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: Scientific Reports

Article Title: TSG6 affects anti cancer drug resistance and angiogenesis in 3D spheroid model of canine mammary gland tumor cells

doi: 10.1038/s41598-025-12950-3

Figure Lengend Snippet: Establishment and confirmation of vascularized TSG6 knockdown spheroids of canine MGT cells with canine ECs. ( A ) Method of forming vascularized TSG6 knockdown spheroids comprising of canine ECs and MGT cells. Cells were seeded onto an ultra-low adhesion plate with a 1:3 ratio. ( B ) Characterization of canine ECs by flow cytometry. Canine ECs were positive for CD31. ( C ) Confocal imaging of vascularized TSG6 knockdown spheroids. The spheroids were stained with APC dye-conjugated IgG1 mouse monoclonal anti-pimonidazole antibody (red), anti-CD31 rabbit monoclonal antibody (green), and Hoechst 33,342 (blue) (scale bar = 100 μm). ( D ) Immunohistology images of vascularized TSG6 knockdown spheroids. The spheroids were stained with anti-CD31 rabbit monoclonal antibody (brown) (scale bar = 20 μm). ( E ) Relative CD31-positive area in vascularized TSG6 knockdown spheroids. The results are presented as the mean ± SD and were of three obtained independent experiments. TSG6, TNF-stimulated gene 6 protein; MGT, mammary gland tumor; EC, endothelial cell; APC, Allophycocyanin; IgG1, Immunoglobulin G1; ctrl, control. * p < 0.05, ** p < 0.01, as determined by one-way ANOVA.

Article Snippet: The sections were washed thrice with cold DPBS (Welgene), blocked with 5% bovine serum albumin (BSA; Sigma-Aldrich) and 0.1% Triton X (Sigma-Aldrich) in DPBS (0.1% PBSTX), and stained with antibodies against TSG6 (1:100; Santa Cruz Biotechnology, Dallas, TX, USA; Cat# sc-377277) and CD31 (1:100; Novus Biologicals, Cat# NB100-2284) overnight at 4 °C.

Techniques: Knockdown, Flow Cytometry, Imaging, Staining, Control

Journal:

Article Title: Species-Specific, Postentry Barriers to Primate Immunodeficiency Virus Infection

doi:

Figure Lengend Snippet: Cell lines used in this study

Article Snippet: CRFK , Cat , Kidney , ATCC.

Techniques:

Journal: Experimental and Therapeutic Medicine

Article Title: miR-223 ameliorates thalamus hemorrhage-induced central poststroke pain via targeting NLRP3 in a mouse model

doi: 10.3892/etm.2022.11280

Figure Lengend Snippet: Effect of NLRP3 siRNA on NLRP3 inflammasome activity and the downstream factors IL-18, IL-1β, GFAP and p-ERK1/2. (A) Transfection with NLRP3 siRNA significantly decreased the protein expression levels of caspase-1, ASC and NLRP3. (B) NLRP3 siRNA also significantly reduced IL-18 and IL-1β protein expression levels. (C) After microinjection of NLRP3 siRNA, the protein expression levels of p-ERK1/2 and GFAP significantly decreased. n=3 mice/group. * P<0.05 vs. saline + scramble; # P<0.05 vs. Coll IV + scramble. NLRP3, NLR family pyrin domain containing 3; siRNA, small interfering RNA; GFAP, glial fibrillary acidic protein; p, phosphorylated; ASC, apoptosis-associated speck-like protein containing a CARD.

Article Snippet: The membrane was blocked with 5% skimmed milk in TBS with 0.1% Tween-20 for 1 h. Subsequently, membranes were incubated overnight at 4˚C with the following primary antibodies: rabbit anti-caspase-1 (1:1,000; Cell Signaling Technology, Inc. cat. no. 24232), rabbit anti-ASC (1:1,000; Cell Signaling Technology, Inc.; cat. no. 67824), rabbit anti-NLRP3 (1:1,000; Cell Signaling Technology, Inc. cat. no. 15101), rabbit anti-IL-18 (1:1,000; Abcam; cat. no. ab243091), mouse anti-IL-1β (1:1,000; Cell Signaling Technology, Inc. cat. no. 63124), rabbit anti-phosphorylated (p)-ERK1/2 (1:2,000; Cell Signaling Technology, Inc. cat. no. 4370), rabbit anti-ERK1/2 (1:2,000; Cell Signaling Technology, Inc. cat. no. 4695), rabbit anti-GAPDH (1:5,000; Cell Signaling Technology, Inc. cat. no. 2118) and mouse anti-glial fibrillary acidic protein (GFAP; 1:2,000; Cell Signaling Technology, Inc. cat. no. 3670).

Techniques: Activity Assay, Transfection, Expressing, Small Interfering RNA

Journal: Experimental and Therapeutic Medicine

Article Title: miR-223 ameliorates thalamus hemorrhage-induced central poststroke pain via targeting NLRP3 in a mouse model

doi: 10.3892/etm.2022.11280

Figure Lengend Snippet: miR-223 targets the NLRP3 3'UTR and affects the protein expression levels of caspase-1, ASC, NLRP3, IL-18, IL-1β, p-ERK1/2 and GFAP. (A) Binding site of miR-223 within the NLRP3 3'-UTR. (B) miR-223 agomir significantly reduced the relative luciferase activity in PC12 cells transfected with the NLRP3 3'-UTR. (C) miR-223 agomir significantly increased miR-223 expression levels. (D) Administration of miR-223 agomir significantly reduced the protein expression levels of caspase-1, ASC and NLRP3. (E) miR-223 agomir also significantly reduced the expression of IL-18 and IL-1β. (F) After microinjection of miR-223 agomir, p-ERK1/2 and GFAP protein expression levels significantly decreased. n=3 mice/cohort. * P<0.05 vs. saline + agomir-scramble; # P<0.05 vs. Coll IV + agomir-scramble. miR, microRNA; NLRP3, NLR family pyrin domain containing 3; UTR, untranslated region; GFAP, glial fibrillary acidic protein; p, phosphorylated; ASC, apoptosis-associated speck-like protein containing a CARD; wt, wild-type; mut, mutant.

Article Snippet: The membrane was blocked with 5% skimmed milk in TBS with 0.1% Tween-20 for 1 h. Subsequently, membranes were incubated overnight at 4˚C with the following primary antibodies: rabbit anti-caspase-1 (1:1,000; Cell Signaling Technology, Inc. cat. no. 24232), rabbit anti-ASC (1:1,000; Cell Signaling Technology, Inc.; cat. no. 67824), rabbit anti-NLRP3 (1:1,000; Cell Signaling Technology, Inc. cat. no. 15101), rabbit anti-IL-18 (1:1,000; Abcam; cat. no. ab243091), mouse anti-IL-1β (1:1,000; Cell Signaling Technology, Inc. cat. no. 63124), rabbit anti-phosphorylated (p)-ERK1/2 (1:2,000; Cell Signaling Technology, Inc. cat. no. 4370), rabbit anti-ERK1/2 (1:2,000; Cell Signaling Technology, Inc. cat. no. 4695), rabbit anti-GAPDH (1:5,000; Cell Signaling Technology, Inc. cat. no. 2118) and mouse anti-glial fibrillary acidic protein (GFAP; 1:2,000; Cell Signaling Technology, Inc. cat. no. 3670).

Techniques: Expressing, Binding Assay, Luciferase, Activity Assay, Transfection, Mutagenesis

Journal: Experimental and Therapeutic Medicine

Article Title: miR-223 ameliorates thalamus hemorrhage-induced central poststroke pain via targeting NLRP3 in a mouse model

doi: 10.3892/etm.2022.11280

Figure Lengend Snippet: Effect of miR-223 antagomir on the nociceptive threshold in naïve mice. Administration of miR-223 antagomir contributed to a significant increase in the paw withdrawal frequency in response to (A) 0.07 g and (B) 0.4 g von Frey filaments and a significant decrease in paw withdrawal latency to (C) thermal and (D) cold stimuli on day 3, 5 and 7 on the contralateral side. (E-G) Administration of miR-223 antagomir or antagomir-scramble did not contribute to behavioral changes on the ipsilateral side. n=8 mice/group. (H) miR-223 antagomir significantly reduced miR-223 expression levels. (I) Administration of miR-223 antagomir significantly elevated caspase-1, ASC and NLRP3 protein expression levels. (J) miR-223 antagomir also significantly elevated IL-18 and IL-1β protein expression levels. (K) Following microinjection of miR-223 antagomir, p-ERK1/2 and GFAP protein expression levels increased. n=3 mice/group. * P<0.05 vs. naïve group. miR, microRNA; NLRP3, NLR family pyrin domain containing 3; GFAP, glial fibrillary acidic protein; p, phosphorylated; ASC, apoptosis-associated speck-like protein containing a CARD.

Article Snippet: The membrane was blocked with 5% skimmed milk in TBS with 0.1% Tween-20 for 1 h. Subsequently, membranes were incubated overnight at 4˚C with the following primary antibodies: rabbit anti-caspase-1 (1:1,000; Cell Signaling Technology, Inc. cat. no. 24232), rabbit anti-ASC (1:1,000; Cell Signaling Technology, Inc.; cat. no. 67824), rabbit anti-NLRP3 (1:1,000; Cell Signaling Technology, Inc. cat. no. 15101), rabbit anti-IL-18 (1:1,000; Abcam; cat. no. ab243091), mouse anti-IL-1β (1:1,000; Cell Signaling Technology, Inc. cat. no. 63124), rabbit anti-phosphorylated (p)-ERK1/2 (1:2,000; Cell Signaling Technology, Inc. cat. no. 4370), rabbit anti-ERK1/2 (1:2,000; Cell Signaling Technology, Inc. cat. no. 4695), rabbit anti-GAPDH (1:5,000; Cell Signaling Technology, Inc. cat. no. 2118) and mouse anti-glial fibrillary acidic protein (GFAP; 1:2,000; Cell Signaling Technology, Inc. cat. no. 3670).

Techniques: Expressing

Journal: Experimental and Therapeutic Medicine

Article Title: miR-223 ameliorates thalamus hemorrhage-induced central poststroke pain via targeting NLRP3 in a mouse model

doi: 10.3892/etm.2022.11280

Figure Lengend Snippet: CY 09 affects IL-18, IL-1β, p-ERK1/2 and GFAP protein expression levels. (A) CY 09 injection caused no alterations in miR-223 expression levels. CY 09 injection (B) significantly reduced IL-18 and IL-1β protein expression levels in the naïve + miR-223 antagomir cohort and (C) significantly lowered the protein expression levels of GFAP and p-ERK1/2. n=3 mice/cohort. * P<0.05 vs. naïve group; # P<0.05 vs. miR-223 antagomir + vehicle. GFAP, glial fibrillary acidic protein; p, phosphorylated; miR, microRNA.

Article Snippet: The membrane was blocked with 5% skimmed milk in TBS with 0.1% Tween-20 for 1 h. Subsequently, membranes were incubated overnight at 4˚C with the following primary antibodies: rabbit anti-caspase-1 (1:1,000; Cell Signaling Technology, Inc. cat. no. 24232), rabbit anti-ASC (1:1,000; Cell Signaling Technology, Inc.; cat. no. 67824), rabbit anti-NLRP3 (1:1,000; Cell Signaling Technology, Inc. cat. no. 15101), rabbit anti-IL-18 (1:1,000; Abcam; cat. no. ab243091), mouse anti-IL-1β (1:1,000; Cell Signaling Technology, Inc. cat. no. 63124), rabbit anti-phosphorylated (p)-ERK1/2 (1:2,000; Cell Signaling Technology, Inc. cat. no. 4370), rabbit anti-ERK1/2 (1:2,000; Cell Signaling Technology, Inc. cat. no. 4695), rabbit anti-GAPDH (1:5,000; Cell Signaling Technology, Inc. cat. no. 2118) and mouse anti-glial fibrillary acidic protein (GFAP; 1:2,000; Cell Signaling Technology, Inc. cat. no. 3670).

Techniques: Expressing, Injection