Journal: British Journal of Nutrition

Article Title: Mitochondrial dysfunction in the liver of type 2 diabetic Goto–Kakizaki rats: improvement by a combination of nutrients

doi: 10.1017/s0007114511000493

Figure Lengend Snippet: Fig. 5. Apoptosis-related factors p53 and p21 in liver tissue: (a) Western blotting images of p21, p53 and a-tubulin, respectively and (b) quantitative results of p53 and p21. Quantitative values were computed as the ratios of the density of the targeted protein to a-tubulin. Results are expressed as fold change of control. Values are means, with their standard errors represented by vertical bars (n 10). Mean value was significantly different from that of the Wistar group: * P,0·05, ** P,0·01. , Wistar; , GK; , GK with nutrients; , GK with pioglitazone.

Article Snippet: Liver tissue proteins were subjected to 10 % SDS-PAGE and detected with primary antibodies against p53 (1:1000, Mouse Ab no. 12 506; Santa Cruz Biotechnology, Santa Cruz, CA, USA), p21 (1:1000, Mouse Ab no. 2946; Cell Signaling Technology, Danvers, MA, USA) or a-tubulin (1:5000).

Techniques: Western Blot, Control

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: Yakugaku zasshi : Journal of the Pharmaceutical Society of Japan

Article Title: Induction of differentiation by panaxydol in human hepatocarcinoma SMMC-7721 cells via cAMP and MAP kinase dependent mechanism.

doi: 10.1248/yakushi.131.993

Figure Lengend Snippet: Fig. 5. Panaxydol Regulates the Expression Levels of p21, p-ERK and Id1 in SMMC-7721 Cells The levels of p21, p-ERK and Id1 were examined by immunoblotting analysis after 5 days incubation with 5, 10 and 20 mM panaxydol. Lane C: control group, Lane 1～3: SMMC-7721 cells treated with 5, 10, 20 mM panaxydol respectively. Data (mean±S.D.) were summarized from three independent experiments (n＝3), p＜0.05, p＜0.01 vs. control group.

Article Snippet: Immunoblotting analysis was carried out as previously described and the following antibodies were used: monoclonal antibody anti-p21 (Protein Tech, 1:1000), poly-anti-p-ERK1 /2 (Cell Signaling, 1:1000) or primary monoclonal antibody anti-Id.

Techniques: Expressing, Western Blot, Incubation, Control

Journal: Cancer Medicine

Article Title: Protective role of cytoplasmic p21Cip1/Waf1 in apoptosis of CDK4/6 inhibitor‐induced senescence in breast cancer cells

doi: 10.1002/cam4.4410

Figure Lengend Snippet: p21 in MDA‐MB‐231 cells treated with abemaciclib and/or ABT‐263. (A, B) Cancer cells were treated with abemaciclib (1.5 µM) and ABT‐263 (1.5 µM) for 2 days. After harvesting, the cytoplasmic and nuclear fractions were separated and subjected to immunoblotting. TBP and GAPDH were used as controls. (C) Immunoblotting was performed similarly using whole lysates. (D) MDA‐MB‐231 cells were treated with abemaciclib (1.5 µM) and ABT‐263 (1.5 µM) with zVAD (10 µM) for 24 h. Flow cytometric analysis was done after staining with annexin V‐FITC and PI. The numbers are proportions of the subsets. (Left) Data of the means ± SD of three cells are shown. * p < 0.05, ** p < 0.01. (E) MDA‐MB‐231 cells were treated similarly with zVAD (10 µM) for 24 h and subjected to immunoblotting. (F) Untreated MDA‐MB‐231 cells were cultured with Hoechst 33342 (5 µg/ml) and stained with anti‐p21 and anti‐caspase‐3 antibodies followed by an Alexa 488‐conjugated anti‐rabbit antibody and Cy5‐conjugated anti‐mouse IgG. Confocal imaging reveals nuclei (blue), p21 (green), and caspse‐3 (white). Scale, 10 µm. (G) Control and p21‐overexpressing MDA‐MB‐231 cells were examined for their p21 expression by immunoblotting. (H) Control and p21‐overexpressing MDA‐MB‐231 cells were treated with abemaciclib (1.5 µM) and ABT‐263 (1.5 µM) for 24 h. Flow cytometric analysis was done after staining with annexin V‐APC. Numbers are the proportions of the subset (Left). The means ± SD of four wells are shown. ** p < 0.01. (I) Cancer cells were treated with abemaciclib (1.5 µM) for 24 h, and the cell lysates were subjected to immunoblot to examine the expression of p21 and c‐Myc

Article Snippet: The following siRNAs were used: p21(I) siRNA (#6456; CST), p21(II) siRNA (#6558; CST), and control siRNA (#6568; CST).

Techniques: Western Blot, Staining, Cell Culture, Imaging, Control, Expressing

Journal: Cancer Medicine

Article Title: Protective role of cytoplasmic p21Cip1/Waf1 in apoptosis of CDK4/6 inhibitor‐induced senescence in breast cancer cells

doi: 10.1002/cam4.4410

Figure Lengend Snippet: Effect of genetic knockdown of p21 in MCF‐7 cells on their sensitivity to drugs. (A) siRNA‐transfected cancer cells were cultured for 48 h and subjected to immunoblotting. (B) MDA‐MB‐231 and MCF‐7 cells transfected with siRNA p21(I) 2 days prior were treated with abemaciclib (1.5 µM) and ABT‐263 (1.5 µM) for 48 h. After staining with annexin V‐FITC and PI, flow cytometric analysis was performed. Data are the means of three wells. * p < 0.05, ** p < 0.01. (C) Representative flow cytometry results; numbers are percentages of the subsets. (D) MDA‐MB‐231 cells were cultured with abemaciclib (1.5 µM) and ABT‐263 (1.5 µM) in the presence of caspase inhibitors (10 µM) for 48 h. Flow cytometric analysis was done after staining with annexin V‐FITC and PI. Data are the means of three wells. ** p < 0.01. (E) siRNA p21(I)‐transfected MDA‐MB‐231 and MCF‐7 cells were cultured with TRAIL for 24 h. Flow cytometric analysis was done after staining with annexin V‐FITC and PI. ** p < 0.01

Article Snippet: The following siRNAs were used: p21(I) siRNA (#6456; CST), p21(II) siRNA (#6558; CST), and control siRNA (#6568; CST).

Techniques: Knockdown, Transfection, Cell Culture, Western Blot, Staining, Flow Cytometry

Journal: Cancer Medicine

Article Title: Protective role of cytoplasmic p21Cip1/Waf1 in apoptosis of CDK4/6 inhibitor‐induced senescence in breast cancer cells

doi: 10.1002/cam4.4410

Figure Lengend Snippet: Poor prognosis of breast cancer patients with p21 high compared with those with p21 low . (A, B, C) Kaplan–Meier plotter univariate analysis of survival time in CDKN1A mRNA expression in breast cancer. Version 2021 of the database was used for analysis. Outlier array data were excluded for array quality control. Patients were split into low‐ and high‐expression groups based on the optimal cutoff. (D, E) TRGAted was used for survival analysis according to p21 protein level in patients with invasive breast carcinoma. All subtypes of TCGA‐BRCA‐L4 were used in the analysis. Patients were split into low‐ and high‐expression groups based on the optimal cutoff

Article Snippet: The following siRNAs were used: p21(I) siRNA (#6456; CST), p21(II) siRNA (#6558; CST), and control siRNA (#6568; CST).

Techniques: Expressing, Control

Journal: Frontiers in Cell and Developmental Biology

Article Title: Stimulation of Cardiomyocyte Proliferation Is Dependent on Species and Level of Maturation

doi: 10.3389/fcell.2022.806564

Figure Lengend Snippet: Stimulation of cardiomyocyte DNA synthesis. (A) Graphical overview of experimental outline. shRNAs are p21shRNA or scrambled shRNA adenoviruses. Other conditions are growth factors that were added on Day 2. (B) Representative images of EdU stained (red) cardiomyocytes (green) in response to various treatments to induce cardiomyocyte proliferation (green: Troponin T, red: EdU, blue: DAPI, scale bar equals 50 µm) (C) Percentage of EdU positive cardiomyocyte nuclei relative to total cardiomyocyte nuclei. *** p < 0.001 vs respective controls, n = 12 for each group except p21 shRNA ( n = 11) and TT-10 ( n = 10). (D) Representative images of pHH3 staining (green: Troponin T, red: pHH3, blue: DAPI, scale bar equals 50 µm) (E) Percentage of pHH3-positive cardiomyocyte nuclei relative to total cardiomyocyte nuclei. *** p < 0.001 vs respective control, n = 12 for each group except p21 shRNA ( n = 11) and TT-10 ( n = 10).

Article Snippet: For p21 knockdown in hiPSC-CMs, cells were transfected with p21 siRNA (Santa Cruz Biotechnology, sc-29427) using lipofectamine RNAiMAX (Thermo Fisher Scientific, 13778075) based on manufacturer’s instructions for 12 h. The cell media was then changed into RPMI1640 with B27 (plus insulin) and corresponding growth factors or small molecules were added to the media based on the treatment.

Techniques: DNA Synthesis, shRNA, Staining, Control