Journal: PLOS ONE

Article Title: Novel Bruton’s tyrosine kinase inhibitor TAS5315 suppresses the progression of inflammation and joint destruction in rodent collagen-induced arthritis

doi: 10.1371/journal.pone.0282117

Figure Lengend Snippet: (a) Phosphorylation of BTK induced by RANKL stimulation (100 ng/mL) for 24 h in M-CSF-induced murine macrophages. Macrophages were simultaneously subjected to TAS5315 and RANKL stimulation. (b) Nuclear translocation of NFATc1 induced by RANKL stimulation (100 ng/mL) for 24 h in M-CSF-induced murine macrophages. M-CSF-induced murine macrophages were simultaneously subjected to TAS5315 and RANKL stimulation. The figures of immunoblot experiments (a, b) represent the data of three independent experiments. (c, d) Human osteoclast differentiation was induced by RANKL (66 ng/mL) and M-CSF (33 ng/mL) in the presence of DMSO, TAS5315, or anti-RANKL antibody (0.1 μg/mL) for 4 days. (c) Representative images of TRAP-positive osteoclasts. (d) TRAP-positive cells with 3 or more nuclei (cell size ≥50 μm) were counted as osteoclasts. (e) CTX-I levels in the supernatant of human osteoclasts treated with M-CSF (33 ng/mL) and RANKL (66 ng/mL) in the presence of DMSO, TAS5315, or anti-RANKL antibody (0.1 μg/mL) for 14 days. (f, g) Pit formation by mouse osteoclasts treated with RANKL (25 ng/mL) and M-CSF (50 ng/mL) in the presence of DMSO, TAS5315, or anti-RANKL antibody (0.1 μg/mL) for 16 days. (f) Representative images of pit formation by mouse osteoclast (DMSO, TAS5315 100 nM, anti-RANKL antibody). (g) Quantitative analysis of the relative pit formation area by mouse osteoclasts. Experiments (c–g) were performed in triplicates and the numerical data (d, f, g) are presented as mean ± SD. * P <0.05, ** P <0.01, *** P <0.001 compared with DMSO group (Dunnett test for TAS5315 groups or Student’s t-test for anti-RANKL antibody).

Article Snippet: The following antibodies were used: rabbit anti-human phosphorylated BTK (tyrosine 223) monoclonal antibody (EP420Y) (ab68217), mouse anti-human p84 monoclonal antibody (ab487) (Abcam, Cambridge, UK), mouse anti-human BTK monoclonal antibody (611117), fluorescein isothiocyanate-conjugated rat anti-mouse CD45R/B220 monoclonal antibody (553088), phycoerythrin-cyanine 7-conjugated anti-mouse CD45R/B220 monoclonal antibody (552772), allophycocyanin-conjugated rat anti-mouse CD86 monoclonal antibody (558703), brilliant violet 421-conjugated rat anti-mouse I-A/I-E monoclonal antibody (562564) (BD Biosciences, San Jose, CA, USA), APC-conjugated Armenian hamster anti-mouse CD69 monoclonal antibody (17-0691-82; Thermo Fisher Scientific, Waltham, MA, USA), rabbit anti-human G3PDH/GAPDH polyclonal antibody (2275-PC-100; Trevigen, Gaithersburg, MD, USA), rabbit anti-mouse BTK monoclonal antibody (8547), rabbit anti-human phosphorylated PLCγ2 (tyrosine 1217) polyclonal antibody (3871), rabbit anti-human PLCγ2 polyclonal antibody (3872), rabbit anti-human phosphorylated AKT (serine 473) monoclonal antibody (4075), rabbit anti-human AKT polyclonal antibody (9272), rabbit anti-human phosphorylated ERK (threonine 185 and tyrosine 187) monoclonal antibody (4370), rabbit anti-human ERK polyclonal antibody (9102) (Cell Signaling Technology, Danvers, MA, USA), mouse anti-human NFATc1 monoclonal antibody (MABS409), and mouse anti-bovine β-tubulin monoclonal antibody (05–661) (Merck Millipore, Darmstadt, Germany).

Techniques: Translocation Assay, Western Blot

Journal: Cancers

Article Title: Genome-Wide Analysis of lncRNA-mRNA Co-Expression Networks in CD133+/CD44+ Stem-like PDAC Cells

doi: 10.3390/cancers15041053

Figure Lengend Snippet: CD133+/CD44+ PDAC cells are highly oncogenic in vivo. 8×105 CD133+/CD44+, CD133−/CD44− or total (unsorted) cells derived from ( A ) MIA PaCa-2 and ( B ) PANC-1 cell lines were inoculated heterotopically into the flanks of NGS mice, and the tumor growth was monitored. CD133+/CD44+ cells show similar growth curves with total cells, whilst the sorted cells fail to grow. Tumor volumes were plotted as mean +/− SEM for each data point retrieved by two independent experiments (N = 8–10 mice/per group/experiment). ( C ) Representative immunohistochemical staining for Ki67 expression in tumors formed by MIA PaCa-2- and PANC-1-derived CD133+/CD44+ and total cells. Scale Bar = 200 μm. ( D ) The corresponding percentages of Ki67-positive cells indicate no statistically significant differences in proliferation rate between CD133+/CD44+ and total cells from both cell lines. Data were collected through three independent experiments.

Article Snippet: Cell proliferation was calculated by IHC, using a mouse anti-human mAb against Ki67 (Cell Signaling Technology, Inc., Danvers, MA, USA) (1:1000 dilution), followed by incubation with an HRP-conjugated anti-mouse secondary Ab (Sigma-Aldrich).

Techniques: In Vivo, Derivative Assay, Immunohistochemical staining, Staining, Expressing

Journal: Frontiers in Immunology

Article Title: Hyaluronan promotes intracellular ROS production and apoptosis in TNFα-stimulated neutrophils

doi: 10.3389/fimmu.2023.1032469

Figure Lengend Snippet: Total and phospho p38 MAPK in TNFα- and HA-stimulated neutrophils. (A) Western blot showing the phosphorylated and total p38 MAPK protein over time in neutrophils primed with HA (200 μg/mL). Data is representative of 3 independent experiments. (B) Graph shows quantification (mean ± SD) of phospho-p38 normalized to total p38 protein (y-axis) over time (x-axis), n = 3. (C) Western blot showing the phosphorylated and total p38 MAPK protein in neutrophils upon fMLF activation (100 nM, 0 – 2 min) at t = 16 min of priming in the presence (HA + fMLF) or absence (fMLF) of HA (200 μg/mL). Data is representative of 3 independent experiments. (D) Graph shows quantification (mean ± SD) of phospho-p38 normalized to total p38 protein (y-axis) over time (x-axis) during activation with fMLF at t = 16 min of priming with (HA + fMLF) and without (fMLF) HA, n = 3. (E) Abundance of phosphorylated and total p38 MAPK protein over time in neutrophils primed with TNFα (10 ng/mL) and HA + TNFα (200 μg/mL HA). Data is representative of 3 independent experiments. (F) Graph shows quantification (mean ± SD) of phospho-p38 normalized to total p38 (y-axis) over time (x-axis) in TNFα-primed and HA+TNFα co-primed neutrophils, n = 3. Statistical analysis for individual time points was performed using unpaired two-tailed t-test, (B, F) ; *p < 0.05.

Article Snippet: The primary antibodies used were the following: rabbit anti-human p38 mAb (1:2,000; Cell Signalling Technology; #8690), mouse anti-human phospho-p38 mAb (Thr180/Tyr182; 1:2,000; Cell Signalling Technology; #9216).

Techniques: Western Blot, Activation Assay, Two Tailed Test

Journal: Cellular and Molecular Gastroenterology and Hepatology

Article Title: Sex Differences in Genomic Features of Hepatitis B–Associated Hepatocellular Carcinoma With Distinct Antitumor Immunity

doi: 10.1016/j.jcmgh.2022.10.009

Figure Lengend Snippet: List of Antibodies and Reagents

Article Snippet: Human/mouse ERα , WB , Cell Signaling Technology (Danvers, MA) , 8644T , 1:1000.

Techniques: Cell Culture

Journal: PLoS ONE

Article Title: New Candidate Biomarkers in the Female Genital Tract to Evaluate Microbicide Toxicity

doi: 10.1371/journal.pone.0110980

Figure Lengend Snippet: (A) LC MS/MS proteomic analysis of mouse vaginal tissue samples down-regulated in response to treatment with N9 (open, black circles) or BZK (black squares). The following proteins are depicted in the graphs: peptidoglycan recognition protein 1 (PGLYRP-1), CD166 antigen (CD166), mucin 5 subtype B (mucin 5B), olfactomedin-4 (OLFM-4), and anterior gradient protein 2 homolog (AGR2). Each data point represents the average of three replicates per individual experimental study. Only proteins shown to change in a minimum of two studies are represented. (B) RT-PCR analysis of mouse mRNA expression. Relative expression levels are normalized to Glyceraldehyde 3-phosphate dehydrogenase (GADPH). Statistical analysis was performed using a two-tailed Student's T-test of N9, BZK, and tenofovir samples compared to vehicle controls (*** = p≤0.001, * = p≤0.05, NS = not significant). (C) Immunoblot of mouse CD166 antigen. Three mice per condition were analyzed. The CD166 signal was normalized to the β-actin signal for quantitation. Statistical analysis was performed using a two-tailed student's T-test of N9 and BZK samples compared to vehicle controls (*** = p≤0.001, ** = p≤0.01).

Article Snippet: Nonoxynol-9 (N9) was purchased from Spectrum Chemicals and Laboratory Products (Gardena, CA); tenofovir was purchased from AK Scientific (Union City, CA); benzalkonium chloride (BZK) and high viscosity sodium carboxymethylcellulose (CMC) were purchased from Sigma Aldrich (Saint Louis, MO); mouse and rabbit apolipoprotein A-I, apolipoprotein C-1, CD166, and rabbit IL-8 ELISA kits were purchased from EIAab (Wuhan, China); mouse fibrinogen ELISA kit was purchased from Abcam (Cambridge, MA); mouse plasminogen ELISA kit was purchased from GenWay (San Diego, CA); mouse IL-8 ELISA kit was purchased from MyBioSource (San Diego, CA); rabbit plasminogen ELISA kit was purchased from Alpco (Salem, NH); rabbit fibrinogen ELISA kit was purchased from Molecular Innovations (Novi, MI); medroxyprogesteron acetate was purchased from Pharmacia (New York, NY); CellTiter-Glo was purchased from Promega (Madison, WI); RNeasy kits were purchased from Qiagen (Valencia, CA); rabbit anti-human olfactomedin 4 (OLFM-4) polyclonal antibody was purchased from Abcam (Cambridge, MA); rabbit anti-human mucin 5B polyclonal antibody was purchased from Bioss USA Antibodies (Woburn, MA); rabbit anti-mouse CD166 was purchased from GeneTex (Irvine, CA); rabbit anti-mouse β-actin was purchased from Abcam (Cambridge, MA); IRDye 800 Goat anti-rabbit IgG was purchased from Li-Cor (Lincoln, NE); mouse anti-human β-actin monoclonal antibody was purchased from Cell Signaling Technology (Danvers, MA); donkey anti-mouse IgG antibody conjugated to IRDye 680 LT and goat anti-rabbit IgG antibody conjugated to IRDye 800CW were purchased from LI-COR biosciences (Lincoln, NE).

Techniques: Liquid Chromatography with Mass Spectroscopy, Reverse Transcription Polymerase Chain Reaction, Expressing, Two Tailed Test, Western Blot, Quantitation Assay