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: Immunity, Inflammation and Disease

Article Title: MAFG Induces the Methylation of CRYAB to Promote the Activation of A1 Astrocyte After Spinal Cord Injury

doi: 10.1002/iid3.70334

Figure Lengend Snippet: The activation of A1 astrocyte was accompanied by MAFG upregulation. (A) In spinal cord tissues of rats, C3‐ and S100A10‐positive cells were checked using immunofluorescence. (B) GSE132242 dataset showed that MAFG was overexpressed in mice after SCI ( n = 4). In spinal cord tissues of rats, (C) RT‐PCR was applied to survey MAFG mRNA expression ( n = 6); (D) MAFG‐positive cells were checked using immunofluorescence; (E) the protein expression of MAFG, C3, and S100A10 was surveyed through Western blot ( n = 6). Data in (B, C, and E) were analyzed using the unpaired t ‑test. * p < 0.05, ** p < 0.01.

Article Snippet: For rat astrocytes, after the indicated treatment, the cells were placed in 4% paraformaldehyde for 15 min. After blocking with 10% goat serum for 1 h, cells were incubated with primary antibodies against MAFG, CRYAB (Abcam); C3, S100A10 (Proteintech, China) at 4°C overnight, maintained with secondary antibodies for 60 min, and treated with 4′,6‐diamidino‐2‐phenylindole (DAPI, Sigma‐Aldrich) for 10 min. After dewaxing, hydration, and antigen retrieval, the spinal cord tissue sections were incubated with primary antibodies against MAFG, C3, and S100A10 at 4°C overnight and treated with secondary antibodies for 1 h. Cells in the tissues were also stained.

Techniques: Activation Assay, Immunofluorescence, Reverse Transcription Polymerase Chain Reaction, Expressing, Western Blot

Journal: Immunity, Inflammation and Disease

Article Title: MAFG Induces the Methylation of CRYAB to Promote the Activation of A1 Astrocyte After Spinal Cord Injury

doi: 10.1002/iid3.70334

Figure Lengend Snippet: Silencing of MAFG inhibited the activation of A1 astrocyte and neuroinflammation. In spinal cord tissues of rats, (A) the mRNA expression of C3 and S100A10 was detected using RT‐PCR ( n = 6); (B) the protein expression of C3 and S100A10 was detected using Western blot ( n = 3); (C) the mRNA expression of TNF‐α, IL‐1β, IL‐6, and IL‐10 was detected using RT‐PCR ( n = 6); (D) the protein expression of TNF‐α, IL‐1β, IL‐6, and IL‐10 was detected using Western blot ( n = 3). All data were analyzed using one‑way ANOVA followed by Tukey's post hoc test. * p < 0.05, ** p < 0.01.

Article Snippet: For rat astrocytes, after the indicated treatment, the cells were placed in 4% paraformaldehyde for 15 min. After blocking with 10% goat serum for 1 h, cells were incubated with primary antibodies against MAFG, CRYAB (Abcam); C3, S100A10 (Proteintech, China) at 4°C overnight, maintained with secondary antibodies for 60 min, and treated with 4′,6‐diamidino‐2‐phenylindole (DAPI, Sigma‐Aldrich) for 10 min. After dewaxing, hydration, and antigen retrieval, the spinal cord tissue sections were incubated with primary antibodies against MAFG, C3, and S100A10 at 4°C overnight and treated with secondary antibodies for 1 h. Cells in the tissues were also stained.

Techniques: Activation Assay, Expressing, Reverse Transcription Polymerase Chain Reaction, Western Blot

Journal: Immunity, Inflammation and Disease

Article Title: MAFG Induces the Methylation of CRYAB to Promote the Activation of A1 Astrocyte After Spinal Cord Injury

doi: 10.1002/iid3.70334

Figure Lengend Snippet: MAFG was highly expressed in the activated A1 astrocyte. (A) In rat astrocytes, MAFG positive cells were checked using immunofluorescence. (B and C) In rat astrocytes, MAFG mRNA expression was detected by RT‐PCR ( n = 3). (D and E) In rat astrocytes, the protein expression of MAFG, Serping1, C3, Sphk1, and S100A10 was detected using Western blot ( n = 3). Data in (B, C, D, and E) were analyzed using the unpaired t ‑test. ** p < 0.01.

Article Snippet: For rat astrocytes, after the indicated treatment, the cells were placed in 4% paraformaldehyde for 15 min. After blocking with 10% goat serum for 1 h, cells were incubated with primary antibodies against MAFG, CRYAB (Abcam); C3, S100A10 (Proteintech, China) at 4°C overnight, maintained with secondary antibodies for 60 min, and treated with 4′,6‐diamidino‐2‐phenylindole (DAPI, Sigma‐Aldrich) for 10 min. After dewaxing, hydration, and antigen retrieval, the spinal cord tissue sections were incubated with primary antibodies against MAFG, C3, and S100A10 at 4°C overnight and treated with secondary antibodies for 1 h. Cells in the tissues were also stained.

Techniques: Immunofluorescence, Expressing, Reverse Transcription Polymerase Chain Reaction, Western Blot

Journal: Immunity, Inflammation and Disease

Article Title: MAFG Induces the Methylation of CRYAB to Promote the Activation of A1 Astrocyte After Spinal Cord Injury

doi: 10.1002/iid3.70334

Figure Lengend Snippet: Silencing of MAFG inhibited the activation of A1 astrocyte and neuroinflammation in rat astrocytes. (A and B) In rat astrocytes, the positive cells of C3 and S100A10 were checked using immunofluorescence. (C and D) In rat astrocytes, the mRNA expression of Serping1, C3, Sphk1, and S100A10 were checked using RT‐PCR ( n = 3). (E and F) In rat astrocytes, the protein expression of Serping1, C3, Sphk1, and S100A10 were checked using Western blot ( n = 3). (G and H) In supernatant of rat astrocytes, the levels of IL‐1β and IL‐6 were examined by ELISA ( n = 3). Data in (C, D, E, F, G, and H) were analyzed using one‑way ANOVA followed by Tukey's post hoc test. * p < 0.05, ** p < 0.01.

Article Snippet: For rat astrocytes, after the indicated treatment, the cells were placed in 4% paraformaldehyde for 15 min. After blocking with 10% goat serum for 1 h, cells were incubated with primary antibodies against MAFG, CRYAB (Abcam); C3, S100A10 (Proteintech, China) at 4°C overnight, maintained with secondary antibodies for 60 min, and treated with 4′,6‐diamidino‐2‐phenylindole (DAPI, Sigma‐Aldrich) for 10 min. After dewaxing, hydration, and antigen retrieval, the spinal cord tissue sections were incubated with primary antibodies against MAFG, C3, and S100A10 at 4°C overnight and treated with secondary antibodies for 1 h. Cells in the tissues were also stained.

Techniques: Activation Assay, Immunofluorescence, Expressing, Reverse Transcription Polymerase Chain Reaction, Western Blot, Enzyme-linked Immunosorbent Assay

Journal: Immunity, Inflammation and Disease

Article Title: MAFG Induces the Methylation of CRYAB to Promote the Activation of A1 Astrocyte After Spinal Cord Injury

doi: 10.1002/iid3.70334

Figure Lengend Snippet: Silencing of MAFG inhibited the methylation of CRYAB in vitro which exerted anti‐inflammatory effects. (A) BSP was performed to detect CRYAB methylation (white represented unmethylation, black represented methylation). (B) In rat astrocytes, Western blot was applied to check the changes of key factors in DNA methylation (DNMT1, DNMT3a, and DNMT3b) ( n = 3). (C) In rat astrocytes, the positive cells of CRYAB were checked using immunofluorescence. (D) In rat astrocytes, CRYAB protein expression was checked using Western blot ( n = 3). (E) In rat astrocytes, the mRNA expression of Serping1, C3, Sphk1, and S100A10 was checked by RT‐PCR ( n = 3). (F) In rat astrocytes, the protein expression of Serping1, C3, Sphk1, and S100A10 was checked using Western blot ( n = 3). (G) In supernatant of rat astrocytes, the levels of IL‐1β and IL‐6 were examined by ELISA ( n = 3). Data in (B, D, E, F, and G) were analyzed using one‑way ANOVA followed by Tukey's post hoc test. * p < 0.05, ** p < 0.01.

Article Snippet: For rat astrocytes, after the indicated treatment, the cells were placed in 4% paraformaldehyde for 15 min. After blocking with 10% goat serum for 1 h, cells were incubated with primary antibodies against MAFG, CRYAB (Abcam); C3, S100A10 (Proteintech, China) at 4°C overnight, maintained with secondary antibodies for 60 min, and treated with 4′,6‐diamidino‐2‐phenylindole (DAPI, Sigma‐Aldrich) for 10 min. After dewaxing, hydration, and antigen retrieval, the spinal cord tissue sections were incubated with primary antibodies against MAFG, C3, and S100A10 at 4°C overnight and treated with secondary antibodies for 1 h. Cells in the tissues were also stained.

Techniques: Methylation, In Vitro, Western Blot, DNA Methylation Assay, Immunofluorescence, Expressing, Reverse Transcription Polymerase Chain Reaction, Enzyme-linked Immunosorbent Assay

Journal: Immunity, Inflammation and Disease

Article Title: MAFG Induces the Methylation of CRYAB to Promote the Activation of A1 Astrocyte After Spinal Cord Injury

doi: 10.1002/iid3.70334

Figure Lengend Snippet: Silencing of MAFG inhibited the activation of A1 astrocyte and neuroinflammation via CRYAB methylation. (A and B) In rat astrocytes, the mRNA expression of Serping1, C3, Sphk1, and S100A10 was checked by RT‐PCR ( n = 3). (C and D) In rat astrocytes, the protein expression of Serping1, C3, Sphk1, and S100A10 was checked using Western blot ( n = 3). (E and F) In supernatant of rat astrocytes, the levels of IL‐1β and IL‐6 were examined by ELISA ( n = 3). All data were analyzed using one‑way ANOVA followed by Tukey's post hoc test. * p < 0.05, ** p < 0.01.

Article Snippet: For rat astrocytes, after the indicated treatment, the cells were placed in 4% paraformaldehyde for 15 min. After blocking with 10% goat serum for 1 h, cells were incubated with primary antibodies against MAFG, CRYAB (Abcam); C3, S100A10 (Proteintech, China) at 4°C overnight, maintained with secondary antibodies for 60 min, and treated with 4′,6‐diamidino‐2‐phenylindole (DAPI, Sigma‐Aldrich) for 10 min. After dewaxing, hydration, and antigen retrieval, the spinal cord tissue sections were incubated with primary antibodies against MAFG, C3, and S100A10 at 4°C overnight and treated with secondary antibodies for 1 h. Cells in the tissues were also stained.

Techniques: Activation Assay, Methylation, Expressing, Reverse Transcription Polymerase Chain Reaction, Western Blot, Enzyme-linked Immunosorbent Assay

Journal: Oncotarget

Article Title: Tumor suppressor p53 regulates insulin receptor ( INSR ) gene expression via direct binding to the INSR promoter

doi: 10.18632/oncotarget.27645

Figure Lengend Snippet: ( A ) Total (100 μg protein), cytoplasmic (40 μg) and nuclear (40 μg) fractions of IGF1R-KD, INSR-KD and control MCF7 cells were electrophoresed through SDS-PAGE gels, blotted onto nitrocellulose membranes and incubated with antibodies against total and phospho-p53. Lamin B was used as a nuclear marker. ( B ) Relative phosphorylation of p53 in total and subcellular fractions was calculated by normalizing phospho- to total p53 levels. Data represent two independent experiments (mean ± SEM; * p < 0.01 versus respective fraction in control cells). ( C ) Effect of IGF1R-KD or INSR-KD on p53 sumoylation. Total and subcellular fractions of IGF1R-KD, INSR-KD and control MCF7 cells were immunoprecipitated with anti-p53, electrophoresed and immunoblotted with a Sumo-1 antibody. IgG was used as a control for the co-IP experiment.

Article Snippet: Protein A-peroxidase conjugated was used for p53 detection (Rockland Immunochemicals Inc, Limerick, PA, USA).

Techniques: Control, SDS Page, Incubation, Marker, Phospho-proteomics, Immunoprecipitation, Co-Immunoprecipitation Assay

Journal: Oncotarget

Article Title: Tumor suppressor p53 regulates insulin receptor ( INSR ) gene expression via direct binding to the INSR promoter

doi: 10.18632/oncotarget.27645

Figure Lengend Snippet: IGF1R-KD, INSR-KD and control MCF7 cells were transiently co-transfected with the pGL3(-877/-2) LUC INSR promoter luciferase reporter along with a wild-type or mutant (MUT143, 248 or 273) p53-encoding expression vector. Cells were harvested after 48 h and luciferase activity was measured. The activity of the INSR promoter is expressed as luciferase values normalized to total protein. A value of 100% was assigned to the promoter activity generated by the INSR promoter construct in control cells. The inset presents the basal INSR promoter activity levels at a large scale. * p < 0.01 versus empty vector-transfected cells. Experiments were performed in triplicates.

Article Snippet: Protein A-peroxidase conjugated was used for p53 detection (Rockland Immunochemicals Inc, Limerick, PA, USA).

Techniques: Control, Transfection, Luciferase, Mutagenesis, Expressing, Plasmid Preparation, Activity Assay, Generated, Construct

Journal: Oncotarget

Article Title: Tumor suppressor p53 regulates insulin receptor ( INSR ) gene expression via direct binding to the INSR promoter

doi: 10.18632/oncotarget.27645

Figure Lengend Snippet: ( A ) IGF1R-KD, INSR-KD and control MCF7 cells were co-transfected with an INSR promoter luciferase reporter along with a wild-type p53 expression vector, as described in the legend to Figure 3. After 48 h cells were harvested and the expression of the endogenous INSR was assessed by Western blots. HSC70 was measured as a loading marker. Panels ( B ) and ( C ) show scanning densitometric analyses of INSR and p53 protein levels, respectively, normalized to HSC70. * p < 0.01 versus empty vector-transfected cells.

Article Snippet: Protein A-peroxidase conjugated was used for p53 detection (Rockland Immunochemicals Inc, Limerick, PA, USA).

Techniques: Control, Transfection, Luciferase, Expressing, Plasmid Preparation, Western Blot, Marker

Journal: Oncotarget

Article Title: Tumor suppressor p53 regulates insulin receptor ( INSR ) gene expression via direct binding to the INSR promoter

doi: 10.18632/oncotarget.27645

Figure Lengend Snippet: ( A ) IGF1R-KD, INSR-KD and control MCF7 cells were co-transfected with the pGL3(-877/-2) LUC INSR promoter luciferase reporter along with an INSR-A expression vector (INSR-GFP) (or empty pEGFP vector) and a wild-type p53 expression vector (or empty pCMV). After 48 h cells were harvested and INSR promoter activity was measured as described in the legend to . The left inset presents the basal INSR promoter activity levels at a large scale. Total, cytoplasmic and nuclear fractions of IGF1R-KD cells were immunoprecipitated with an INSR antibody, electrophoresed and immunoblotted with anti-INSR and anti-p53 (right inset). IgG was used as a control for the co-IP experiment. * p < 0.01 versus control cells. § p < 0.01 versus empty pCMV-transfected cells. ( B ) Cells were transfected with the INSR promoter reporter along with an IGF1R expression vector (or empty pEGFP) and a p53 expression vector (or empty pCMV). Cells were processed as described above. Total, cytoplasmic and nuclear fractions of INSR-KD cells were immunoprecipitated with an IGF1R antibody, electrophoresed and immunoblotted with anti-IGF1R and anti-p53 (inset).

Article Snippet: Protein A-peroxidase conjugated was used for p53 detection (Rockland Immunochemicals Inc, Limerick, PA, USA).

Techniques: Control, Transfection, Luciferase, Expressing, Plasmid Preparation, Activity Assay, Immunoprecipitation, Co-Immunoprecipitation Assay

Journal: Oncotarget

Article Title: Tumor suppressor p53 regulates insulin receptor ( INSR ) gene expression via direct binding to the INSR promoter

doi: 10.18632/oncotarget.27645

Figure Lengend Snippet: ( A ) A genomic fragment extending from nt -540 to -18 of the INS R promoter was labeled using a 5′-biotinylated antisense primer. The labeled fragment was bound to streptavidin magnetic beads, incubated with nuclear extracts of IGF1R-KD, INSR-KD or control cells, and eluted with a high-salt buffer. Western blots were performed on nuclear extracts (NE) and eluted material (E) using a p53 antibody. DNA affinity chromatography was also employed to assess the binding of endogenous INSR protein ( B ) and endogenous IGF1R protein ( C ) to the INSR promoter region.

Article Snippet: Protein A-peroxidase conjugated was used for p53 detection (Rockland Immunochemicals Inc, Limerick, PA, USA).

Techniques: Labeling, Magnetic Beads, Incubation, Control, Western Blot, Affinity Chromatography, Binding Assay

Journal: Oncotarget

Article Title: Tumor suppressor p53 regulates insulin receptor ( INSR ) gene expression via direct binding to the INSR promoter

doi: 10.18632/oncotarget.27645

Figure Lengend Snippet: IGF1R-KD, INSR-KD and control MCF7 cells were transiently co-transfected with the pGL3(-877/-2) LUC INSR promoter luciferase reporter along an Sp1 expression vector (or empty pEGFP plasmid) and a wild-type p53 vector (or empty pCMV). Cells were harvested after 48 h and promoter activity was measured. * p < 0.01 versus control cells. § p < 0.01 versus empty pCMV-transfected cells. The left inset presents the basal INSR promoter activity levels in all three cell lines at a large scale. The right inset presents the endogenous levels of Sp1 and p53 proteins in control and KD cells.

Article Snippet: Protein A-peroxidase conjugated was used for p53 detection (Rockland Immunochemicals Inc, Limerick, PA, USA).

Techniques: Control, Transfection, Luciferase, Expressing, Plasmid Preparation, Activity Assay

Journal: Oncotarget

Article Title: Tumor suppressor p53 regulates insulin receptor ( INSR ) gene expression via direct binding to the INSR promoter

doi: 10.18632/oncotarget.27645

Figure Lengend Snippet: IGF1R-KD, INSR-KD and control MCF7 cells were plated in 96-well plates in quadruplicate and, after 24 h, were transfected with p53-WT or empty vector (pCMV). Proliferation was measured after an additional 72 h using an XTT assay. A value of 100% was given to the cell number of control, untreated cells at the end of the incubation period. * p < 0.01 versus respective pCMV-transfected cells.

Article Snippet: Protein A-peroxidase conjugated was used for p53 detection (Rockland Immunochemicals Inc, Limerick, PA, USA).

Techniques: Control, Transfection, Plasmid Preparation, XTT Assay, Incubation

Journal: Oncotarget

Article Title: Tumor suppressor p53 regulates insulin receptor ( INSR ) gene expression via direct binding to the INSR promoter

doi: 10.18632/oncotarget.27645

Figure Lengend Snippet: IGF1R-KD, INSR-KD and control MCF7 cells were seeded in triplicate onto 6-well plates and, after 24 h, were transfected with p53-WT (or empty pCMV vector). After an additional 24 h, the cells were tripsinized, counted and plated again (in 6-well plates in triplicate, 10 5 cells/well) for 72 h. Cells were then permeabilized with Triton X-100, stained with propidium iodide and analyzed using a FacsCalibur system. * p < 0.01 versus respective pCMV-transfected cells.

Article Snippet: Protein A-peroxidase conjugated was used for p53 detection (Rockland Immunochemicals Inc, Limerick, PA, USA).

Techniques: Control, Transfection, Plasmid Preparation, Staining