Journal: Journal of Molecular Signaling

Article Title: Coexpression of human somatostatin receptor-2 (SSTR2) and SSTR3 modulates antiproliferative signaling and apoptosis

doi: 10.1186/1750-2187-7-5

Figure Lengend Snippet: p21 and p27 Kip1 mediated cytostatic effects upon activation of SSTR2/SSTR3. Cotransfected cells were treated with SST (1, 50 nM and 1 μM), L-779976 (10 and 100 nM) and L-796778 (25 and 50 nM) for 24 h with or without serum starvation and processed for p21 ( top panel ) and p27 Kip1 ( bottom panel ) expression using Western blot analysis. Induction of p21 and p27 Kip1 in response to SST and receptor-selective agonists suggests a cytostatic role for both receptors. The high basal levels of p21 observed in serum-deprived cells were maintained upon treatment with SST and specific agonists. Conversely, SST decreased p27 Kip1 expression in cotransfected cells by ~50% in FBS-deficient conditions. β-Tubulin was used as a loading control. Results are expressed as mean ± S.D of three independent experiments.

Article Snippet: Mouse monoclonal antibodies against p21 and PARP-1 were purchased from BD-Biosciences, Mississauga, ON, Canada.

Techniques: Activation Assay, Expressing, Western Blot

Journal: PLoS ONE

Article Title: Polyomavirus-Associated Trichodysplasia Spinulosa Involves Hyperproliferation, pRB Phosphorylation and Upregulation of p16 and p21

doi: 10.1371/journal.pone.0108947

Figure Lengend Snippet: Sections of healthy epidermis ( A1–A4 ), healthy hair follicle ( B1–B4 ), TS epidermis ( C1–C4 ) and TS follicle ( D1–D4 ) are stained for p16 ink4a (first panel), p21 waf (second panel), pRB (third panel) and TSPyV (fourth panel). Insets in the fourth panel depict the same region with Hoechst DNA staining (blue). Dotted lines indicate the dermoepidermal junction. Bar depicts 100 µm.

Article Snippet: p21 waf (mouse) , 6B6 , 1∶250 , BD Biosciences, USA.

Techniques: Staining

Journal: PLoS ONE

Article Title: Polyomavirus-Associated Trichodysplasia Spinulosa Involves Hyperproliferation, pRB Phosphorylation and Upregulation of p16 and p21

doi: 10.1371/journal.pone.0108947

Figure Lengend Snippet: An oversimplified cell-cycling scenario is shown that envisions the TSPyV LT-antigen involvement in regulation of pRB pathway activity. In a normal physiological condition, hypophosphorylated pRB is complexed with transcription factor E2F during early G1 (rest) phase of the cell cycle. When pRB is hyperphosphorylated at specific residues by Cyclin-dependent kinases (CDK) coupled to Cyclin-D1, E2F is released that activates expression of growth stimulatory genes needed for the cells to enter the S (DNA synthesis) phase. pRB phosphorylation is under tight regulation of p16 ink4a and p21 waf . Hypothetically, through its conserved LXCXE motif TSPyV LT-antigen interacts with pRB/E2F complex to dissociate these proteins via pRB hyperphosphorylation, resulting into S phase entry and subsequent increased expression of p16 ink4a and p21 waf as a negative cell cycling feedback (red arrows).

Article Snippet: p21 waf (mouse) , 6B6 , 1∶250 , BD Biosciences, USA.

Techniques: Activity Assay, Expressing, DNA Synthesis

Journal: Journal of Veterinary Research

Article Title: Immunoreactivity of p21, MMP-1 and CB2 receptor proteins in cutaneous canine mast cell tumours: an association with the three-tier grading system

doi: 10.2478/jvetres-2023-0066

Figure Lengend Snippet: Immunohistochemistry with primary anti-p21 antibody with positive reaction visible as brown staining. A – weak nuclear reaction in a grade 1 mast cell tumour (MCT); B – moderate nuclear reaction in a grade 2 MCT; C – strong nuclear reaction in a grade 3 MCT

Article Snippet: The primary antibodies used in this study, all canine-specific, were as follows: anti-CB2R (Cat. No. TA317640; OriGene Technologies, Rockville, MD, USA), anti-p21 (Cat. No. M7202; clone SX118, Agilent Dako, Santa Clara, CA, USA) and anti-MMP-1 (Cat. No. ABIN2777120; antibodies-online, Aachen, Germany), diluted 1 : 200, 1 : 50 and 1 : 500, respectively.

Techniques: Immunohistochemistry, Staining

Journal: Journal of Veterinary Research

Article Title: Immunoreactivity of p21, MMP-1 and CB2 receptor proteins in cutaneous canine mast cell tumours: an association with the three-tier grading system

doi: 10.2478/jvetres-2023-0066

Figure Lengend Snippet: Frequency of immunoreactivity for p21 protein in grade 1, 2 and 3 mast cell tumours (MCTs). * – P-value < 0.05; **_ P-value < 0.01; *** P-value < 0.001 compared to grade 1 MCTs; (−) – no reaction; (+) – weak reaction; (++) – moderate reaction; (+++) – strong reaction

Article Snippet: The primary antibodies used in this study, all canine-specific, were as follows: anti-CB2R (Cat. No. TA317640; OriGene Technologies, Rockville, MD, USA), anti-p21 (Cat. No. M7202; clone SX118, Agilent Dako, Santa Clara, CA, USA) and anti-MMP-1 (Cat. No. ABIN2777120; antibodies-online, Aachen, Germany), diluted 1 : 200, 1 : 50 and 1 : 500, respectively.

Techniques:

Journal: International Journal of Medical Sciences

Article Title: SSRP1 influences colorectal cancer cell growth and apoptosis via the AKT pathway

doi: 10.7150/ijms.38439

Figure Lengend Snippet: SSRP1 exerts proliferation and apoptosis of colorectal cancer cells by activating the AKT pathway. (A) Graphs with quantitative data for the flow cytometric cell cycle distribution assay in HCT15 and SW620 cells. (B, C) FITC Annexin V/PI staining indicated increased apoptosis in HCT15 and SW620 cells with SSRP1 inhibiting. (D, E) Western blot displayed reduced P-AKT, CyclinD1, BCL-2 and MDM2, accompanying increased p27, p21, p53 and BAX in transfected HCT15 and SW620 cells. (F, G) Apoptosis ratio in HCT15 cells with SSRP1 or AKT inhibitors through flow cytometry. (H, I) The protein level of P-AKT, AKT, BAX and BCL-2 in HCT15 cells with SSRP1 or AKT inhibitors. * P < 0.05, ** P < 0.01 vs NC group.

Article Snippet: Nonspecific binding sites were blocked with 5% low-fat milk and 0.1% Tween-20 at room temperature for 2 h. Subsequently, the membranes were incubated overnight at 4°C with the following primary antibodies: Proteintech (Wuhan, China): anti-SSRP1 (1:1000; 15696-1-AP), anti-p27 (1:1000; 25614-1-AP), anti-p21 (1:1000; 10355-1-AP), anti-CyclinD1 (1:1000; 60186-1-Ig), anti-BCL2 (1:1000; 26593-1-AP), anti-BAX (1:1000; 50599-2-Ig), anti-MDM2 (1:1000; 19058-1-AP), anti-p53 (1:1000; 10442-1-AP), anti-AKT (1:1000; 60203-2-Ig), anti-β-actin (1:2000; 66009-1-Ig) and arigo (Taiwan, China): anti-P-AKT (1:1000; ARG51559); secondary antibody: goat anti-Mouse (1:2000; 10828-I-AP) and goat anti-Rabbit IgG (H+L) (1:2000; SA0000I-2) from Proteintech Group Inc.

Techniques: Staining, Western Blot, Transfection, Flow Cytometry

Journal: International Journal of Medical Sciences

Article Title: SSRP1 influences colorectal cancer cell growth and apoptosis via the AKT pathway

doi: 10.7150/ijms.38439

Figure Lengend Snippet: SSRP1 inhibition blocks the growth of colorectal cancer in vivo . (A) Treatment for transplanted xenogeneic models using HCT15 cells. (B) Image of the tumor tissues of NC group and siSSRP1 group. (C) Tumor growth curve of NC group and siSSRP1 group for 34 d treatment. (D) Immunofluorescent staining showing that SSRP1 expression was reduced in siSSRP1 group, compared with NC group. (E) Validation of SSPR1 protein levels through western blot. (F) TUNEL assay of tumor tissue. (G) Immunofluorescent staining of PCNA, BAX, Bcl2, MMP-2 and MMP-9. (H) AKT, P-AKT, p27, p21, CyllinD1, p53, MDM2, BAX, and Bcl2 protein levels were determined by western blot analysis. * P <0.05, ** P <0.01 vs NC group.

Article Snippet: Nonspecific binding sites were blocked with 5% low-fat milk and 0.1% Tween-20 at room temperature for 2 h. Subsequently, the membranes were incubated overnight at 4°C with the following primary antibodies: Proteintech (Wuhan, China): anti-SSRP1 (1:1000; 15696-1-AP), anti-p27 (1:1000; 25614-1-AP), anti-p21 (1:1000; 10355-1-AP), anti-CyclinD1 (1:1000; 60186-1-Ig), anti-BCL2 (1:1000; 26593-1-AP), anti-BAX (1:1000; 50599-2-Ig), anti-MDM2 (1:1000; 19058-1-AP), anti-p53 (1:1000; 10442-1-AP), anti-AKT (1:1000; 60203-2-Ig), anti-β-actin (1:2000; 66009-1-Ig) and arigo (Taiwan, China): anti-P-AKT (1:1000; ARG51559); secondary antibody: goat anti-Mouse (1:2000; 10828-I-AP) and goat anti-Rabbit IgG (H+L) (1:2000; SA0000I-2) from Proteintech Group Inc.

Techniques: Inhibition, In Vivo, Staining, Expressing, Western Blot, TUNEL Assay

Journal: Journal of Biological Chemistry

Article Title: Inactivation of Dnmt3b in Mouse Embryonic Fibroblasts Results in DNA Hypomethylation, Chromosomal Instability, and Spontaneous Immortalization

doi: 10.1074/jbc.m413246200

Figure Lengend Snippet: FIG. 6. Analysis of the G1 to S-phase checkpoint and p53 func- tion in Dnmt3b-deficient MEFs. A, wild-type, Dnmt3b/, and p53/ MEF lines were treated with or without -irradiation (10 grays), and 25 h later, cell proliferation (bromodeoxyuridine incorpora- tion) was analyzed by FACScan. B, the indicated MEF lines were treated with or without -irradiation, and 2 h later, the cells were lysed and analyzed by immunoblotting with p53 and tubulin antibodies. C, the indicated MEF lines were treated with or without -irradiation, and 18 h later, the cells were lysed and analyzed by immunoblotting with p21 and tubulin antibodies.

Article Snippet: Antibodies against p21 (F-5) and p53 (Pab240) were obtained from Santa Cruz Biotechnology and Chemicon International, Inc., respectively.

Techniques: Irradiation, Western Blot

Journal: Communications Biology

Article Title: Cardiomyocyte-specific disruption of the circadian BMAL1–REV-ERBα/β regulatory network impacts distinct miRNA species in the murine heart

doi: 10.1038/s42003-023-05537-z

Figure Lengend Snippet: a Diurnal variations in miR-1a-2-5p, miR-181a-1-3p, miR-499-3p, let-7c-1-3p, miR-23b-5p, miR-31-5p, miR-34a-5p, miR-215-5p, and miR-741-3p levels for CBK and CON hearts collected at six distinct times of the day ( n = 3–8). b Diurnal variations in cdkn1a , npas2 , nte5 , armc2 , herpud1 , mid1ip1 , rhobtb1 , npc1 , and mylk4 mRNA levels for CBK and CON hearts collected at six distinct times of the day ( n = 4–8). c Western blot of p21 protein levels in CBK and littermate control (CON) hearts collected from 12wk old mice at ZT0 ( n = 7). Data are presented as mean ± SEM. Main effects of genotype, time-of-day, and/or interaction are reported at the top of the figure panels. p -values are either reported as numbers, or as symbols: * p < 0.05 for CBK versus CON hearts.

Article Snippet: Membranes were probed for with anti- BMAL1 and CDKN1A antibodies (Abcam ab3350 and Santa Cruz sc-6246, respectively).

Techniques: Western Blot, Control