.1016/j.psj.2026.106765

Figure Lengend Snippet: RNA sequencing analysis reveals aberrant expression of genes related to cell growth, cell cycle, and apoptosis induced by NRF2 inhibition. GSEA of genes related to cell growth (A), cell cycle (B), and apoptosis (C and D) in ML385-PGCs and Ctrl-PGCs. Heatmap (E) depicting the expression of apoptosis pathway genes between ML385-PGCs and Ctrl-PGCs. Comparative analysis (F) of normalized FPKM values of apoptosis pathway genes between ML385-PGCs and Ctrl-PGCs. Statistical significance is indicated by * ( p < 0.05).

Article Snippet: Apoptosis was assessed using the Annexin V‐FITC/PI Apoptosis Detection Kit (40302ES50, Yeasen, Shanghai, China) according to the manufacturer's instructions.

Techniques: RNA Sequencing, Expressing, Inhibition

Journal: Poultry Science

Article Title: NRF2 deficiency impairs proliferation and survival of chicken primordial germ cells via oxidative stress, mitochondrial dysfunction and apoptosis

doi: 10.1016/j.psj.2026.106765

Figure Lengend Snippet: Schematic representation illustrating the impact of NRF2 deficiency on chicken PGCs. NRF2 plays a critical role in maintaining the normal growth of PGCs during in vitro culture by activating antioxidant pathways. NRF2 deficiency impairs antioxidant defense, leading to oxidative stress, mitochondrial damage, and increased apoptosis. Additionally, mitophagy and autophagy are activated to remove damaged organelles. Furthermore, elevated ferroptosis markers, including oxidative stress‑induced lipid peroxidation and Fe²⁺ accumulation, are observed, contributing to reduced PGC viability. Δψm denotes mitochondrial membrane potential.

Article Snippet: Apoptosis was assessed using the Annexin V‐FITC/PI Apoptosis Detection Kit (40302ES50, Yeasen, Shanghai, China) according to the manufacturer's instructions.

Techniques: In Vitro, Membrane

Journal: International Journal of Pharmaceutics: X

Article Title: A pH-responsive dual-drug nanoplatform for stromal remodeling and enhanced chemotherapy via MMP3/TGF- β inhibition

doi: 10.1016/j.ijpx.2026.100489

Figure Lengend Snippet: In vitro therapeutic efficacy and synergy analysis of RPAE-QM in 4 T1 cells. (A) Cell viability of 4 T1 cells incubated with different formulations for 48 h determined by MTT assay. (B) The corresponding IC 50 values of the respective treatments. (C) Representative flow cytometry plots of Annexin V-FITC/PI staining for apoptosis analysis in 4 T1 cells. (D) Quantitative analysis of the total apoptotic rate. (E) Dose-response curves of free Que., free DM1, and their combination used for quantitative synergy determination. (F) The Combination Index (CI) plot as a function of Fraction affected (Fa) generated using the Chou-Talalay method; the reference line at CI = 1 indicates an additive effect, while CI < 1 indicates synergism. Data are presented as mean ± SD (n = 3). * P < 0.05, ** P < 0.01, *** P < 0.001 and **** P < 0.0001.

Article Snippet: After 24 h, the cells were harvested and stained with Annexin V-FITC/PI Cell Apoptosis Detection Kit (Beyotime, China) for flow cytometer analysis.

Techniques: In Vitro, Drug discovery, Incubation, MTT Assay, Flow Cytometry, Staining, Generated

Journal: Cell Insight

Article Title: Capicua mediates interleukin-2 withdrawal–induced death of activated CD8 + T cells

doi: 10.1016/j.cellin.2026.100323

Figure Lengend Snippet: CRISPR screens identify CIC as a key mediator of CWID . (A) IL-2 deprivation induces apoptosis in CTLL-2 cells. Cell death was assessed by annexin-V/propidium iodide (PI) assay in the presence or absence of IL-2 (100 U/mL) for 24 h. (B) Schematic of CRISPR screens to identify the regulators of CWID. CTLL-2 cells were transduced with a mouse CRISPR knockout pooled library, deprived of IL-2 for 7 days, and then expanded under IL-2-sufficient conditions for 7 days before being subjected to next-generation deep sequencing. (C) Waterfall plots for hits from the genome-wide CRISPR screen. Genes were ranked for resistance to CWID based on their beta scores. (D) Effects of CIC-deficiency on IL-2 withdrawal-induced cell death in CTLL-2 cells. Expression of Capicua in Cic -edited CTLL-2 cells was determined by immunoblotting analysis (left). The indicated edited and control CTLL-2 cells were deprived of IL-2 for the indicated times before cell viability measurement (right). Experiments were performed three independent times with similar results. Data are shown as mean ± SEM from one representative experiment performed in triplicate. ∗∗ P < 0.01.

Article Snippet: Reagents and antibodies used in this study were obtained from the indicated sources: Recombinant human IL-2 (SL Pharm, S19991010); Annexin V-FITC/PI Apoptosis Kit (Elabscience, E-CK-A211); Actinomycin D (MCE, HY-17559); TrueCut Cas9 Protein (Invitrogen, A36498); Nuclear and Cytoplasmic Extraction Reagents (Thermo, 78835); Zombie VioletTM Fixable Viability Kit (BioLegend, 423113); Pacific Blue anti-mouse CD3 antibody (BioLegend, 100214); FITC anti-mouse CD8a antibody (BioLegend, 100706); APC anti-mouse CD8a antibody (BD Pharmingen, 553035); PE/Cyanine7 anti-mouse CD3 antibody (BioLegend, 100220); APC anti-mouse IFN-γ antibody (Invivogen, 17-7311-82); PE-Labeled Mouse H-2Kb&B2M&OVA (SIINFEKL) Tetramer Protein (Acrobiosystems, H2A-MP2H7); Capicua antibody (Novusbio, NB110-59906); anti-FLAG antibody (Sigma-Aldrich, F3165); β-actin (Sigma-Aldrich, A2228); Bim antibody (Cell Signaling Technology, 2933T); Bcl2 antibody (Cell Signaling Technology, 3498T); Bax antibody (Cell Signaling Technology, 2772T); Bcl2 antibody (Proteintech, 68103-1-Ig); BAX antibody (Proteintech, 60267-1-Ig); KEAP1 antibody (Proteintech, 10503-2-AP); Anti-mouse Ig HRP (Rockland Immunochemicals, 18-8817-33); Anti-rabbit IgG HRP (Rockland Immunochemicals, 18-8816-33); HRP-conjugated Mouse anti-Rabbit IgG Light Chain (ABclonal, AS061); Prestained Protein Ladder 10-180 kDa (Thermo, 26616); Prestained Protein Ladder 25-400 kDa (Sevenbio, SW179).

Techniques: CRISPR, Transduction, Knock-Out, Sequencing, Genome Wide, Expressing, Western Blot, Control

Journal: Cell Insight

Article Title: Capicua mediates interleukin-2 withdrawal–induced death of activated CD8 + T cells

doi: 10.1016/j.cellin.2026.100323

Figure Lengend Snippet: CIC is required for activated CD8 + T cell death in vivo . (A) Schematic presentation of the Listeria monocytogenes infection model. Age- and sex-matched WT ( n = 3/group) and Cic -cKO ( n = 3/group) mice were intravenously administered with 1 × 10 5 CFU of LmOVA in 100 μL PBS and were euthanized at the indicated days post-infection. Splenocytes were prepared and subjected to flow cytometry for cell counting or apoptosis analysis. (B) Quantification of the number of total IFNγ–producing CD8 + T cells in the spleen. (C) Quantification of the number of total OVA-specific CD8 + T cells in the spleen. (D) Analysis of cell death in splenic CD8 + T cells at day 14 post-infection. Experiments (B-D) were repeated twice independently with similar results. Representative data are shown. The data shown in (B-C) are mean ± SEM of one representative experiment. ∗ P < 0.05.

Article Snippet: Reagents and antibodies used in this study were obtained from the indicated sources: Recombinant human IL-2 (SL Pharm, S19991010); Annexin V-FITC/PI Apoptosis Kit (Elabscience, E-CK-A211); Actinomycin D (MCE, HY-17559); TrueCut Cas9 Protein (Invitrogen, A36498); Nuclear and Cytoplasmic Extraction Reagents (Thermo, 78835); Zombie VioletTM Fixable Viability Kit (BioLegend, 423113); Pacific Blue anti-mouse CD3 antibody (BioLegend, 100214); FITC anti-mouse CD8a antibody (BioLegend, 100706); APC anti-mouse CD8a antibody (BD Pharmingen, 553035); PE/Cyanine7 anti-mouse CD3 antibody (BioLegend, 100220); APC anti-mouse IFN-γ antibody (Invivogen, 17-7311-82); PE-Labeled Mouse H-2Kb&B2M&OVA (SIINFEKL) Tetramer Protein (Acrobiosystems, H2A-MP2H7); Capicua antibody (Novusbio, NB110-59906); anti-FLAG antibody (Sigma-Aldrich, F3165); β-actin (Sigma-Aldrich, A2228); Bim antibody (Cell Signaling Technology, 2933T); Bcl2 antibody (Cell Signaling Technology, 3498T); Bax antibody (Cell Signaling Technology, 2772T); Bcl2 antibody (Proteintech, 68103-1-Ig); BAX antibody (Proteintech, 60267-1-Ig); KEAP1 antibody (Proteintech, 10503-2-AP); Anti-mouse Ig HRP (Rockland Immunochemicals, 18-8817-33); Anti-rabbit IgG HRP (Rockland Immunochemicals, 18-8816-33); HRP-conjugated Mouse anti-Rabbit IgG Light Chain (ABclonal, AS061); Prestained Protein Ladder 10-180 kDa (Thermo, 26616); Prestained Protein Ladder 25-400 kDa (Sevenbio, SW179).

Techniques: In Vivo, Infection, Flow Cytometry, Cell Counting

Journal: Materials Today Bio

Article Title: Mannosylated graphene oxide nanotherapeutics co-delivering docetaxel and a STING agonist reprogram myeloid cells and potentiate antitumor immunity

doi: 10.1016/j.mtbio.2026.103086

Figure Lengend Snippet: GO-EDM-DTX-Vad combined with NIR induces TNBC cell death, apoptosis and immunogenic cell death. (A, B) Calcein-AM/PI live/dead staining of 4T1 (A) and MDA-MB-231 (B) cells (live, green; dead, red; scale bar, 200 μm). (C, D) Annexin V/PI apoptosis plots for 4T1 (C) and MDA-MB-231 (D) cells. (E, F) Quantification of viable and early/late apoptotic 4T1 cells. (H, I) Quantification of viable and early/late apoptotic MDA-MB-231 cells. (G, J) Dose–response viability curves after 48 h treatment with free DTX or GO-EDM-DTX-Vad in 4T1 (G) and MDA-MB-231 (J). (K, L) Immunofluorescence of CRT exposure and HMGB1 release after 24 h treatment in 4T1 (K) and MDA-MB-231 (L) cells (nuclei, blue; CRT/HMGB1, green; scale bar, 100 μm). (M) Schematic of the ICD-to-DC maturation assay. (N–P) Frequencies of CD40 + , CD80 + and CD86 + DCs after co-culture. Data are mean ± SD (n = 3). ∗ P < 0.05, ∗∗ P < 0.01, ∗∗∗ P < 0.001; ns, not significant.

Article Snippet: Annexin V-FITC/PI apoptosis detection kits were purchased from Elabscience Biotechnology Co., Ltd. (Wuhan, China).

Techniques: Staining, Immunofluorescence, Co-Culture Assay

Journal: Poultry Science

Article Title: Fermentation alleviates the adverse effect of peony seed meal on hepatocytes

doi: 10.1016/j.psj.2026.106807

Figure Lengend Snippet: The effect of aPSM on hepatocytes. (A) Cell viability as measured by CCK-8 assay. (B-C) Apoptosis was determined by flow cytometry after 24 h treatment. (D) The relative mRNA abundances of apoptosis genes were determined by real-time PCR after 24 h treatment. Data are presented as mean ± SD. * P < 0.05, ** P < 0.01, and *** P < 0.001. n = 6 in each group. aPSM: Alcohol extract of peony seed meal.

Article Snippet: The apoptosis of hepatocytes was detected by flow cytometry using annexin V-FITC/PI apoptosis kit (KGA107, KeyGen BioTech, Jiangsu, China).

Techniques: CCK-8 Assay, Flow Cytometry, Real-time Polymerase Chain Reaction

Journal: Poultry Science

Article Title: High genotoxicity of CRISPR/Cas9 versus limited efficacy of CRISPRi in chicken primordial germ cells

doi: 10.1016/j.psj.2026.106722

Figure Lengend Snippet: CRISPR/Cas9 induces DNA damage and apoptosis in PGCs. (A) Flow cytometry analysis 24 h after electroporation, quantifying the proportion of Annexin V + /PI + cells. The horizontal axis indicates PI and the vertical axis Annexin V. The upper-left quadrant (Annexin V + /PI + ) represents late apoptotic cells, and the lower-right quadrant (Annexin V + only) represents early apoptotic cells. Upper panels: results after electroporation with Cas9 + various gRNAs; lower panels: results with dCas9 + various gRNAs. (B) Bar graph of Annexin V + /PI + percentages across groups. Cas9 editing induced a highly significant increase in late apoptosis. (C) γ-H 2 AX foci (green) detected by immunofluorescence 24 h after electroporation. Foci appear as discrete nuclear puncta; nuclei are counterstained with DAPI (blue). Scale bar = 10 µm. (D) Quantification of γ-H 2 AX foci per cell. Cas9 targeting resulted in a significant increase in γ-H 2 AX foci per cell, whereas dCas9 with sgRNA did not. Statistical significance determined by one-way ANOVA (p-values are indicated in the figure).

Article Snippet: After another centrifugation, the supernatant was removed and cells were resuspended in 500 μL of 1× Annexin V Binding Buffer (from Annexin V-FITC/PI Apoptosis Kit, Elabscience E-CK-A211).

Techniques: CRISPR, Flow Cytometry, Electroporation, Immunofluorescence

Journal: Poultry Science

Article Title: High genotoxicity of CRISPR/Cas9 versus limited efficacy of CRISPRi in chicken primordial germ cells

doi: 10.1016/j.psj.2026.106722

Figure Lengend Snippet: PGCs are more sensitive to DNA double-strand breaks than somatic cells. (A) Cell viability curves after 24 h treatment with increasing concentrations of etoposide (ETP; x-axis shows log 10 [ETP]). PGC viability dropped sharply even at low ETP doses, whereas CEF cells were more tolerant (viability data are mean ± SD of triplicates). (B) Apoptosis detection in PGCs treated with low-dose ETP. The proportion of Annexin V + /PI + cells (late apoptosis) was significantly elevated even at 0.03 µM ETP. (C) Western blot analysis of γ-H 2 AX protein in PGCs treated with varying ETP concentrations (left panel; β-actin as loading control). The bar graph (right) shows the ratio of γ-H 2 AX to β-actin band intensity, with a marked increase at 3 µM ETP (p-values are indicated in the figure). (D) Quantification of γ-H 2 AX foci per cell in THP-1 cells, male PGCs, and female PGCs after exposure to X-ray doses of 0, 2, 4, 6, and 8 Gy. After 48 h recovery, γ-H 2 AX foci increased significantly with higher radiation in THP-1, male PGCs, and female PGCs. Female PGC data points are red squares; male PGCs are blue squares; THP-1 are black circles. (E) Cell-cycle distribution of male vs. female PGCs after DNA damage. PGCs were irradiated (2, 4, 6 Gy), cultured 48 h, and analyzed by flow cytometry for cell-cycle phase (propidium iodide staining). Stacked bars show the percentage of cells in G 0 G 1 , S, and G 2 /M phases in untreated vs. irradiated cells. After damage, female PGCs predominantly arrested in G 2 /M (increased G 2 fraction), whereas male PGCs accumulated in S phase. Statistical significance by one-way ANOVA (p-values are indicated in the figure).

Article Snippet: After another centrifugation, the supernatant was removed and cells were resuspended in 500 μL of 1× Annexin V Binding Buffer (from Annexin V-FITC/PI Apoptosis Kit, Elabscience E-CK-A211).

Techniques: Western Blot, Control, Irradiation, Cell Culture, Flow Cytometry, Staining