Journal: Frontiers in Physiology

Article Title: Oxidative stress-mediated responses in endometrial cancer cells: contrasting effects of doxorubicin and menadione

doi: 10.3389/fphys.2026.1733194

Figure Lengend Snippet: Validation of primary antibodies against SESN2, SESN3, and SOD1 using ICW assay. (A) AN3CA and KLE cells were probed with anti-SESN2 antibody (dilutions 1:1200–1:150). (B) AN3CA and KLE cells were probed with anti-SESN3 antibody (dilutions 1:1000–1:100). (C) AN3CA and KLE cells were probed with anti-SOD1 antibody (dilutions 1:1000–1:100). Secondary antibodies (1:800, LI-COR) were applied, with CellTag™ 520 (1:500, LI-COR) as a loading control for signal normalization. Data are shown as mean ± SD of three technical replicate wells from a single antibody-optimization experiment. Statistical significance was assessed by two-way ANOVA (factors: cell line and antibody dilution), followed by Sidak’s multiple-comparisons test comparing AN3CA vs. KLE within each dilution. **p < 0.005.

Article Snippet: PrimePCRTM Probe Assay was used for SOD1 (ID: qHsaCIP0026883, Bio-Rad), while TaqMan® Gene Expression Assays were used for SESN2 (ID: Hs00230241_m1), SESN3 (ID: Hs00914870_m1), and UBC (ID: Hs00824723_m1) (Thermo Fisher Scientific, Waltham, MA, United States).

Techniques: Biomarker Discovery, Control

Journal: Frontiers in Physiology

Article Title: Oxidative stress-mediated responses in endometrial cancer cells: contrasting effects of doxorubicin and menadione

doi: 10.3389/fphys.2026.1733194

Figure Lengend Snippet: Differential effects of DOX and menadione on SESN2 expression in AN3CA and KLE cells assessed by ICW assay. (A) In AN3CA cells, menadione (1–25 µM) induced a modest upward trend in normalized SESN2 levels. (B) Raw fluorescence signals corresponding to SESN2 (700 nm) and CellTag™ 520 (520 nm). (C) In KLE cells, menadione increased normalized SESN2 levels at 1–5 μM, followed by a marked decrease at 25 µM. (D) Raw fluorescence signals corresponding to SESN2 (700 nm) and CellTag™ 520 (520 nm). (E) In AN3CA cells treated with DOX (0.125–4 µM), SESN2 exhibited a biphasic response, peaking at 0.125 µM and declining sharply from 1 µM onward. (F) Raw fluorescence signals corresponding to SESN2 (700 nm) and CellTag™ 520 (520 nm). (G) In KLE cells, DOX induced modest SESN2 upregulation at 0.125–0.25 µM, followed by progressive downregulation with the lowest levels at 4 µM. (H) Raw fluorescence signals corresponding to SESN2 (700 nm) and CellTag™ 520 (520 nm). Normalized protein levels were calculated as SESN2/CellTag™ 520 ratios (normalized signal). Untreated control (medium-only) wells were included in each experiment and served as the reference group for comparisons. Statistical significance was determined by one-way ANOVA followed by Dunnett’s post hoc test comparing each treatment condition to the untreated control. For DOX, the figure shows a representative ICW run (n = 6 technical replicate wells per condition), and the experiment was repeated independently once with consistent results (n = 3). For menadione, data are from one ICW experiment (n = 6 technical replicate wells per condition). *p < 0.05; **p < 0.005; ***p < 0.0005.

Article Snippet: PrimePCRTM Probe Assay was used for SOD1 (ID: qHsaCIP0026883, Bio-Rad), while TaqMan® Gene Expression Assays were used for SESN2 (ID: Hs00230241_m1), SESN3 (ID: Hs00914870_m1), and UBC (ID: Hs00824723_m1) (Thermo Fisher Scientific, Waltham, MA, United States).

Techniques: Expressing, Fluorescence, Control

Journal: Frontiers in Physiology

Article Title: Oxidative stress-mediated responses in endometrial cancer cells: contrasting effects of doxorubicin and menadione

doi: 10.3389/fphys.2026.1733194

Figure Lengend Snippet: SESN2, SESN3, and SOD1 gene expression profiles in AN3CA and KLE cells analyzed by qRT-PCR. (A) SESN2 mRNA was significantly upregulated in both cell lines following 4 µM DOX treatment. Menadione induced robust SESN2 upregulation at 25 µM in KLE cells. (B) SESN3 mRNA was strongly upregulated by DOX in both lines, whereas menadione downregulated SESN3 in both cell lines. (C) SOD1 mRNA showed significant upregulation in KLE cells under 4 µM DOX, while AN3CA cells exhibited no significant changes. Under menadione, AN3CA cells displayed significant downregulation of SOD1 at 10 µM and 25 μM, while KLE cells showed downregulation at 25 µM. RT–qPCR data were normalized to UBC as the endogenous reference gene and are presented relative to the untreated control (medium-only). Each condition was analyzed in three technical replicate wells. Statistical significance was determined by one-way ANOVA followed by Dunnett’s post hoc test comparing each treatment condition to the untreated control. Key findings were confirmed in an independent repeat experiment. *p < 0.05; **p < 0.005; ***p < 0.0005.

Article Snippet: PrimePCRTM Probe Assay was used for SOD1 (ID: qHsaCIP0026883, Bio-Rad), while TaqMan® Gene Expression Assays were used for SESN2 (ID: Hs00230241_m1), SESN3 (ID: Hs00914870_m1), and UBC (ID: Hs00824723_m1) (Thermo Fisher Scientific, Waltham, MA, United States).

Techniques: Gene Expression, Quantitative RT-PCR, Control