Journal: Antioxidants

Article Title: Saposhnikovia divaricata Inhibits Inflammation, Oxidative Stress, and Ferroptosis to Alleviate DSS-Induced Ulcerative Colitis

doi: 10.3390/antiox15020258

Figure Lengend Snippet: Network pharmacology analysis identifies p53 as a core ferroptosis-related target of FF in UC. ( A ) Venn diagram illustrating the intersection of FF compound targets with ferroptosis- and UC-related targets. ( B ) Protein–protein interaction (PPI) network of the common targets. Node size and color intensity represent the degree of connectivity, with TP53 (p53) identified as the core target. ( C ) Compound-target-pathway network diagram. The inner pink nodes represent the 38 intersecting targets linking FF, UC, and ferroptosis. ( D ) Gene Ontology (GO) enrichment analysis of the common targets, categorized into Biological Process (BP, red), Cellular Component (CC, green), and Molecular Function (MF, blue). ( E ) Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis.

Article Snippet: The p53 antibody was obtained from Boster Biological Technology Co., Ltd. (Pleasanton, CA, USA, Item #BM0101).

Techniques:

Journal: Antioxidants

Article Title: Saposhnikovia divaricata Inhibits Inflammation, Oxidative Stress, and Ferroptosis to Alleviate DSS-Induced Ulcerative Colitis

doi: 10.3390/antiox15020258

Figure Lengend Snippet: FF modulates the expression of ferroptosis-related proteins in colon tissue via the p53 pathway. ( A ) Representative immunohistochemical (IHC) images of p53, SLC7A11, and GPX4 expression in colon sections (scale bar = 50 μm). ( B – D ) Quantitative analysis of the relative protein expression levels of p53 (B), SLC7A11 (C), and GPX4 (D). Data are presented as the mean ± SD ( n = 3 independent experiments). ### p < 0.001 versus the control (CON) group; * p < 0.05, ** p < 0.01, *** p < 0.001 versus the DSS model group.

Article Snippet: The p53 antibody was obtained from Boster Biological Technology Co., Ltd. (Pleasanton, CA, USA, Item #BM0101).

Techniques: Expressing, Immunohistochemical staining, Control

Journal: Aging cell

Article Title: Positive crosstalk between arginase-II and S6K1 in vascular endothelial inflammation and aging.

doi: 10.1111/acel.12001

Figure Lengend Snippet: Fig. 1 Silencing Arg-II in senescent cells inhibits eNOS uncoupling, reverses endothelial senescent phenotypic changes, and suppresses endothelial inflammation. Senescent human umbilical vein endothelial cells (HUVECs) were transduced with rAd ⁄ U6-LacZshRNA as control (con) or rAd ⁄ U6-Arg-IIshRNA to silence Arg-II gene. (A) Immunoblotting shows Arg-II silencing in senescent cells. (B) DHE staining for detection of O 2 and DAF-2DA staining for detection of NO. Quantifications of DHE and DAF-2DA signals are shown below. (C) SA-b-gal staining. Bar graphs show quantifications of SA-b-gal-positive cells. (D) Immunoblotting analysis of senescence markers p53-S15, p53, and p21Cip1

Article Snippet: Antibody against p21Cip1 (OP64) was purchased from Calbiochem (Genève, Switzerland); antibody against phosphor-p53-S15 (#9284s) was from Cell Signalling (Allschwil, Switzerland); Antibodies against S6K1 (#9205s) were from BD Transduction laboratories (Allschwil, Switzerland); antibodies against arginase-II (sc-20151) and p53 (sc-6243) were from SantaCruz (Nunningen, Switzerland); anti-SOD1 (TA302692) was from OriGene Technologies, Inc (Nunningen, Switzerland); Monoclonal antibody against HA-tag (12CA5) was obtained from Dr Brian A. Hemmings (Friedrich Miescher Institute for Biomedical Research, Basel, Switzerland); Alexa Fluor680-conjugated anti-mouse IgG (A21057) and dihydroethidium (DHE) were from Molecular Probes ⁄ Invitrogen (Lucerne, Switzerland); IRDye800-conjugated anti-rabbit IgG (926-32211) were from LI-COR Biosciences (Bad Homburg, Germany); the membrane-permeable 4,5- diaminofluoresceine acetate (DAF-2DA) was from VWR international SA (Dietikon, Switzerland); X-gal was from Promega (Dübendorf, Switzerland); Endothelial cell growth supplement (ECGS) pack was from PromoCell GmbH (Allschwil, Switzerland) and all cell culture media and materials were purchased from Gibco BRL (Lucerne, Switzerland).

Techniques: Transduction, Control, Western Blot, Staining

Journal: Aging cell

Article Title: Positive crosstalk between arginase-II and S6K1 in vascular endothelial inflammation and aging.

doi: 10.1111/acel.12001

Figure Lengend Snippet: Fig. 2 Co-expression of superoxide dismutase-1 (SOD1) in young endothelial cells prevents Arg-II-induced eNOS-uncoupling, endothelial senescence and inflammation. Young endothelial cells were transduced with empty rAd ⁄ CMV vector as control (con) or rAd ⁄ CMV-Arg-II alone or rAd ⁄ CMV-Arg-II plus rAd ⁄ CMV-SOD1. (A) Immunoblotting analysis to confirm overexpression of Arg-II and SOD1. (B) DHE staining for detection of O 2 and DAF-2DA staining for detection of NO and effect of SOD1. Bar graphs show quantifications of DHE and DAF-2DA signals. (C) SA-b-gal staining and effect of SOD1. Bar graphs show quantifications of percentage of SA-b-gal positive cells. (D) Immunoblotting analysis of senescence markers p53-S15, p53, and p21Cip1 levels, and endothelial inflammation markers vascular adhesion molecule-1 (VCAM1) and intercellular adhesion molecule-1 (ICAM1) expression. Tubulin served as loading control. Bar graphs show quantifications of the markers. (E) CFDA-SE fluorescence labeled THP-1 monocyte adhesion to endothelial cells that were transduced with rAd expressing transgenes as indicated. Bar graphs show quantifications of the adhered monocytes. *P < 0.05, **P < 0.01 and ***P < 0.005 vs. control (con); ††<0.01 and †††P < 0.005 vs. Arg-II. Scale bar = 0.2 mm.

Article Snippet: Antibody against p21Cip1 (OP64) was purchased from Calbiochem (Genève, Switzerland); antibody against phosphor-p53-S15 (#9284s) was from Cell Signalling (Allschwil, Switzerland); Antibodies against S6K1 (#9205s) were from BD Transduction laboratories (Allschwil, Switzerland); antibodies against arginase-II (sc-20151) and p53 (sc-6243) were from SantaCruz (Nunningen, Switzerland); anti-SOD1 (TA302692) was from OriGene Technologies, Inc (Nunningen, Switzerland); Monoclonal antibody against HA-tag (12CA5) was obtained from Dr Brian A. Hemmings (Friedrich Miescher Institute for Biomedical Research, Basel, Switzerland); Alexa Fluor680-conjugated anti-mouse IgG (A21057) and dihydroethidium (DHE) were from Molecular Probes ⁄ Invitrogen (Lucerne, Switzerland); IRDye800-conjugated anti-rabbit IgG (926-32211) were from LI-COR Biosciences (Bad Homburg, Germany); the membrane-permeable 4,5- diaminofluoresceine acetate (DAF-2DA) was from VWR international SA (Dietikon, Switzerland); X-gal was from Promega (Dübendorf, Switzerland); Endothelial cell growth supplement (ECGS) pack was from PromoCell GmbH (Allschwil, Switzerland) and all cell culture media and materials were purchased from Gibco BRL (Lucerne, Switzerland).

Techniques: Expressing, Transduction, Plasmid Preparation, Control, Western Blot, Over Expression, Staining, Labeling

Journal: Aging cell

Article Title: Positive crosstalk between arginase-II and S6K1 in vascular endothelial inflammation and aging.

doi: 10.1111/acel.12001

Figure Lengend Snippet: Fig. 5 Silencing Arg-II prevents S6K1-induced eNOS-uncoupling, senescence and inflammation in young endothelial cells. The transduction procedure of young cells was the same as in Fig. 5A. (A) DHE staining for detection of O 2 and DAF-2DA staining for detection of NO and effect of Arg-II silencing. Bar graphs show quantifications of DHE and DAF-2DA signals. (B) SA-b-gal staining and effect of Arg-II silencing. Bar graphs show quantifications of percentage of SA-b-gal positive cells. (C) Immunoblotting analysis of senescence markers p53-S15, p53, and p21Cip1 levels, and endothelial inflammation markers vascular adhesion molecule-1 (VCAM1) and intercellular adhesion molecule-1 (ICAM1). Tubulin served as loading control. Bar graphs show quantifications of the markers. (D) CFDA-SE fluorescence labeled THP-1 monocyte adhesion to endothelial cells that were transduced with rAd expressing transgenes and shRNA as indicated. Bar graphs show quantifications of the adhered monocytes. **P < 0.01, ***P < 0.005 vs. control (con ⁄ LacZ); †P < 0.05, ††P < 0.01, †††P < 0.005 vs. S6K1ca ⁄ LacZ group. Scale bar = 0.2 mm.

Article Snippet: Antibody against p21Cip1 (OP64) was purchased from Calbiochem (Genève, Switzerland); antibody against phosphor-p53-S15 (#9284s) was from Cell Signalling (Allschwil, Switzerland); Antibodies against S6K1 (#9205s) were from BD Transduction laboratories (Allschwil, Switzerland); antibodies against arginase-II (sc-20151) and p53 (sc-6243) were from SantaCruz (Nunningen, Switzerland); anti-SOD1 (TA302692) was from OriGene Technologies, Inc (Nunningen, Switzerland); Monoclonal antibody against HA-tag (12CA5) was obtained from Dr Brian A. Hemmings (Friedrich Miescher Institute for Biomedical Research, Basel, Switzerland); Alexa Fluor680-conjugated anti-mouse IgG (A21057) and dihydroethidium (DHE) were from Molecular Probes ⁄ Invitrogen (Lucerne, Switzerland); IRDye800-conjugated anti-rabbit IgG (926-32211) were from LI-COR Biosciences (Bad Homburg, Germany); the membrane-permeable 4,5- diaminofluoresceine acetate (DAF-2DA) was from VWR international SA (Dietikon, Switzerland); X-gal was from Promega (Dübendorf, Switzerland); Endothelial cell growth supplement (ECGS) pack was from PromoCell GmbH (Allschwil, Switzerland) and all cell culture media and materials were purchased from Gibco BRL (Lucerne, Switzerland).

Techniques: Transduction, Staining, Western Blot, Control, Labeling, Expressing, shRNA

Journal: Aging cell

Article Title: Positive crosstalk between arginase-II and S6K1 in vascular endothelial inflammation and aging.

doi: 10.1111/acel.12001

Figure Lengend Snippet: Fig. 6 Deficiency in Arg-II gene in mice (Arg-II) ⁄ )) protects against vascular inflammation and aging. Aortas of young (2–3 months) and old (23–24 months) wild-type (WT) and Arg-II) ⁄ ) mice were cleaned of perivascular tissues and subjected to en face staining or Immunoblotting analysis. (A) qRT-PCR analysis of Arg-II mRNA levels. (B) Confocal microscopic en face detection of endothelial vascular adhesion molecule-1 (VCAM1) and intercellular adhesion molecule-1 (ICAM1), vWF (the endothelial marker), followed by counterstaining with DAPI. Shown are representative images of each group. (C) Immunoblotting analyses of VCAM1, ICAM1, p21 levels in the aortas and p53-Ser15 and p53 levels in the heart of young and old WT and Arg-II) ⁄ ) mice. (D and E) Quantifications of the above results. Tubulin is taken as loading control. n = 4 mice in each group. **P < 0.01, ***P < 0.001 vs. young WT mice; †P < 0.05, ††P < 0.01, †††P < 0.001 vs. old WT mice. Scale bar = 100 lm.

Article Snippet: Antibody against p21Cip1 (OP64) was purchased from Calbiochem (Genève, Switzerland); antibody against phosphor-p53-S15 (#9284s) was from Cell Signalling (Allschwil, Switzerland); Antibodies against S6K1 (#9205s) were from BD Transduction laboratories (Allschwil, Switzerland); antibodies against arginase-II (sc-20151) and p53 (sc-6243) were from SantaCruz (Nunningen, Switzerland); anti-SOD1 (TA302692) was from OriGene Technologies, Inc (Nunningen, Switzerland); Monoclonal antibody against HA-tag (12CA5) was obtained from Dr Brian A. Hemmings (Friedrich Miescher Institute for Biomedical Research, Basel, Switzerland); Alexa Fluor680-conjugated anti-mouse IgG (A21057) and dihydroethidium (DHE) were from Molecular Probes ⁄ Invitrogen (Lucerne, Switzerland); IRDye800-conjugated anti-rabbit IgG (926-32211) were from LI-COR Biosciences (Bad Homburg, Germany); the membrane-permeable 4,5- diaminofluoresceine acetate (DAF-2DA) was from VWR international SA (Dietikon, Switzerland); X-gal was from Promega (Dübendorf, Switzerland); Endothelial cell growth supplement (ECGS) pack was from PromoCell GmbH (Allschwil, Switzerland) and all cell culture media and materials were purchased from Gibco BRL (Lucerne, Switzerland).

Techniques: Staining, Western Blot, Quantitative RT-PCR, Marker, Control

Journal: Journal of Cancer

Article Title: Mutant Pattern of p53 as a Feasible Predictor of Distant Metastasis Following Curative Gastrectomy for Advanced-stage Gastric Cancer

doi: 10.7150/jca.98563

Figure Lengend Snippet: Clinical characteristics of the patients with p53 staining patterns.

Article Snippet: IHC was performed using primary antibody for p53 (clone DO-7, cat. no. ZM0408, 1:200 dilution; Zhongshan GoldenBridge Biotechnology, Beijing, China) and BOND-III autostainer (Leica Biosystems Nussloch GmbH, Nussloch, Germany).

Techniques: Staining, Mutagenesis

Journal: Journal of Cancer

Article Title: Mutant Pattern of p53 as a Feasible Predictor of Distant Metastasis Following Curative Gastrectomy for Advanced-stage Gastric Cancer

doi: 10.7150/jca.98563

Figure Lengend Snippet: Recurrence patterns involving both p53 mutant and wild-type patterns.

Article Snippet: IHC was performed using primary antibody for p53 (clone DO-7, cat. no. ZM0408, 1:200 dilution; Zhongshan GoldenBridge Biotechnology, Beijing, China) and BOND-III autostainer (Leica Biosystems Nussloch GmbH, Nussloch, Germany).

Techniques: Mutagenesis

Journal: Journal of Cancer

Article Title: Mutant Pattern of p53 as a Feasible Predictor of Distant Metastasis Following Curative Gastrectomy for Advanced-stage Gastric Cancer

doi: 10.7150/jca.98563

Figure Lengend Snippet: Overall survival and recurrence-free survival of patients with both the p53 wild-type and mutant patterns. Kaplan-Meier curves for (A) overall survival and (B) recurrence-free survival over a period of 5 years with the p53 wild-type and p53 mutant pattern. Patients with the p53 mutant pattern had low (A) overall survival and (B) recurrence-free survival rates in all patients. In subgroup analysis, the recurrence-free survival rate was lower in patients with the p53 mutant pattern than in those with the wild-type pattern as regards both (C) pN0 or (D) pN+, and (E) early and (F) advanced-stage gastric cancer.

Article Snippet: IHC was performed using primary antibody for p53 (clone DO-7, cat. no. ZM0408, 1:200 dilution; Zhongshan GoldenBridge Biotechnology, Beijing, China) and BOND-III autostainer (Leica Biosystems Nussloch GmbH, Nussloch, Germany).

Techniques: Mutagenesis

Journal: Journal of Cancer

Article Title: Mutant Pattern of p53 as a Feasible Predictor of Distant Metastasis Following Curative Gastrectomy for Advanced-stage Gastric Cancer

doi: 10.7150/jca.98563

Figure Lengend Snippet: Comparison of the characteristics of patients with early- and advanced-stage gastric cancer in association with the p53 wild-type and p53 mutant pattern.

Article Snippet: IHC was performed using primary antibody for p53 (clone DO-7, cat. no. ZM0408, 1:200 dilution; Zhongshan GoldenBridge Biotechnology, Beijing, China) and BOND-III autostainer (Leica Biosystems Nussloch GmbH, Nussloch, Germany).

Techniques: Comparison, Mutagenesis

Journal: Journal of Cancer

Article Title: Mutant Pattern of p53 as a Feasible Predictor of Distant Metastasis Following Curative Gastrectomy for Advanced-stage Gastric Cancer

doi: 10.7150/jca.98563

Figure Lengend Snippet: Clinical characteristics of the patients with p53 staining patterns before and after matching on the propensity score.

Article Snippet: IHC was performed using primary antibody for p53 (clone DO-7, cat. no. ZM0408, 1:200 dilution; Zhongshan GoldenBridge Biotechnology, Beijing, China) and BOND-III autostainer (Leica Biosystems Nussloch GmbH, Nussloch, Germany).

Techniques: Staining, Mutagenesis

Journal: Journal of Cancer

Article Title: Mutant Pattern of p53 as a Feasible Predictor of Distant Metastasis Following Curative Gastrectomy for Advanced-stage Gastric Cancer

doi: 10.7150/jca.98563

Figure Lengend Snippet: Comparison of patient characteristics between pN0 and pN+ gastric cancer in p53 wild-type and mutant pattern.

Article Snippet: IHC was performed using primary antibody for p53 (clone DO-7, cat. no. ZM0408, 1:200 dilution; Zhongshan GoldenBridge Biotechnology, Beijing, China) and BOND-III autostainer (Leica Biosystems Nussloch GmbH, Nussloch, Germany).

Techniques: Comparison, Mutagenesis

Journal: Journal of cellular and molecular medicine

Article Title: Functional role of RRS1 in breast cancer cell proliferation.

doi: 10.1111/jcmm.13922

Figure Lengend Snippet: FIGURE 5 P53 and p21 are induced by RRS1 knockdown. MCF‐ 7 cells were infected with a retrovirus expressing RRS1 (shRRS1) or with a Ctrl vector (shctrl) for 2 days. Whole‐cell lysates were analysed by Western blot. P53 and p21 expression levels were increased by RRS1 knockdown (*P < 0.05 vs shctrl)

Article Snippet: For western blotting, xenograft tumors and cell lines were lysed, and protein samples were harvested as previously described.29 Equal amounts of protein were resolved by SDS‐PAGE and blotted using antibodies specific to RRS1 (1:1000, Abcam), p53 (1:500, OriGene), RPL11 (1:1000, Abcam) and β‐actin (1:1000, Bioss, Beijing, China).

Techniques: Knockdown, Infection, Expressing, Plasmid Preparation, Western Blot

Journal: The Journal of thoracic and cardiovascular surgery

Article Title: Loss of p53, rather than beta-catenin overexpression, induces survivin-mediated resistance to apoptosis in an esophageal cancer cell line.

doi: 10.1016/j.jtcvs.2009.11.038

Figure Lengend Snippet: FIGURE 4. TE7 cells exhibit a loss of p53 with resulting increased survi- vin transcription. A, Representative immunoblots of p53 protein expression in nhEso and TE7 cells. B, Schematic representation of the survivin pro- moter-luciferase reporter construct that included 2 TCF-b-catenin binding

Article Snippet: Co-transfection assays were carried out in nhEso and TE7 cells using 0.3 mg of survivin luciferase reporter gene plasmid, 0.3 mg of either an empty expression vector or an overexpression vector encoding the tumor protein p53 (OriGene Technologies Inc, Rockville, Md), in 0.7 mL Lipofectamine reagent.

Techniques: Western Blot, Expressing, Luciferase, Construct, Binding Assay

Journal: The Journal of thoracic and cardiovascular surgery

Article Title: Loss of p53, rather than beta-catenin overexpression, induces survivin-mediated resistance to apoptosis in an esophageal cancer cell line.

doi: 10.1016/j.jtcvs.2009.11.038

Figure Lengend Snippet: FIGURE 5. Effect of p53 overexpression on survivin promoter activity and survivin mRNA transcription in nhEso and TE7 cells. A, Reporter gene activity in nhEso and TE7 cells after overexpression of p53. Cells were co-transfected with the survivin reporter construct and the expression vector containing human p53 cDNA (p53) or control vector lacking p53 (control) using Lipofectamine (Invitrogen, Carlsbad, Calif). Twenty-four

Article Snippet: Co-transfection assays were carried out in nhEso and TE7 cells using 0.3 mg of survivin luciferase reporter gene plasmid, 0.3 mg of either an empty expression vector or an overexpression vector encoding the tumor protein p53 (OriGene Technologies Inc, Rockville, Md), in 0.7 mL Lipofectamine reagent.

Techniques: Over Expression, Activity Assay, Transfection, Construct, Expressing, Plasmid Preparation, Control

Journal: Archives of Biological Sciences

Article Title: Quercetin and lithium chloride modulate Wnt signaling in pluripotent embryonal carcinoma NT2/D1 cells

doi: 10.2298/abs1301201m

Figure Lengend Snippet: Fig. 2. LiCl decreases the proliferation rate of NT2/D1 cells in a p53-dependent manner. (A) MTT proliferation assay of NT2/D1 treated with lithium chloride-7, 10 and 20 mM, for 24 h. The results are shown as per- centages of the negative control, untreated NT2/D1 cells. Values are presented as the means ±S.E.M. of at least three independent experiments. Mean values of relative proliferation rates were compared using Student’s t test. Values of p<0, 05 are presented by *. (B) Western blot analysis of p53 protein expression in NT2/ D1 cells treated with 7, 10 and 20 mM lithium chloride for 24 h. The level of GAPDH was used as a control for equal amounts of input proteins.

Article Snippet: Western blots were performed using anti c-myc (9E10) (Santa Cruz Biotechnology), anti-p53 (DO1) (Gene Spin), anti α-tubulin (DM1A) (Calbi- ochem), and anti-GAPDH (AM20337PU-S) (Acris Antibodies, Inc).

Techniques: Proliferation Assay, Negative Control, Western Blot, Expressing, Control

Journal: Cellular physiology and biochemistry : international journal of experimental cellular physiology, biochemistry, and pharmacology

Article Title: RASSF10 is Epigenetically Inactivated and Suppresses Cell Proliferation and Induces Cell Apoptosis by Activating the p53 Signalling Pathway in Papillary Thyroid Carcinoma Cancer.

doi: 10.1159/000464386

Figure Lengend Snippet: Fig. 5. RASSF10 and p53 regulated PTC cell apoptosis. Apoptosis was determined using flow cytometry analysis of Annexin V/PI double-stained K1 cells. *P < 0.05 versus control group, #P < 0.05 versus RASSF10 group.

Article Snippet: Expression vector construction and transfection The open reading frame (ORF) of the RASSF10 gene was generated by RT-PCR, and p53 siRNA and shRASSF10 were purchased from OriGene Technologies (Rockville, USA).

Techniques: Flow Cytometry, Staining, Control

Journal: Translational Cancer Research

Article Title: ALOX12 suppresses colon cancer progression by promoting p53-mediated ferroptosis through upregulating ROS-induced stress

doi: 10.21037/tcr-2026-0503

Figure Lengend Snippet: ALOX12 is essential for p53-mediated ferroptosis in CC. (A) Western blotting and qRT-PCR analysis of HCT116 and DLD-1 cells incubated with Nutlin-3 (0, 10, 15, 20, 30, 40 µM) for 24 h. Mean ± SD, n=3 independent experiments. (B) Representative phase-contrast images of HCT116 and DLD-1 cells. HCT116 cells were incubated with Nutlin-3 (10 µM), TBH (200 µM), ML355 (2 µM) or Fer-1 (2 µM) for 24 h. DLD-1 cells were incubated with Nutlin-3 (10 µM), TBH (100 µM), ML355 (2 µM) or Fer-1 (2 µM) for 24 h. Scale bars, 100 µm. N=3 independent experiments. (C) Cell death analysis of HCT116 and DLD-1 cells. HCT116 cells incubated with Nutlin-3 (10 µM), TBH (200 µM), ML355 (2 µM), Fer-1 (2 µM), 3-MA (2 mM), Z-VAD (10 µM), or Nec-1 (10 µM) for 24 h, while DLD-1 cells were incubated with Nutlin-3 (10 µM), TBH (100 µM), ML355 (2 µM), Fer-1 (2 µM), 3-MA (2 mM), Z-VAD (10 µM), or Nec-1 (10 µM) for 24 h. Mean ± SD, n=3 independent experiments. (D-F) ROS (D), MDA (E) and Fe 2+ (F) detection of HCT116 and DLD-1 cells. HCT116 cells were incubated with Nutlin-3 (10 µM), TBH (200 µM), ML355 (2 µM) or Fer-1 (2 µM) for 24 h, while DLD-1 cells were incubated with Nutlin-3 (10 µM), TBH (100 µM), ML355 (2 µM) or Fer-1 (2 µM) for 24 h. Mean ± SD, n=3 independent experiments. ** P<0.01; ****, P<0.0001; ns, not significant. CC, colon cancer; MDA, malondialdehyde; qRT-PCR, quantitative reverse transcription polymerase chain reaction; ROS, reactive oxygen species; SD, standard deviation; TBH, tert-Butyl hydroperoxide.

Article Snippet: Protein extracts were analyzed by western blotting according to standard protocols, using primary antibodies against p53 (A0263, Abclonal, Wuhan, China, 1:1,000), SLC7A11 (CY7046, Abways, Shanghai, China, 1:1,000), ALOX12 (CY8070, Abways, 1:1,000), GAPDH (AB0037, Abways, 1:5,000), and GPX4 (A11243, Abclonal, 1:1,000), followed by an HRP-conjugated goat anti-rabbit secondary antibody (AS014, Abclonal, 1:5,000).

Techniques: Western Blot, Quantitative RT-PCR, Incubation, Reverse Transcription, Polymerase Chain Reaction, Standard Deviation

Journal: Translational Cancer Research

Article Title: ALOX12 suppresses colon cancer progression by promoting p53-mediated ferroptosis through upregulating ROS-induced stress

doi: 10.21037/tcr-2026-0503

Figure Lengend Snippet: Mechanisms into the regulation of ALOX12 in p53-mediated ferroptosis. (A) Western blotting analysis of HCT116, DLD-1 cells transfected with ALOX12 lentivirus or si-ALOX12 for 24 h, the expression of ALOX12, SLC7A11 and p53 was detected. Mean ± SD, n=3 independent experiments. (B) Western blotting analysis of HCT116 and DLD-1 cells transfected with si-p53 for 24 h, the expression of ALOX12, SLC7A11 and p53 was detected. Mean ± SD, n=3 independent experiments. (C) Western blotting analysis of HCT116 and DLD-1 cells treated with Nutlin-3 (10 µM) or co-transfected with si-p53 for 24 h, the expression of ALOX12, SLC7A11 and p53 was detected. Mean ± SD, n=3 independent experiments. (D) Cell death analysis of HCT116 and DLD-1 cells. HCT116 cells were incubated with erastin (40 µM) or co-treated with Fer-1 (2 µM) for 24 h, while DLD-1 cells were treated with erastin (60 µM) or co-treated with Fer-1 (2 µM) for 24 h. Mean ± SD, n=3 independent experiments. (E-G) ROS (E), MDA (F) and Fe 2+ (G) detection of HCT116 and DLD-1 cells. HCT116 cells were incubated with erastin (40 µM) or co-treated with Fer-1 (2 µM) for 24 h, while DLD-1 cells were treated with erastin (60 µM) or co-treated with Fer-1 (2 µM) for 24 h. Mean ± SD, n=3 independent experiments. (H) Cell death analysis of HCT116 and DLD-1 cells when considering ALOX12 status. HCT116 cells were incubated with erastin (40 µM) or co-treated with Fer-1 (2 µM) for 24 h, while DLD-1 cells were treated with erastin (60 µM) or co-treated with Fer-1 (2 µM) for 24 h. Mean ± SD, n=3 independent experiments. (I) Co-IP assay of HCT116 and DLD-1 cells using SLC7A11 antibody (see methods). N=3 independent experiments. (J) Co-IP assay of HCT116 and DLD-1 cells using anti-Flag antibody (see Methods). N=3 independent experiments. (K) ALOX12 enzyme activity analysis of HCT116 and DLD-1 cells by ELISA (see Methods), cells were pre-treated with ML355 (2 µM), Nutlin-3 (10 µM) or co-transfected with si-p53 for 24 h. Mean ± SD, n=3 independent experiments. (L,M) MDA (L) and Fe 2+ (M) detection of HCT and DLD-1 cells. HCT116 cells were incubated with Nutlin-3 (10 µM), TBH (200 µM), Lip-1 (2 µM) or transfected with si-p53 for 24 h, while DLD-1 cells were treated with Nutlin-3 (10 µM), TBH (100 µM), Lip-1 (2 µM) or transfected with si-p53 for 24 h. Mean ± SD, n=3 independent experiments. *, P<0.05; ** P<0.01; *** P<0.001; ****, P<0.0001; ns, not significant. Co-IP, co-immunoprecipitation; ELISA, enzyme-linked immunosorbent assay; MDA, malondialdehyde; ROS, reactive oxygen species; SD, standard deviation; TBH, tert-Butyl hydroperoxide.

Article Snippet: Protein extracts were analyzed by western blotting according to standard protocols, using primary antibodies against p53 (A0263, Abclonal, Wuhan, China, 1:1,000), SLC7A11 (CY7046, Abways, Shanghai, China, 1:1,000), ALOX12 (CY8070, Abways, 1:1,000), GAPDH (AB0037, Abways, 1:5,000), and GPX4 (A11243, Abclonal, 1:1,000), followed by an HRP-conjugated goat anti-rabbit secondary antibody (AS014, Abclonal, 1:5,000).

Techniques: Western Blot, Transfection, Expressing, Incubation, Co-Immunoprecipitation Assay, Activity Assay, Enzyme-linked Immunosorbent Assay, Immunoprecipitation, Standard Deviation

Journal: Evidence-based complementary and alternative medicine : eCAM

Article Title: Curcumin Alleviates D-Galactose-Induced Cardiomyocyte Senescence by Promoting Autophagy via the SIRT1/AMPK/mTOR Pathway.

doi: 10.1155/2022/2990843

Figure Lengend Snippet: Figure 1: Curcumin inhibited D-galactose-induced senescence and decreased ROS in cardiomyocytes. (a) Primary neonatal rat car- diomyocytes were treated with D-gal (10 g/l). )e suppression effect of curcumin on primary neonatal rat cardiomyocyte senescence shown by SA-β-galactosidase staining (n 5). Scale bar: 100 μm. (b) SA-β-galactosidase-positive cells. (c) Curcumin inhibited the expression of the senescence markers p53 and p16 in senescent cardiomyocytes, as observed using western blot (n 5). (d) p53 expression. (e) p16 expression. (f) Curcumin reduces intracellular ROS levels in cardiomyocytes as observed using the DCFH-DA probe staining, n 5, scale bar: 100 μm. (g) Statistical results for ROS production. ∗∗P < 0.01 vs. control. #P < 0.05, # #P < 0.01, vs. D-gal. )e data are expressed as mean ± SEM.

Article Snippet: See the T erm s and C onditions (https://onlinelibrary.w iley.com /term s-and-conditions) on W iley O nline L ibrary for rules of use; O A articles are governed by the applicable C reative C om m ons L icense Technology, USA), phospho-AMPK (1 :1000; Cell Signaling Technology, USA), SIRT1 (1 : 1000; Cell Signaling Technology, USA), P53 (1 : 500; Abclonal, China, A19585), P16 (1 : 2000; Abcam, USA), LC3I/II (1 : 2000; Abcam, USA), P62 (1 : 1000; Cell Signaling Technology, USA), and GAPDH (1 :1000; Cell Signaling Technology, USA).)e immunoreactive proteins were observed using the enhanced chemiluminescence method (ECL) with Millipore’s electroluminescent solution (USA) and analyzed using ImageJ (USA).

Techniques: Staining, Expressing, Western Blot, Control

Journal: Evidence-based complementary and alternative medicine : eCAM

Article Title: Curcumin Alleviates D-Galactose-Induced Cardiomyocyte Senescence by Promoting Autophagy via the SIRT1/AMPK/mTOR Pathway.

doi: 10.1155/2022/2990843

Figure Lengend Snippet: Figure 4: siSIRT1 blocked the antiaging and antioxidant effect of curcumin in D-galactose-induced senescent cardiomyocytes. (a) Repre- sentative SA-β-galactosidase staining images showing that SIRT1 knockdown inhibited the antiaging effects of curcumin in senescent car- diomyocytes, as observed through the increased number of SA-β-galactosidase positive cells (n 5). Scale bar: 100 μm. (b) Results of the statistics of SA-β-galactosidase staining. (c) Representative western blot of P53 and p16 (n 5). (d) Quantitation results of p53. (e) Quantitation results of p16. (f) SIRT1 siRNA blocked the antioxidant effect of curcumin in D-galactose-induced senescent cardiomyocytes. Representative images of DCFH-DA staining indicated that ROS production in senescent cardiomyocytes is reduced because of the effect of curcumin (n 5). Scale bar: 100 μm. (g) Statistical results for ROS production. #P < 0.05, # #P < 0.01, vs. control siRNA. )e data are expressed as the mean ± SEM.

Article Snippet: See the T erm s and C onditions (https://onlinelibrary.w iley.com /term s-and-conditions) on W iley O nline L ibrary for rules of use; O A articles are governed by the applicable C reative C om m ons L icense Technology, USA), phospho-AMPK (1 :1000; Cell Signaling Technology, USA), SIRT1 (1 : 1000; Cell Signaling Technology, USA), P53 (1 : 500; Abclonal, China, A19585), P16 (1 : 2000; Abcam, USA), LC3I/II (1 : 2000; Abcam, USA), P62 (1 : 1000; Cell Signaling Technology, USA), and GAPDH (1 :1000; Cell Signaling Technology, USA).)e immunoreactive proteins were observed using the enhanced chemiluminescence method (ECL) with Millipore’s electroluminescent solution (USA) and analyzed using ImageJ (USA).

Techniques: Staining, Knockdown, Western Blot, Quantitation Assay, Control