p21waf1 cip1  (Cell Signaling Technology Inc)

Journal: Bioactive Materials

Article Title: Microenvironment-educated MSC-EVs loaded injectable smart hydrogel for targeting senescent nucleus pulposus cells and inhibiting ferroptosis against intervertebral disc degeneration

doi: 10.1016/j.bioactmat.2026.02.030

Figure Lengend Snippet: Senescent Microenvironment-Educated Mesenchymal Stem Cells Release High-Affinity Senescent NPC Domesticated Extracellular Vesicles. (A) Schematic diagram of the experimental setup for educating MSCs with SASP-CM to generate D-EVs versus N-EVs. (B) Confocal microscopy images showing different EVs internalization by senescent NPCs after 12 h in vitro. (C) Flow cytometry and quantification analysis of different EVs uptake by senescent NPCs. (D) In vivo validation of the senescent niche. Representative fluorescence images following injection of senescence-tracer (Red). (E) In vivo PKH26-labeled D-EVs tracking. (F) Representative SA-β-Gal images and quantification of MSCs treated with SASP-CM or not. (G) Gene Ontology (GO) analysis confirming enrichment of external encapsulating structure organization and cytokine production in Biological Process (BP) categories. (H) Heatmap indicating gene expression associated with EVs biogenesis within D-MSCs and N-MSCs. (I) Heatmap indicating gene expression associated with cytokine production within D-MSCs and N-MSCs. (J and L) Gene Ontology (GO) analysis confirming enrichment of terms related to vesicle organization and transport in the Cellular Component (CC) categories. (K) Western blot analysis confirmed core senescence markers p16 and p21 and DNA damage marker γ-H2AX in N-MSC and D-MSC. (M) Western blot analysis confirmed the expression of CD9, CD63, TSG101, Calnexin, and GM130 in MSC-EVs, N-EVs, or D-EVs. (N) TEM images showing the morphology and size of MSC-derived EVs, N-EVs, and D-EVs. (O) NTA shows size distribution in MSC-EVs, N-EVs, or D-EVs. The data were presented as mean ± SD. n = 3, ns, not significant; ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001.

Article Snippet: After blocked with 5% non-fat milk for 2 h at room temperature, the membranes were incubated with primary antibodies against GAPDH (1:5000, 104941-AP, Proteintech), TSG101 (1:1000, DF8427, Affinity), CD9 (1:1000, AF5139, Affinity), CD63 (1:2000, 25682-1-AP, Proteintech), Calnexin (1:5000, 10427-2-AP, Proteintech), GM130 (1:20000, 11308-1-AP, Proteintech), CXCR3 (1:5000, 26756-1-AP, Proteintech), CXCL10 (1:2000, 10937-1-AP, Proteintech), MMP3 (1:2000, 17873-1-AP, Proteintech), ADAMTS5 (DF13268, Affinity), P16 (AF5484, Affinity), P21 (10355-1-AP, Proteintech), GPX4 (1:1000, 381958, Zen-bio), SLC7A11 (1:1000, 26864-1-AP, Proteintech), ACSL4 (1:5000, 22401-1-AP, Proteintech) and Tubulin (1:10000, T40103 , Abmart) overnight at 4 °C.

Techniques: Confocal Microscopy, In Vitro, Flow Cytometry, In Vivo, Biomarker Discovery, Fluorescence, Injection, Labeling, Gene Expression, Western Blot, Marker, Expressing, Derivative Assay

Journal: Bioactive Materials

Article Title: Microenvironment-educated MSC-EVs loaded injectable smart hydrogel for targeting senescent nucleus pulposus cells and inhibiting ferroptosis against intervertebral disc degeneration

doi: 10.1016/j.bioactmat.2026.02.030

Figure Lengend Snippet: D-EVs Alleviate Cellular Senescence and Restore ECM anabolic/catabolic metabolism in Senescent NPCs. (A) The CCK8 assay was used to determine D-EVs concentrations on cell viability. (B) Flow cytometry analysis of proliferative capacity in the above group, and (C) quantitative analysis. (D) Representative ROS images of senescent NPCs treated with N-EVs, D-EVs, or D-EVs + GW4869. (E) Representative SA-β-Gal images of senescent NPCs treated with N-EVs, D-EVs, or D-EVs + GW4869, and (F) quantitative analysis. (G) Confocal analysis of γ-H2A with IF staining depicting DNA damage in the control, TBHP, N-Evs, or D-EVs group. (H) WB analysis of ECM metabolism–related and aging-related proteins in NPCs following treatment with Control, TBHP, N-Evs, or D-EVs. (I) Western blot analysis of p53, p21, and p16 in senescent NPCs treated with D-EVs, D-CM, or D-CM EV-dep . (J) Confocal analysis of COL2 with IF staining in the control, TBHP, N-EVs, or D-EVs group. The data were presented as mean ± SD. n = 3, ns, not significant; ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001.

Article Snippet: After blocked with 5% non-fat milk for 2 h at room temperature, the membranes were incubated with primary antibodies against GAPDH (1:5000, 104941-AP, Proteintech), TSG101 (1:1000, DF8427, Affinity), CD9 (1:1000, AF5139, Affinity), CD63 (1:2000, 25682-1-AP, Proteintech), Calnexin (1:5000, 10427-2-AP, Proteintech), GM130 (1:20000, 11308-1-AP, Proteintech), CXCR3 (1:5000, 26756-1-AP, Proteintech), CXCL10 (1:2000, 10937-1-AP, Proteintech), MMP3 (1:2000, 17873-1-AP, Proteintech), ADAMTS5 (DF13268, Affinity), P16 (AF5484, Affinity), P21 (10355-1-AP, Proteintech), GPX4 (1:1000, 381958, Zen-bio), SLC7A11 (1:1000, 26864-1-AP, Proteintech), ACSL4 (1:5000, 22401-1-AP, Proteintech) and Tubulin (1:10000, T40103 , Abmart) overnight at 4 °C.

Techniques: CCK-8 Assay, Flow Cytometry, Staining, Control, Western Blot

Journal: Bioactive Materials

Article Title: Microenvironment-educated MSC-EVs loaded injectable smart hydrogel for targeting senescent nucleus pulposus cells and inhibiting ferroptosis against intervertebral disc degeneration

doi: 10.1016/j.bioactmat.2026.02.030

Figure Lengend Snippet: D-EVs Counteract NPC Senescence by Suppressing Ferroptosis. (A) KEGG pathway analysis of DEGs in senescent NPCs following treatment with D-EVs or not. (B-C) GSEA plots showing significant enrichment of ferroptosis and cell cycle in senescent NPCs. (D-E) Heatmap quantification of key genes involved in ferroptosis and cell cycle. (F) Western blot analysis of key ferroptosis (GPX4, SLC7A11, ACSL4) and senescence (p21, P16) markers in NPCs following treatment with different experimental conditions. (G) Representative images of C11-BODIPY 581/591 staining to detect lipid peroxidation (green) in the control, TBHP, Era, Era + Fer-1, or TBHP + Fer-1 groups. (H-I) Quantitative assessment of malondialdehyde (MDA) levels (H) and glutathione (GSH) levels (I) in the control, TBHP, N-EVs, D-EVs, or D-EVs + Era groups. (J) Western blot analysis of key ferroptosis (GPX4, SLC7A11, ACSL4) and senescence (p21, P16) markers in NPCs following treatment with PBS, N-EVs, D-EVs, or D-EVs + Era. (K) Confocal analysis of GPX4 with IF staining in the control, TBHP, N-Evs, D-EVs, and D-EVs + Era group. (L) Flow cytometry analysis of cell cycle distribution in the above experimental conditions. Statistical comparisons were performed between the experimental group and the TBHP-induced group. The data were presented as mean ± SD. n = 3, ns, not significant; ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001.

Article Snippet: After blocked with 5% non-fat milk for 2 h at room temperature, the membranes were incubated with primary antibodies against GAPDH (1:5000, 104941-AP, Proteintech), TSG101 (1:1000, DF8427, Affinity), CD9 (1:1000, AF5139, Affinity), CD63 (1:2000, 25682-1-AP, Proteintech), Calnexin (1:5000, 10427-2-AP, Proteintech), GM130 (1:20000, 11308-1-AP, Proteintech), CXCR3 (1:5000, 26756-1-AP, Proteintech), CXCL10 (1:2000, 10937-1-AP, Proteintech), MMP3 (1:2000, 17873-1-AP, Proteintech), ADAMTS5 (DF13268, Affinity), P16 (AF5484, Affinity), P21 (10355-1-AP, Proteintech), GPX4 (1:1000, 381958, Zen-bio), SLC7A11 (1:1000, 26864-1-AP, Proteintech), ACSL4 (1:5000, 22401-1-AP, Proteintech) and Tubulin (1:10000, T40103 , Abmart) overnight at 4 °C.

Techniques: Western Blot, Staining, Control, Flow Cytometry

Journal: Bioactive Materials

Article Title: Microenvironment-educated MSC-EVs loaded injectable smart hydrogel for targeting senescent nucleus pulposus cells and inhibiting ferroptosis against intervertebral disc degeneration

doi: 10.1016/j.bioactmat.2026.02.030

Figure Lengend Snippet: D-EVs Deliver GPX4 to Inhibit Ferroptosis in Senescent NPCs. (A) Representative Senescent-Tracker images of NPCs treated with N-EVs, D-EVs, Era, and D-Evs sh-CXCL10 . (B) Volcano plot of transcriptomic data comparing D-MSC and N-MSC. (C) KEGG pathway analysis of DEGs in D-MSCs versus N-MSCs. (D) Volcano plot of proteomic data comparing D-EVs and N-EVs. (E) KEGG pathway analysis of transcriptomic and proteomic data integration. (F) A Venn diagram illustrating the intersection of genes from the D-MSC transcriptome, the D-EVs proteome, and the ferroptosis-related gene set. (G) Bar graph showing the relative expression levels of core overlapping genes identified in (F). (H) MS analysis revealed that GPX4 is enriched in the D-EVs proteome. (I) Western blot analysis confirming GPX4 protein in D-EVs and N-EVs. (J) Western blot analysis of key senescence (p21, P16) markers in NPCs following treatment with PBS or N-EVs with CXCL10 or GPX4 knockout. (K) Representative images of EdU depicting cell proliferation ability in the control, TBHP, D-EVs, D-EVs sh-CXCL10 , D-EVs sh-GPX4 , and D-EVs sh-CXCL10+GPX4 groups. (L-M) Confocal images showing GPX4 delivery from different EVs to senescent NPCs at 12h and 24h co-culture, and (N) colocalization analysis. The data were presented as mean ± SD. n = 3, ns, not significant; ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001.

Article Snippet: After blocked with 5% non-fat milk for 2 h at room temperature, the membranes were incubated with primary antibodies against GAPDH (1:5000, 104941-AP, Proteintech), TSG101 (1:1000, DF8427, Affinity), CD9 (1:1000, AF5139, Affinity), CD63 (1:2000, 25682-1-AP, Proteintech), Calnexin (1:5000, 10427-2-AP, Proteintech), GM130 (1:20000, 11308-1-AP, Proteintech), CXCR3 (1:5000, 26756-1-AP, Proteintech), CXCL10 (1:2000, 10937-1-AP, Proteintech), MMP3 (1:2000, 17873-1-AP, Proteintech), ADAMTS5 (DF13268, Affinity), P16 (AF5484, Affinity), P21 (10355-1-AP, Proteintech), GPX4 (1:1000, 381958, Zen-bio), SLC7A11 (1:1000, 26864-1-AP, Proteintech), ACSL4 (1:5000, 22401-1-AP, Proteintech) and Tubulin (1:10000, T40103 , Abmart) overnight at 4 °C.

Techniques: Expressing, Western Blot, Knock-Out, Control, Co-Culture Assay

Journal: iScience

Article Title: Identification and validation of key PANoptosis-related genes via integrative machine learning and single-cell sequencing in AILI

doi: 10.1016/j.isci.2026.115183

Figure Lengend Snippet: Expression and functional exploration of the key genes in single-cell sequencing data (A) The UMAP plot shows the total sample composition, tissue sources, and cell subtypes. (B) The stacked graph shows the proportion of each type of cell in the control group and the AILI group. (C) Bubble plots of marker gene expression demonstrating the accuracy of the cell annotations. (D) Bubble chart showing the expression of Cdkn1a and Pdk1 in various cells in the control group. (E) Bubble chart showing the expression of Cdkn1a and Pdk1 in various cells in the AILI group. (F) Circle plot and heatmap showing the cell communication weights and numbers of all cell subtypes. (G–J) Receptor‒ligand communication weights between AILI and control samples.

Article Snippet: p21 Polyclonal antibody , Proteintech , Cat No.28248-1-AP; RRID: AB_2881097.

Techniques: Expressing, Functional Assay, Single Cell, Sequencing, Control, Marker, Gene Expression

Journal: iScience

Article Title: Identification and validation of key PANoptosis-related genes via integrative machine learning and single-cell sequencing in AILI

doi: 10.1016/j.isci.2026.115183

Figure Lengend Snippet: Validation of key PANoptosis-related genes in animal models (A) H&E staining of liver tissues from WT and AILI mice (scale bars, 100 μm; n = 5). (B) mRNA expression of Cdkn1a and Pdk1 by RT-qPCR. (C–F) Correlation analyses between hepatic Cdkn1a and Pdk1 mRNA levels and serum ALT and AST levels at 24 h after AILI. (G) Detection and statistical analysis of key PANoptosis-related gene and marker protein expression in liver tissues from WT and AILI mice. (H) Immunohistochemical staining for P21 and PDK1 in liver tissues from WT and AILI mice (scale bars, 50 μm; n = 5). All the data are presented as the means ± SDs. One-way ANOVA with Tukey’s test and a two-tailed Student’s t test were used for statistical analysis. Spearman’s rank correlation was used to assess the associations between relative mRNA expression levels of Cdkn1a and Pdk1 and serum ALT and AST levels. ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001.

Article Snippet: p21 Polyclonal antibody , Proteintech , Cat No.28248-1-AP; RRID: AB_2881097.

Techniques: Biomarker Discovery, Staining, Expressing, Quantitative RT-PCR, Marker, Immunohistochemical staining, Two Tailed Test

Journal: International Journal of Oncology

Article Title: Overcoming acquired doxorubicin resistance of ovarian carcinoma cells by verapamil-mediated promotion of DNA damage-driven cytotoxicity

doi: 10.3892/ijo.2026.5861

Figure Lengend Snippet: Effect of Topo II inhibitors on DNA damage formation and activation of DDR-related mechanisms in A2780 and A2780ADR cells. (A) At 24 h after treatment of logarithmically growing cells with the indicated concentrations of Doxo or Eto, the number of nuclear γH2AX-foci, 53BP1-foci, γH2AX/53BP1 co-localized foci and γH2AX pan-stained cells was analyzed. The upper part of the figure shows representative images (total magnification, ×1,000). Quantitative data depicted in the histogram are the mean ± SD from n=3 independent experiments with each five images being analyzed per experimental condition. * P≤0.05; ** P≤0.01; *** P≤0.001 (A2780 vs. A2780ADR); # P≤0.05; ## P≤0.01; ### P≤0.001 (treated vs. untreated control). Control experiments performed by use of 1st or 2nd antibody only or no antibody at all did not interfere with the signal of main interest (that is, nuclear foci; data not shown). (B) Logarithmically growing cells were treated with the indicated concentrations of Doxo for 24 or 72 h. Afterwards, the protein expression of DDR-related factors was analyzed by western blotting using EKR2 protein expression as loading control. Doxo, doxorubicin; Eto, etoposide; p-, phosphorylated; Chk1/2, checkpoint kinase 1/2; ERK2, extracellular regulated kinase; γH2AX, Ser139 phosphorylated histone H2AX; 2; Kap1, KRAB-associated protein 1; PARP, poly (ADP-ribose) polymerase; p21, cyclin-dependent kinase inhibitor 1; p53, tumor suppressor p53.

Article Snippet: The following primary antibodies were used: ATP7A (cat. no. PA5-103110; Invitrogen; Thermo Fisher Scientific; Inc.), Caspase-7 cleaved (Asp198; cat. no. 9491S; Cell Signaling Technology, Inc.), Chk1phospho (Ser345; cat. no. 2341, Cell Signaling Technology, Inc.), Chk2phosphoT68 (Y171; cat. no. ab32148; Abcam), CTR1/SLC31A1 (EPR7936; cat. no. ab129067; Abcam), Cyclin B1 (cat. no. 4138, Cell Signaling Technology, Inc.), ERK2 [PA5-32396, Invitrogen/Thermo Fisher Scientific; Carlsbad, USA], Galactosidase beta (E2U2I) (cat. no. 27198, Cell Signaling Technology, Inc.), GAPDH (14C10; cat. no. 2118S; Cell Signaling Technology, Inc.), H2AX phospho (Ser139, cloneJBW301; cat. no. 05-636, Merck KGaA), MDR1/ABCB1 (D3H1Q; cat. no. 12683; Cell Signaling Technology, Inc.), OCT2 (cat. no. MBS9600162, Biozol Diagnostics Vertrieb GmbH), P16 (F-12; cat. no. sc-1661; Santa Cruz Biotechnology, Inc.), p21 (C-19; cat. no. sc-397; Santa Cruz Biotechnology, Inc.), P53 phospho (S15; cat. no. 9284S; Cell Signaling Technology, Inc.), PARP (cat. no. 9542S, Cell Signaling Technology, Inc.), Rad51 (cat. no. ab63801; Abcam), RPA32 phospho (S4/S8; cat. no. ICH-00422; Bethyl Laboratories Inc.), TopBP1 (D8G4L; cat. no. 14342, Cell Signaling Technology, Inc.), Topoisomerase II alpha (D10G9; cat. no. 12286, Cell Signaling Technology, Inc.).

Techniques: Activation Assay, Staining, Control, Expressing, Western Blot

Journal: International Journal of Oncology

Article Title: Overcoming acquired doxorubicin resistance of ovarian carcinoma cells by verapamil-mediated promotion of DNA damage-driven cytotoxicity

doi: 10.3892/ijo.2026.5861

Figure Lengend Snippet: Influence of combined treatment of A2780ADR cells with Doxo and selected inhibitors on mechanism of the DDR and mRNA expression of selected susceptibility-related genes. (A) Logarithmically growing A2780ADR cells were co-treated with the indicated concentrations of Doxo and selected pharmacological inhibitors (concentrations see ) for 24 or 72 h. Afterwards, the protein expression of DDR-related factors was analyzed by western blotting. For loading control, blots were reprobed with ERK2 antibody. (B) Reverse transcription-quantitative PCR of the mRNA expression of selected factors known to contribute to different mechanisms of drug sensitivity. Data shown are mean ± SD from triplicate determinations as described in methods. Relative mRNA level in untreated A2780ADR cells was set to 1.0. Doxo, doxorubicin; DDR, DNA damage response; p-, phosphorylated; nd, not detectable; Bax, Bcl-2 associated protein X; Bcl-2, B-cell lymphoma; BBC3, Bcl-2 binding component 2; BRCA1, 2, breast cancer associated gene 1,2; Cl casp-7, cleaved caspase 7; Chk, checkpoint kinase; CXCL8, chemokine ligand 8 (interleukin 8); p21, CDK inhibitor 1; p16, CDK inhibitor 2; CDKN1A/2A, cyclin dependent kinae inhibitor 1A/2A; CCNB1, Cyclin B1; b-Gal, beta-galactosidase; FASL, FAS ligand; FASR, FAS receptor; GADD, growth arrest and DNA damage inducible GPX1, glutathione peroxidase 1; GSTM1, glutathione S-transferase 1; HMOX1, heme oxygenase 1; γH2AX, Ser139 phosphorylated histone H2AX; p53, tumor suppressor p53; PARP, poly (ADP-ribose) polymerase; PCNA-proliferating cell nuclear antigen; PGC1A, PPARG coactivator 1; PPARGC1A, peroxisome proliferator-activated receptor gamma coactivator 1-alpha; RAD51, radiation damage gene 51; RPAreplication protein A; SOD1, superoxide dismutase 1; Ver, verapamil.

Article Snippet: The following primary antibodies were used: ATP7A (cat. no. PA5-103110; Invitrogen; Thermo Fisher Scientific; Inc.), Caspase-7 cleaved (Asp198; cat. no. 9491S; Cell Signaling Technology, Inc.), Chk1phospho (Ser345; cat. no. 2341, Cell Signaling Technology, Inc.), Chk2phosphoT68 (Y171; cat. no. ab32148; Abcam), CTR1/SLC31A1 (EPR7936; cat. no. ab129067; Abcam), Cyclin B1 (cat. no. 4138, Cell Signaling Technology, Inc.), ERK2 [PA5-32396, Invitrogen/Thermo Fisher Scientific; Carlsbad, USA], Galactosidase beta (E2U2I) (cat. no. 27198, Cell Signaling Technology, Inc.), GAPDH (14C10; cat. no. 2118S; Cell Signaling Technology, Inc.), H2AX phospho (Ser139, cloneJBW301; cat. no. 05-636, Merck KGaA), MDR1/ABCB1 (D3H1Q; cat. no. 12683; Cell Signaling Technology, Inc.), OCT2 (cat. no. MBS9600162, Biozol Diagnostics Vertrieb GmbH), P16 (F-12; cat. no. sc-1661; Santa Cruz Biotechnology, Inc.), p21 (C-19; cat. no. sc-397; Santa Cruz Biotechnology, Inc.), P53 phospho (S15; cat. no. 9284S; Cell Signaling Technology, Inc.), PARP (cat. no. 9542S, Cell Signaling Technology, Inc.), Rad51 (cat. no. ab63801; Abcam), RPA32 phospho (S4/S8; cat. no. ICH-00422; Bethyl Laboratories Inc.), TopBP1 (D8G4L; cat. no. 14342, Cell Signaling Technology, Inc.), Topoisomerase II alpha (D10G9; cat. no. 12286, Cell Signaling Technology, Inc.).

Techniques: Expressing, Western Blot, Control, Reverse Transcription, Real-time Polymerase Chain Reaction, Binding Assay

Journal: International Journal of Oncology

Article Title: Overcoming acquired doxorubicin resistance of ovarian carcinoma cells by verapamil-mediated promotion of DNA damage-driven cytotoxicity

doi: 10.3892/ijo.2026.5861

Figure Lengend Snippet: Effect of Topo II inhibitors on DNA damage formation and activation of DDR-related mechanisms in A2780 and A2780ADR cells. (A) At 24 h after treatment of logarithmically growing cells with the indicated concentrations of Doxo or Eto, the number of nuclear γH2AX-foci, 53BP1-foci, γH2AX/53BP1 co-localized foci and γH2AX pan-stained cells was analyzed. The upper part of the figure shows representative images (total magnification, ×1,000). Quantitative data depicted in the histogram are the mean ± SD from n=3 independent experiments with each five images being analyzed per experimental condition. * P≤0.05; ** P≤0.01; *** P≤0.001 (A2780 vs. A2780ADR); # P≤0.05; ## P≤0.01; ### P≤0.001 (treated vs. untreated control). Control experiments performed by use of 1st or 2nd antibody only or no antibody at all did not interfere with the signal of main interest (that is, nuclear foci; data not shown). (B) Logarithmically growing cells were treated with the indicated concentrations of Doxo for 24 or 72 h. Afterwards, the protein expression of DDR-related factors was analyzed by western blotting using EKR2 protein expression as loading control. Doxo, doxorubicin; Eto, etoposide; p-, phosphorylated; Chk1/2, checkpoint kinase 1/2; ERK2, extracellular regulated kinase; γH2AX, Ser139 phosphorylated histone H2AX; 2; Kap1, KRAB-associated protein 1; PARP, poly (ADP-ribose) polymerase; p21, cyclin-dependent kinase inhibitor 1; p53, tumor suppressor p53.

Article Snippet: The following primary antibodies were used: Copper transporting ATPase (ATP7A), extracellular regulated kinase 2 (ERK2), phosphorylated (p)-histone H3 (Ser10) from Thermo Fisher Scientific Inc., cleaved caspase-7 (Asp198), p-Chk1 (Ser 345), cyclin B1, galactosidase β (E2U2I), GAPDH (14C10), MDR1/ABCB1 (D3H1Q), p-P53 (S15), PARP, TopBP1(D8G4L), topoisomerase IIa (D10G9), 53BP1 and Ki67 were from Cell Signaling Technology Inc., pChk2 (T68) [Y171], copper uptake protein 1 (CTR1/SLC31A1) [EPR7936] and Rad51 from Abcam, γH2AX (Ser 139) clone JBW301, p-KAP-1 (S824) and p-RPA32 (S4/S8) from Bethyl Laboratories Inc., organic cation transporter-2 (OCT2) from Biozol Diagnostics Vertrieb GmbH, p16 (F-12) and p21 (C-19) from Santa Cruz Biotechnology, Inc. As secondary antibodies, horseradish peroxidase-conjugated secondary antibodies goat anti-mouse IgG and mouse anti-rabbit IgG were used (Rockland Immunochemicals Inc.).

Techniques: Activation Assay, Staining, Control, Expressing, Western Blot

Journal: International Journal of Oncology

Article Title: Overcoming acquired doxorubicin resistance of ovarian carcinoma cells by verapamil-mediated promotion of DNA damage-driven cytotoxicity

doi: 10.3892/ijo.2026.5861

Figure Lengend Snippet: Influence of combined treatment of A2780ADR cells with Doxo and selected inhibitors on mechanism of the DDR and mRNA expression of selected susceptibility-related genes. (A) Logarithmically growing A2780ADR cells were co-treated with the indicated concentrations of Doxo and selected pharmacological inhibitors (concentrations see ) for 24 or 72 h. Afterwards, the protein expression of DDR-related factors was analyzed by western blotting. For loading control, blots were reprobed with ERK2 antibody. (B) Reverse transcription-quantitative PCR of the mRNA expression of selected factors known to contribute to different mechanisms of drug sensitivity. Data shown are mean ± SD from triplicate determinations as described in methods. Relative mRNA level in untreated A2780ADR cells was set to 1.0. Doxo, doxorubicin; DDR, DNA damage response; p-, phosphorylated; nd, not detectable; Bax, Bcl-2 associated protein X; Bcl-2, B-cell lymphoma; BBC3, Bcl-2 binding component 2; BRCA1, 2, breast cancer associated gene 1,2; Cl casp-7, cleaved caspase 7; Chk, checkpoint kinase; CXCL8, chemokine ligand 8 (interleukin 8); p21, CDK inhibitor 1; p16, CDK inhibitor 2; CDKN1A/2A, cyclin dependent kinae inhibitor 1A/2A; CCNB1, Cyclin B1; b-Gal, beta-galactosidase; FASL, FAS ligand; FASR, FAS receptor; GADD, growth arrest and DNA damage inducible GPX1, glutathione peroxidase 1; GSTM1, glutathione S-transferase 1; HMOX1, heme oxygenase 1; γH2AX, Ser139 phosphorylated histone H2AX; p53, tumor suppressor p53; PARP, poly (ADP-ribose) polymerase; PCNA-proliferating cell nuclear antigen; PGC1A, PPARG coactivator 1; PPARGC1A, peroxisome proliferator-activated receptor gamma coactivator 1-alpha; RAD51, radiation damage gene 51; RPAreplication protein A; SOD1, superoxide dismutase 1; Ver, verapamil.

Article Snippet: The following primary antibodies were used: Copper transporting ATPase (ATP7A), extracellular regulated kinase 2 (ERK2), phosphorylated (p)-histone H3 (Ser10) from Thermo Fisher Scientific Inc., cleaved caspase-7 (Asp198), p-Chk1 (Ser 345), cyclin B1, galactosidase β (E2U2I), GAPDH (14C10), MDR1/ABCB1 (D3H1Q), p-P53 (S15), PARP, TopBP1(D8G4L), topoisomerase IIa (D10G9), 53BP1 and Ki67 were from Cell Signaling Technology Inc., pChk2 (T68) [Y171], copper uptake protein 1 (CTR1/SLC31A1) [EPR7936] and Rad51 from Abcam, γH2AX (Ser 139) clone JBW301, p-KAP-1 (S824) and p-RPA32 (S4/S8) from Bethyl Laboratories Inc., organic cation transporter-2 (OCT2) from Biozol Diagnostics Vertrieb GmbH, p16 (F-12) and p21 (C-19) from Santa Cruz Biotechnology, Inc. As secondary antibodies, horseradish peroxidase-conjugated secondary antibodies goat anti-mouse IgG and mouse anti-rabbit IgG were used (Rockland Immunochemicals Inc.).

Techniques: Expressing, Western Blot, Control, Reverse Transcription, Real-time Polymerase Chain Reaction, Binding Assay