Journal: PloS one

Article Title: Crosstalk between nuclear factor I-C and transforming growth factor-β1 signaling regulates odontoblast differentiation and homeostasis.

doi: 10.1371/journal.pone.0029160

Figure Lengend Snippet: Figure 1. Patterns of gene expression during odontoblast differentiation. MDPC-23 cells were cultured in differentiation medium for up to 3 weeks. (A) The expression of DSP was evaluated by western blot analysis. GAPDH was used as a loading control. (B) Mineralized nodules stained with alizarin red-S were photographed. (C) Four stages of differentiation were identified: confluent (preodontoblast; 0 days); early odontoblast differentiation (,7 days); late odontoblast differentiation (7,14 days); and mineralization (14,21 days). Solid gray bars indicate the periods of elevated expression of genes indicated during culture. (D) The expression of NFI-C was evaluated by western blot analysis and the results were quantified using ImageJ. (E) TGFb-RI, TGFb-RII, p-Smad2/3, Runx2, Osx, and p21 were evaluated by western blot analysis. doi:10.1371/journal.pone.0029160.g001

Article Snippet: All other antibodies against TGFb-RI (sc-398), TGFb-RII (sc-400), p-Smad2/3 (sc-11769), p21 (sc-6246), and Osterix (sc-22538) were purchased from Santa Cruz Biotechnology (Santa Cruz, CA).

Techniques: Gene Expression, Cell Culture, Expressing, Western Blot, Control, Staining

Journal: PloS one

Article Title: Crosstalk between nuclear factor I-C and transforming growth factor-β1 signaling regulates odontoblast differentiation and homeostasis.

doi: 10.1371/journal.pone.0029160

Figure Lengend Snippet: Figure 2. NFI-C is degraded by TGF-b1 in MDPC-23 cells. (A) The protein levels of NFI-C, p-Smad2/3, and p21 protein in TGF-b1-treated MDPC- 23 cells were analyzed by western blot (left panel), and the results were quantified using ImageJ (right panel). GAPDH used as a loading control. (B) MDPC-23 cells were incubated with TGF-b1 (10 ng/ml) in the presence or absence of the proteasome inhibitor, MG132 (10 mM) for 1 hr. Cytoplasmic and nuclear fractions were isolated and subjected to western blot analysis. GAPDH and lamin B served as cell fractionation controls. (C) The subcellular localization of NFI-C was analyzed by immunostaining with NFI-C specific antibody. DAPI staining was used to detect nuclei. doi:10.1371/journal.pone.0029160.g002

Article Snippet: All other antibodies against TGFb-RI (sc-398), TGFb-RII (sc-400), p-Smad2/3 (sc-11769), p21 (sc-6246), and Osterix (sc-22538) were purchased from Santa Cruz Biotechnology (Santa Cruz, CA).

Techniques: Western Blot, Control, Incubation, Isolation, Cell Fractionation, Immunostaining, Staining

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: 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

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