Journal: Drug Design, Development and Therapy

Article Title:

Tranilast Inhibits Pulmonary Fibrosis by Suppressing TGFβ/SMAD2 Pathway

doi: 10.2147/dddt.s264715

Figure Lengend Snippet: Figure 4 Tranilast attenuated BLM-induced ECM production and phosphorylation of SMAD2 in mice. (A) TGFβ1 concentration in BAL fluid was evaluated by ELISA. (B) Effects of tranilast on collagen contents. Data are presented as mean ± SD in each group of 10 mice. **p < 0.05. (C) Effect of tranilast on expression of fibronectin in the murine lungs by immunohistochemistry (IHC). Bar = 200 µm. (D) The average of the percentage of fibronectin positive ratio in each of the four groups was calculated by dividing the average of each group with that of the control group. Data are presented as mean ± SD in each group of 10 mice. *p < 0.01. (E) IHC staining for phospho-SMAD2 in the murine lungs. (F) The phospho- Smad2-positive cells were counted in 10 fields at × 200 magnification. Bar = 200 µm. The average of the percentage of phospho-SMAD2-positive cells in each of the four groups was calculated by dividing the average of each group with that of the control group. Data are presented as mean ± SD in each group of 10 mice. *p < 0.01, **p < 0.05. (G) Levels of phospho- and total-SMAD2 in the whole lung lysates of mice by Western blotting analysis. (H) Fold changes were analyzed by setting the ratios of the phospho/total protein band intensities.

Article Snippet: Enzyme-Linked ImmunoSorbent Assay (ELISA) for Anti-TGFβ1 Antibody Detection TGFβ1 concentration in BAL fluid was analyzed using ELISA sandwich, specifically developed with antibodies against canine TGFβ1 (Mouse/Rat/Porcine/Canine TGFβ1, MB100B, R&D Systems, Minneapolis, USA).

Techniques: Phospho-proteomics, Concentration Assay, Enzyme-linked Immunosorbent Assay, Expressing, Immunohistochemistry, Control, Western Blot

Journal: ImmunoHorizons

Article Title: Characterization of Canine Peyer’s Patches by Multidimensional Analysis: Insights from Immunofluorescence, Flow Cytometry, and Single-Cell RNA Sequencing

doi: 10.4049/immunohorizons.2300091

Figure Lengend Snippet: Structural organization of canine Peyer’s patches, according to immunofluorescence data. ( A ) Sample collection and preparation. Intestinal tissues from two dogs were sampled, and regions of ∼2 cm in length were identified as Peyer’s patches (PPs). The PPs were isolated, sectioned on a cryostat, and prepared for microscopy, with the various structures within the intestine identified before DAPI staining. ( B ) Visualization of PPs with H&E show different staining intensities within the follicles. The staining with Ki67, for proliferating cells, and CD21, for B cells, allows the visualization of proliferating B cells within the GC. ( C ) Immunofluorescence staining of PPs. PPs were stained with DAPI in combination with Abs against CD8 and one of the following markers: CD21, CD4, or FOXP3. Epifluorescence microscopy is shown at original magnification ×10, visualizing specific structures, including the subepithelial dome (SED), T cell areas, and B cell follicles. ( D ) Myeloid cell staining. Immunostaining of the CD14 and CD11c markers, along with CD21, was used to identify dendritic cells within the SED. Different images from the lower and upper parts of the sample were captured to adequately show the CD11c staining in proximity to both the epithelium and the follicle.

Article Snippet: We used the following Abs: mouse anti-dog CD3 Alexa Fluor 700 (Bio-Rad, MCA1774A700), rat anti-dog CD4 PE-Cy7 (Bio-Rad, MCA1038PeCy7), rat anti-dog CD8 Pacific Blue (Bio-Rad, MCA1039PB), and mouse anti-dog CD21 Alexa Fluor 647 (Bio-Rad, MCA1781A647).

Techniques: Immunofluorescence, Isolation, Microscopy, Staining, Epifluorescence Microscopy, Immunostaining

Journal: ImmunoHorizons

Article Title: Characterization of Canine Peyer’s Patches by Multidimensional Analysis: Insights from Immunofluorescence, Flow Cytometry, and Single-Cell RNA Sequencing

doi: 10.4049/immunohorizons.2300091

Figure Lengend Snippet: Characterization of T and B cell populations in canine Peyer’s patches by flow cytometry. ( A ) Peyer’s patches (PPs) from two dogs were dissociated, and single-cell suspensions were stained for flow cytometry in a Cytek Northern Lights flow cytometer. ( B ) Proportions of T and B cells. T cells accounted for 41.9% and B cells for 22.2% of the cell population analyzed. Within the B cell population, 11.6% of the cells were positive for Bcl-6. T cells were further separated into CD4 + , CD8 + , and CD4 + CD8 + subsets, revealing high levels of heterogeneity in the expression of the regulatory marker FOXP3. ( C ) FOXP3 expression in T cell subsets. The percentage of FOXP3 + cells was significantly higher among CD3 + CD4 + CD8 + cells than among CD3 + CD4 + or CD3 + CD8 + cells. Statistical analysis was performed by two-way ANOVA. * p ≤ 0.05, **** p ≤ 0.001. dp, double-positive.

Article Snippet: We used the following Abs: mouse anti-dog CD3 Alexa Fluor 700 (Bio-Rad, MCA1774A700), rat anti-dog CD4 PE-Cy7 (Bio-Rad, MCA1038PeCy7), rat anti-dog CD8 Pacific Blue (Bio-Rad, MCA1039PB), and mouse anti-dog CD21 Alexa Fluor 647 (Bio-Rad, MCA1781A647).

Techniques: Flow Cytometry, Staining, Northern Blot, Expressing, Marker

Journal: ImmunoHorizons

Article Title: Characterization of Canine Peyer’s Patches by Multidimensional Analysis: Insights from Immunofluorescence, Flow Cytometry, and Single-Cell RNA Sequencing

doi: 10.4049/immunohorizons.2300091

Figure Lengend Snippet: Gene expression distribution, B and T cell subpopulations, and the Peyer’s patch atlas. ( A ) Marker expression. The expression of markers such as ICOS, TOP2A, JCHAIN, CCR10, and RORC was visualized across the clusters, facilitating cell subtype identification. ( B ) T cell subpopulations. The distribution of different T cell subpopulations is presented as a pie chart, with the corresponding percentages. ( C ) B cell subpopulations. The distribution of different B cell subpopulations is shown as a pie chart, with the corresponding percentages. ( D ) We propose an annotation of the immune cells identified within clusters, based on the specific expression patterns of marker genes. Bcl-6, B cell lymphoma 6; CD62L, CD62 L-selectin; DZ, dark zone; GC, germinal center; ILC3, innate lymphoid cells group 3; LZ, light zone; PD-1, programmed cell death protein 1; Tfh, T follicular helper cell.

Article Snippet: We used the following Abs: mouse anti-dog CD3 Alexa Fluor 700 (Bio-Rad, MCA1774A700), rat anti-dog CD4 PE-Cy7 (Bio-Rad, MCA1038PeCy7), rat anti-dog CD8 Pacific Blue (Bio-Rad, MCA1039PB), and mouse anti-dog CD21 Alexa Fluor 647 (Bio-Rad, MCA1781A647).

Techniques: Gene Expression, Marker, Expressing

Journal: Communications Biology

Article Title: ARP-T1-associated Bazex–Dupré–Christol syndrome is an inherited basal cell cancer with ciliary defects characteristic of ciliopathies

doi: 10.1038/s42003-021-02054-9

Figure Lengend Snippet: a , c , e , g mRNA expression of ACTRT1 during differentiation of keratinocytes, NHEK ( a N = 5) and HaCaT ( c N = 3), and epithelial cells, ARPE19 ( e N = 3) and hTERT-RPE1 ( g N = 5). Data are presented as means of the fold change compared to the value of undifferentiated samples. Each open circle represents one independent experiment. b , d , f , h Representative images of ARP-T1 expression during differentiation of keratinocytes, NHEK ( b ) and HaCaT ( d ), and epithelial cells, ARPE19 ( f ) and hTERT-RPE1 ( h ). ARP-T1 was detected using guinea pig anti-ARP-T1 antisera ( b ) or mouse antibody ( d , f , h ). * indicates polymers of ARP-T1 confirmed by mass spectrometry analysis. Keratin 10 and IFT88 were used as markers of cell differentiation in keratinocytes and epithelial cells, respectively, actin and tubulin as loading controls.

Article Snippet: Membranes were incubated overnight at 4 °C with the following antibodies: anti-ARP-T1 (1:1000, SAB1408334, Sigma-Aldrich/1:2000, GP-SH6, Progen), anti-keratin 10 (1:200, MS-611-P0, Thermo Scientific), anti-IFT88 (1:1000, 13967-1-AP, Proteintech, UK), anti-actin (loading control, 1:5000, A2066, Sigma-Aldrich) and anti-alpha tubulin (loading control, 1:5000, T9026, Sigma-Aldrich).

Techniques: Expressing, Mass Spectrometry, Cell Differentiation

Journal: Experimental and Therapeutic Medicine

Article Title: Poly(ADP-ribose) polymerase-1 inhibitor ameliorates dextran sulfate sodium-induced colitis in mice by regulating the balance of Th17/Treg cells and inhibiting the NF- κ B signaling pathway

doi: 10.3892/etm.2020.9566

Figure Lengend Snippet: Expression of IL-1β, TNF-α, IκB-α, NF-κB p65 and phospho-NF-κB p65 in colonic tissues. The mRNA levels of (A) IL-1β and (B) TNF-α were measured by reverse transcription-quantitative PCR. (C) The expression levels of IκB-α, NF-κB p65 and phospho-NF-κB p65 were measured by western blot analysis. GAPDH was used as an internal control for grayscale analyses. The results are presented as the mean ± SD (n=10). * P<0.05 vs. control; # P<0.05 vs. DSS group. 5-AIQ, 5-aminoisoquinolinone; DSS, dextran sodium sulfate; phospho, phosphorylated.

Article Snippet: The membranes were then incubated with the following primary antibodies at 4˚C overnight: Anti-NF-κB p65 (cat. no. 10745-1-AP; 1:2,000), anti-STAT3 (cat. no. 10253-2-AP; 1:1,000), anti-PARP (cat. no. 66520-1-IG; 1:1,000), anti-Foxp3 (cat. no. 22173-1-AP; 1:1,000; all, ProteinTech Group, Inc.), anti-phosphorylated (p)-STAT3 (cat. no. AF3293; 1:500; Affinity Biosciences, Inc.), anti-phosphorylated (p)-NF-κB p65 (cat. no. 3033; 1:1,000; Cell Signaling Technology, Inc.), anti-RORγt (cat. no. bs-23110; 1:1,000), anti-IκB-α (cat. no. bs-1287; 1:1,000; all from BIOSS), anti-GAPDH (cat. no. AB-P-R001; 1:1,000; Hangzhou Goodhere Biotech Co., Ltd.).

Techniques: Expressing, Real-time Polymerase Chain Reaction, Western Blot