Journal: Antioxidants (Basel, Switzerland)

Article Title: Inhibition of Oxidative Stress and ALOX12 and NF-κB Pathways Contribute to the Protective Effect of Baicalein on Carbon Tetrachloride-Induced Acute Liver Injury.

doi: 10.3390/antiox10060976

Figure Lengend Snippet: Figure 2. Protein expression levels involved ferroptosis were detected in the liver tissues of mice challenged with CCl4. (A) Protein expression levels of ALOX12, HO-1, COX-2, p21 and Nrf2 at 6 and 24 h identified by Western blotting analysis of liver tissues of mice treated with CCl4; the quantitative analysis is showed in (B) (n = 4). 6 h vs. ctrl, * p < 0.05, ** p < 0.01; 24 h vs. ctrl, # p < 0.05, ## p < 0.01. Ctrl: control.

Article Snippet: The following primary antibodies were used to probe the membranes: primary rabbit antibodies against p21 (1:1000), COX2 (1:1000), Nrf2 (1:1000), and HO-1 (1:1000) (ProteinTech Group, Inc., Chicago, IL, USA), mouse monoclonal antibody against ALOX12 (1:1000) (Abcam, Cabridge, MA, USA) and β-actin (1:1000) (Santa Cruz Biotechnology, Dallas, TX, USA).

Techniques: Expressing, Western Blot, Control

Journal: Antioxidants (Basel, Switzerland)

Article Title: Inhibition of Oxidative Stress and ALOX12 and NF-κB Pathways Contribute to the Protective Effect of Baicalein on Carbon Tetrachloride-Induced Acute Liver Injury.

doi: 10.3390/antiox10060976

Figure Lengend Snippet: Figure 6. Effects of baicalein supplementation on apoptotic pathway in the liver tissues of mice challenged with CCl4. (A) Representative TUNEL-stained sections showing apoptosis in the liver tissue of mice. (B) Quantitative analysis of TUNEL positive rates (n = 4). (C,D) Activities of (C) caspases-3 and -9 (D) (n = 6). (E) The relative expression of Bax, GADD45a and p21 mRNAs in the liver tissues (n = 5). ** p < 0.01, compared to the control group; # p < 0.05 or ## p < 0.01, compared to the CCl4 only group. Bar = 100 µm. Bai: baicalein.

Article Snippet: The following primary antibodies were used to probe the membranes: primary rabbit antibodies against p21 (1:1000), COX2 (1:1000), Nrf2 (1:1000), and HO-1 (1:1000) (ProteinTech Group, Inc., Chicago, IL, USA), mouse monoclonal antibody against ALOX12 (1:1000) (Abcam, Cabridge, MA, USA) and β-actin (1:1000) (Santa Cruz Biotechnology, Dallas, TX, USA).

Techniques: TUNEL Assay, Staining, Expressing, Control

Journal: Radiotherapy and oncology : journal of the European Society for Therapeutic Radiology and Oncology

Article Title: Impact of DNA repair and reactive oxygen species levels on radioresistance in pancreatic cancer.

doi: 10.1016/j.radonc.2021.03.038

Figure Lengend Snippet: Fig. 3. Cell cycle distribution in parental and RR cell lines. A. Cell cycle distribution was determined in unirradiated cells. Cells were stained with PI and cell cycle distribution was measured by flow cytometry. Quantification of cells in G2/M phase is shown for Panc-1 and Panc–1 RR, MIA PaCa-2 and MIA PaCa-2 RR. Data are expressed as mean ± SD of at least 3 independent experiments (Student’s t-test; *p 0.05, ****p 0.0001). B. Representative western blot showing the expression of p21, CDC25C, CDC2, WEE1, Cyclin B1 and ß-actin in parental and RR Panc-1 and MIA PaCa-2 cells. C. Quantification of Western blot analysis. Image Lab (Bio-Rad) was used to quantify the bands in relation to the loading control ß-actin. The graph represents the quantification of proteins relative to ß-actin and normalized to the respective parental cell line. Data are expressed as mean ± SD of 3 independent experiments (Student’s t-test; *p 0.05, **p 0.01, ***p 0.001, ****p 0.0001). D. Cell cycle distribution was determined 24 h after irradiation with 8 Gy. Cells were stained with PI and cell cycle distribution was measured by flow cytometry. Relative induction of cells in G2/M phase 24 h after irradiation with 8 Gy in relation to unirradiated cells is shown for Panc-1 and Panc–1 RR, MIA PaCa-2 and MIA PaCa-2 RR. Data are expressed as mean ± SD of at least 3 independent experiments (Student’s t-test; *p 0.05, **p 00.1).

Article Snippet: Immunoblot analysis was performed using anti-p21 (#2947, Cell Signaling), anti-CDC2 (#9116, Cell Signaling), anti-Cyclin B1 (#4135, Cell Signaling), anti-CDC25C (#4688, Cell Signaling), anti-WEE1 (#4936, Cell Signaling), anti-ß-actin (A5316, Sigma), anti-phospho-NRF2 (phospho S40) (#ab76026, Abcam), anti-NRF2 (#ab62352, Abcam), antiSOD2/MnSOD (#ab13533, abcam), anti-SOD1 (#ab13498, abcam), anti-TRX (#ab133524, abcam), and anti-GAPDH (#5174, cell signaling).

Techniques: Staining, Cytometry, Western Blot, Expressing, Control, Irradiation

Journal: PloS one

Article Title: Sterigmatocystin-induced DNA damage triggers G2 arrest via an ATM/p53-related pathway in human gastric epithelium GES-1 cells in vitro.

doi: 10.1371/journal.pone.0065044

Figure Lengend Snippet: Figure 4. The p53-p21 pathway is activated in ST-treated GES-1 cells. GES-1 cells were treated with different concentrations of ST (0.075, 0.3, 1.5, and 3 mM) or solvent for 48 h. (A) Representative immunoblots show the effect of ST treatment on the phosphorylation of p53 (Ser-15) and the expression of p53 and p21. b-actin was used as the normalization control. (B) Intensities of the immunoreactive bands were quantified by densitometric scanning and compared with those of the control (considered ‘‘1’’). The values shown represent the means 6 SD. *P,0.05 compared with the solvent-treated control group. doi:10.1371/journal.pone.0065044.g004

Article Snippet: The primary antibodies used for the Western blot analysis were mouse anti-human Cyclin B1 antibody (eBioscience, CA, USA), rabbit anti-human Cdc2, Cdc25C, ATM, phospho-ATM (Ser-1981), and phospho-Chk2 (Thr-68) monoclonal antibodies (Epitomics, CA, USA), rabbit anti-human Chk2 monoclonal antibodies (Millipore, MA, USA), rabbit anti-human phospho-Cdc2 (Tyr15) and phospho-Cdc25C (Ser216) monoclonal antibodies (Cell Signaling Technology, MA, USA), mouse antihuman phospho-p53 (Ser15) monoclonal antibody (Cell Signaling Technology, MA, USA), rabbit anti-human p53, Bax, and caspase-3 antibodies, and mouse anti-human p21 waf1 and Bcl-2 antibodies (Santa Cruz, CA, USA).

Techniques: Solvent, Western Blot, Phospho-proteomics, Expressing, Control

Journal: PloS one

Article Title: Sterigmatocystin-induced DNA damage triggers G2 arrest via an ATM/p53-related pathway in human gastric epithelium GES-1 cells in vitro.

doi: 10.1371/journal.pone.0065044

Figure Lengend Snippet: Figure 6. Silencing of p53 by specific p53 siRNA inhibited ST-induced G2 arrest. Cells were either not transfected or transfected with 100 nM p53 siRNA and then treated with 3 mM ST for 48 h. (A) Cells were subjected to immunoblot analysis for p-p53 (Ser15), p53, p21, and (C) the regulators related to G2 arrest. NC: cells transfected with the same concentration of negative control siRNA. b-actin was used as the loading control. (B, D) Intensities of the immunoreactive bands in ‘‘A’’ and ‘‘C’’ were quantified by densitometric scanning and compared with those of the control (considered ‘‘1’’). (E) The cell cycle phases of the cells were analyzed by FCM. The values shown represent the means 6 SD, *P,0.05 compared with the solvent-treated control group. mP,0.05 compared with the ST-treated groups. #P,0.05 compared with the p53 siRNA-treated groups. doi:10.1371/journal.pone.0065044.g006

Article Snippet: The primary antibodies used for the Western blot analysis were mouse anti-human Cyclin B1 antibody (eBioscience, CA, USA), rabbit anti-human Cdc2, Cdc25C, ATM, phospho-ATM (Ser-1981), and phospho-Chk2 (Thr-68) monoclonal antibodies (Epitomics, CA, USA), rabbit anti-human Chk2 monoclonal antibodies (Millipore, MA, USA), rabbit anti-human phospho-Cdc2 (Tyr15) and phospho-Cdc25C (Ser216) monoclonal antibodies (Cell Signaling Technology, MA, USA), mouse antihuman phospho-p53 (Ser15) monoclonal antibody (Cell Signaling Technology, MA, USA), rabbit anti-human p53, Bax, and caspase-3 antibodies, and mouse anti-human p21 waf1 and Bcl-2 antibodies (Santa Cruz, CA, USA).

Techniques: Transfection, Western Blot, Concentration Assay, Negative Control, Control, Solvent

Journal: Integrative cancer therapies

Article Title: Scutellaria Barbata D Don Inhibits Colorectal Cancer Growth via Suppression of Multiple Signaling Pathways.

doi: 10.1177/1534735413508811

Figure Lengend Snippet: Figure 3. Effect of ethanol extract of Scutellaria barbata D Don (EESB) on cell proliferation and the expression of Cyclin D1, CDK4, and p21 in colorectal cancer (CRC) xenograft mice. (A) At the end of the study, tumor tissues were processed for immunohistochemical (IHC) staining for proliferating cell nuclear antigen (PCNA). The photographs are representative images taken at a magnification of 400×. Quantification of IHC assay was represented as percentage of positively stained cells. Data shown are mean ± standard deviation (error bars) of 6 individual mice in each group. *P < .05, versus controls. (B, C) The mRNA or protein expression levels of Cyclin D1, CDK4, and p21 were determined by reverse transcriptase–polymerase chain reaction (RT-PCR) and Western blot analyses. GAPDH and β-actin were used as the internal controls for the RT-PCR and Western blotting, respectively. Images are representatives of 3 individual mice in each group. (D, E) Quantitation of mRNA and protein expression was assayed by densitometric analysis. The data were normalized to the mean mRNA or protein expression of untreated control (100%). *P < .01, versus controls.

Article Snippet: Phospho-STAT3 (p-STAT3), total STAT3, Bcl-2, Bax, p21, cyclinD1, CDK4 antibodies, horseradish peroxidase (HRP)–conjugated secondary antibodies were obtained from Cell Signaling (Beverly, MA).

Techniques: Expressing, Immunohistochemical staining, Immunohistochemistry, Staining, Standard Deviation, Reverse Transcription, Polymerase Chain Reaction, Reverse Transcription Polymerase Chain Reaction, Western Blot, Quantitation Assay, Control

Journal: Oncology reports

Article Title: Epigenetic silencing of DUSP9 induces the proliferation of human gastric cancer by activating JNK signaling.

doi: 10.3892/or.2015.3998

Figure Lengend Snippet: Figure 4. DUSP9 is a negative regulator of cell cycle-related molecules activity in GC cells. (A and B) Quantitative PCR analysis of CCND1, CDK6, and p21 in MKN-1 cells, induced by the transient transfection of the DUSP/Si-DUSP9 gene. (C and D) Western blot analyses of c-Jun, CCND1, CDK4, CDK6 and p21 in the indicated cell groups. DUSP9, dual‑specificity phosphatase 9; GC, gastric cancer.

Article Snippet: Protein (20 μg) was loaded onto a 10% SDS-PAGE gel (Bio-Rad, Hercules, CA, USA) that was then transferred onto a PVDF membrane (Amersham Biosciences, Buckinghamshire, UK) and incubated with rabbit anti-DUSP9 (1:2,000 diluted, cat no. ab54941-100; Abcam, Cambridge, UK), rabbit anti-p21 (1:1,000 diluted; Cell Signaling Technology, Danvers, MA, USA), rabbit anti-CDK4 (1:2,000 diluted), rabbit anti-CDK6 (1:3,000 diluted), rabbit anti-CCND1 (1:2,000 diluted) and rabbit anti-c-Jun (1:1,000 diluted) (all from Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA) at 4 ̊C overnight in blocking buffer (3% non-fat dry milk/BSA in TBS) followed by incubation with HRP-conjugated secondary anti-mouse anti-body (1:5,000 diluted; ZSGB-Bio, China).

Techniques: Activity Assay, Real-time Polymerase Chain Reaction, Transfection, Western Blot

Journal: Nutrients

Article Title: ER-Stress and Senescence Coordinately Promote Endothelial Barrier Dysfunction in Diabetes-Induced Atherosclerosis

doi: 10.3390/nu14142786

Figure Lengend Snippet: Hyperglycaemia-induced senescence promotes barrier disruption in endothelial cells. ( A ) Experimental design and treatment conditions. ( B , C ) Dot plot summarizing data of barrier integrity measured by FITC dextran leakage ( B ) and TEER ( C ). ( D – G ) Exemplary immunoblot of p21, p16, and p53 (( D ), loading control: GAPDH) and dot plot summarizing p21 ( E ), p16 ( F ) and p53 ( G ) densitometric quantifications of immunoblots. ( H – L ) Representative immunofluorescence images showing endothelial cell staining for p21 (( H ), top panel, red; nuclear counterstain: DAPI, blue), p16 (( H ), first middle panel, red; nuclear counterstain: DAPI, blue), p53 (( H ), second middle panel, red; nuclear counterstain: DAPI, blue) and senescence-associated beta galactosidase staining (( H ), bottom panel, blue). Dot plots summarizing data for p21 ( I ), p16 ( J ), p53 ( K ) and SAβ gal ( L ). Scale bar: 20 µm ( H ). HCAEC maintained under control (non-treated, C), high glucose (25 mM; HG) or oxidized low density lipoprotein (oxLDL 50 µg/mL, oxLDL) conditions. Each dot represents data obtained from one biological specimen; * p < 0.05, ** p < 0.01; ANOVA.

Article Snippet: The following reagents and antibodies were used in this study: p21 Waf1/Cip1 (12D1) rabbit, p16 INK4A (D7C1M) rabbit, p53 (1C12) mouse (Cell Signaling Technology, Frankfurt, Germany); anti-phospho-IRE1 (S724) rabbit monoclonal antibody (Boster, Germany); human XBP1 antibody (R & D System, Wiesbaden, Germany); anti ATF6 alpha (Rockland, Germany); alexa fluor FITC goat anti-rabbit IgG, alexa fluor TRITC goat anti-rabbit IgG, alexa fluor TRITC goat anti-mouse IgG and alexa fluor FITC goat anti-mouse IgG (Invitrogen, Karlsruhe, Germany).

Techniques: Disruption, Western Blot, Control, Immunofluorescence, Staining

Journal: Nutrients

Article Title: ER-Stress and Senescence Coordinately Promote Endothelial Barrier Dysfunction in Diabetes-Induced Atherosclerosis

doi: 10.3390/nu14142786

Figure Lengend Snippet: Senescence is induced within plaques of diabetic ApoE −/− mice. ( A ) Representative immunofluorescence images showing staining of brachiocephalic arteries for p21 (( A ), top panel, p21, green; nuclear counterstain: DAPI, blue), p16 (( A ), middle panel, p16, red; nuclear counterstain: DAPI, blue) and p53 (( A ), bottom panel, p16, red; nuclear counterstain: DAPI, blue). ( B – D ) Dot plots summarizing immunofluorescence data for p21 ( B ) p16 ( C ), p53 ( D ) and Scale bar: 20 µm ( A ). ApoE −/− control mice (Cont, normal chow diet, citrate instead of streptozotocin injections), DM mice (normal chow diet, streptozotocin injections) or HFD mice (fed high fat diet). Each dot represents data obtained from one mouse specimen; ** p < 0.01; ANOVA.

Article Snippet: The following reagents and antibodies were used in this study: p21 Waf1/Cip1 (12D1) rabbit, p16 INK4A (D7C1M) rabbit, p53 (1C12) mouse (Cell Signaling Technology, Frankfurt, Germany); anti-phospho-IRE1 (S724) rabbit monoclonal antibody (Boster, Germany); human XBP1 antibody (R & D System, Wiesbaden, Germany); anti ATF6 alpha (Rockland, Germany); alexa fluor FITC goat anti-rabbit IgG, alexa fluor TRITC goat anti-rabbit IgG, alexa fluor TRITC goat anti-mouse IgG and alexa fluor FITC goat anti-mouse IgG (Invitrogen, Karlsruhe, Germany).

Techniques: Immunofluorescence, Staining, Control

Journal: Nutrients

Article Title: ER-Stress and Senescence Coordinately Promote Endothelial Barrier Dysfunction in Diabetes-Induced Atherosclerosis

doi: 10.3390/nu14142786

Figure Lengend Snippet: aPC reduces glucose-induced senescence. ( A – C ) Representative immunoblots showing p21 and p16 expressions (( A ), loading control: GAPDH). Dot plots summarize densitometric quantifications of immunoblotting results for p21 ( B ) and p16 ( C ). ( D – F ) Representative immunofluorescence images showing endothelial cells staining for p21 (( D ), upper panel, p21, red; nuclear counterstain: DAPI, blue) and p16 (( D ), lower panel, p16, red; nuclear counterstain: DAPI, blue). Dot plots summarizing immunofluorescence data for p21 ( E ) and p16 ( F ). Scale bar: 20 µm ( D ). ( G , H ) Dot plot summarizing barrier integrity data measured by FITC dextran leakage ( G ) and TEER ( H ). HCAECs maintained under control ( C ), high glucose (25 mM; HG), or HG+aPC (25 mM glucose + 20nM of exogenous activated protein C) conditions. Each dot represents data obtained from one biological specimen. ** p < 0.01; ANOVA.

Article Snippet: The following reagents and antibodies were used in this study: p21 Waf1/Cip1 (12D1) rabbit, p16 INK4A (D7C1M) rabbit, p53 (1C12) mouse (Cell Signaling Technology, Frankfurt, Germany); anti-phospho-IRE1 (S724) rabbit monoclonal antibody (Boster, Germany); human XBP1 antibody (R & D System, Wiesbaden, Germany); anti ATF6 alpha (Rockland, Germany); alexa fluor FITC goat anti-rabbit IgG, alexa fluor TRITC goat anti-rabbit IgG, alexa fluor TRITC goat anti-mouse IgG and alexa fluor FITC goat anti-mouse IgG (Invitrogen, Karlsruhe, Germany).

Techniques: Western Blot, Control, Immunofluorescence, Staining