Journal: bioRxiv

Article Title: Regulation of heterotopic ossification through local inflammatory monocytes in a mouse model of aberrant wound healing

doi: 10.1101/871574

Figure Lengend Snippet: a-c Injury site homogenates harvested from burn/tenotomy mice on day 0, 3, and 7 post burn/tenotomy. a ) Monocyte/Macrophage associated factors. b ) Monocyte/Macrophage and neutrophil maturation factors. c ) Cytokines stimulated by monocyte factors. d ) TGF family members. e ) Stem cell maintaining factors. Levels of cytokine and chemokines in pg/ug of Total protein, data represented as the median with interquartile range. Changes in cytokines and chemokines across day 3 and day 7 vs day 0 were analyzed by an analysis of variance (ANOVA) with post-hoc Dunnett test (n=3 mice/time point) significance. Non-heteroscedastic data identified by Levene’s test for homogeneity of variances were alternatively analyzed by Welch statistic and post-hoc Dunnett T3. Degrees of freedom (df or df1) across samples = 2. F statistic and significant post-hoc p-values respectively: CXCL1: 30.359, p(D0 vs. D7)=0.036, CXCL2: 8.504, CCL2: 268.773, p(D0 vs. D3)=0.000, p(D0 vs. D7)=0.000, CCL3: 16.430, CCL4: 22.441, p(D0 vs. D3)=0.014, G-CSF: 12.579, GM-CSF: 4.988, IL-1b: 3.486, IL-6: 13.019, TNF-α: 38.435, p(D0 vs. D7)=0.019, TGF-β1: 9.156, TGF-β2: 11.376, TGF-β3: 7.362, CCL5: 0.825, CXCL5: 0.825, LIF: 25.368, p(D0 vs. D3)=0.001, p(D0 vs. D7)=0.002 *p<.05 **p<.01. f) t-SNE dimensionality reduction analysis of single cell sequencing from day 3 cells harvested at the extremity injury site revealed 14 distinct cell clusters (representative, performed in triplicate). g,h) Feature plots displaying the single cell gene expression of g) monocyte/macrophage cytokines and chemokines increased in the homogenates and h) their receptors.

Article Snippet: After re-blocking the slides, the slides were incubated with a mouse anti-human TGF-β1 antibody (Cat No. MAB240, R&D Systems) at a dilution of 1:50 overnight.

Techniques: Sequencing, Expressing

Journal: bioRxiv

Article Title: Regulation of heterotopic ossification through local inflammatory monocytes in a mouse model of aberrant wound healing

doi: 10.1101/871574

Figure Lengend Snippet: a) Left: GSEA analysis of microarray data collected from buffy coat of human burn injury patients at increased risk of HO compared to post-surgical control patients. Right: GSEA analysis of RNAseq performed of tendon injury site 3 weeks after burn/tenotomy in mice. b) Western blot of whole tissue protein collected from the injury site of C57BL/6J mice after burn/tenotomy at indicated time points. A western blot for p-SMAD2 and H3 was performed on the nuclear fraction and SMAD2 and alpha-Tubulin were performed on the cytosolic fraction. n=5 were pooled for each time point. c) Co-localization of F4/80+ and TGF-β1 at tendon injury site 1 week after burn/tenotomy. Scale bars correspond to 100um. d) Left: Co-localization of CD68+ and TGF-β1 in early human HO. Right: Co-localization of p-SMAD2 and PDGFRα in human HO.

Article Snippet: After re-blocking the slides, the slides were incubated with a mouse anti-human TGF-β1 antibody (Cat No. MAB240, R&D Systems) at a dilution of 1:50 overnight.

Techniques: Microarray, Western Blot

Journal: bioRxiv

Article Title: Regulation of heterotopic ossification through local inflammatory monocytes in a mouse model of aberrant wound healing

doi: 10.1101/871574

Figure Lengend Snippet: a) Representative Safranin O stain of tendon injury site 3 weeks after burn/tenotomy in p7N3 (CD47 agonist) treated and PBS control mice. n=3/group. b) MicroCT analysis of tenotomy site 9 weeks after burn/tenotomy in PBS and p7N3 (CD47 agonist) treated mice. Left: Representative 3D reconstruction. Right: Quantification of total HO, floating HO and proximal HO.n=7/group. Total HO: t=3.415, df=7.840, p=0.009; Floating HO: t=2.201, df=12, p=0.048; Proximal HO: t=2.686, df=8.549, p=0.026. c) Levels of TGF-β1, TGFβ2, and TGFβ3 in pg/ug total protein and represented as median with interquartile range from Top: homogenates from the extremity injury (TGF-β1: t=-0.635, df=4, p=0.560; TGF-β2: t=-0.643, df=4, p=0.555; TGF-β3: t=-1.272, df=2.186, p=0.322) and Bottom: plasma from PBS and p7N3 (CD47) peptide treated mice 3days after burn/tenotomy (TGF-β1: t=1.544, df=2.037, p=0.260; TGF-β2: t=2.747, df=4, p=0.052; TGF-β3: t=-1.492, df=4, p=0.210). n=3 mice per treatment group. d) qPCR analysis of M1 ( iNos ) and M2 ( Arg1 and Mrc1 ) macrophage markers and Tgfb1 in macrophages isolated from the extremity injury site of naive (day 0), burn/tenotomy day 3, burn/tenotomy day 3 treated with PBS, and burn/tenotomy day 3 treated with p7N3 (CD47) peptide. Day 0 vs. Day 3 – iNOS : t=2.020, df=2, p=0.181; Arg1 : t=-6.084, df=3, p=0.009; Mrc1 : t=0.703, df=4, p=0.521; Tgfb1 : t=0.253, df=4, p=0.812. PBS vs. CD47 – iNOS : t=-0.834, df=2.043, p=0.491; Arg1 : t=0.895, df=4, p=0.421; Mrc1 : t=1.176, df=4, p=0.305; Tgfb1 : t=1.186, df=4, p=0.301. e) Representative images of phagocytosis assay using macrophages isolated from the extremity injury at day 3 after burn/tenotomy in mice treated with PBS or p7N3 (CD47). PBS n=3, CD47 n=4 approximately 25 cells/mouse. f) Measurement of cellular circularity Circularity: t=6.119, df=55.537, p=0.000 and quantification of mean fluorescent intensity phagocytosed by each macrophage. MFI: t=-0.357, df=111, p=0.722.

Article Snippet: After re-blocking the slides, the slides were incubated with a mouse anti-human TGF-β1 antibody (Cat No. MAB240, R&D Systems) at a dilution of 1:50 overnight.

Techniques: Staining, Isolation, Phagocytosis Assay

Journal: The Journal of Biological Chemistry

Article Title: Interleukin-1β Processing Is Dependent on a Calcium-mediated Interaction with Calmodulin *

doi: 10.1074/jbc.M115.680694

Figure Lengend Snippet: Identification of pro-IL-1β interacting proteins on a human proteome microarray. A human proteome microarray was incubated with human recombinant pro-IL-1β (+) or with reagent diluent alone as a negative control (−) and probed with a mouse anti-human IL-1β antibody. An SNR value was generated for each probe set, with an SNR of >3 considered to be a hit. A comprehensive list of all identified pro-IL-1β interacting proteins (hits) is shown, including one representative negative result (for brain protein 44-like) and a positive control (anti-biotin mouse monoclonal IgG). The identity of each protein, the corresponding images for each duplicate set of proteins on the array, and the mean SNR for the duplicate protein spots are shown.

Article Snippet: Two HuProt human protein microarray slides (v.2.0) containing 19,951 probe sets spotted in duplicate were purchased from CDI Laboratories (Mayaguez, PR).

Techniques: Microarray, Incubation, Recombinant, Negative Control, Generated, Positive Control

Journal: Cell Cycle

Article Title: Relationship between RUNX1 and AXIN1 in ER-negative versus ER-positive Breast Cancer

doi: 10.1080/15384101.2016.1237325

Figure Lengend Snippet: Association between RUNX1 and AXIN1 in ER− breast cancer tumors. Breast cancer tumor microarray TMA-1007 from Protein Biotechnologies, Inc. was immunostained for RUNX1 and AXIN1. The ER− invasive ductal carcinomas were designated as positive or negative for RUNX1 and AXIN1. (A) RUNX1 and AXIN1 status, and the odds ratio and 95% confidence intervals for the association between AXIN1 status and RUNX1 status in the ER− tumors in the TMA. Association between the RUNX1 status and AXIN1 status was tested using the Pearson chi-square test for the 2 × 2 table. (B) RUNX1 and AXIN1 immunohistochemical staining of 2 representative ER− tumors from the TMA illustrating the association between RUNX1 and AXIN1 expression.

Article Snippet: Tissue microarray analysis Breast cancer tissue microarray (TMA) slides used in this study were purchased from Protein Biotechnologies, Inc. (TM-1007).

Techniques: Microarray, Immunohistochemical staining, Staining, Expressing

Journal: Journal of Cancer

Article Title: Cadherin Related Family Member 2 Acts As A Tumor Suppressor By Inactivating AKT In Human Hepatocellular Carcinoma

doi: 10.7150/jca.27663

Figure Lengend Snippet: Involvement of CDHR2 in dephosphorylation of AKT and COX2 downregulation of downstream. High-throughput protein microarray analysis (A) and Western blot detection (B) of p-AKT and total AKT in LV-CDHR2 or LV-luc infected SMMC-7721 cells. (C) The effect of myr-Akt on doubling time of SMMC-7721 cells stably expressing CDHR2. (D) Real-time PCR analysis of mRNA expression of COX2 in SMMC-7721 cells stably expressing CDHR2. (E) The effect of myr-Akt on mRNA expression of COX2 in CDHR2-expressing SMMC-7721 cells.

Article Snippet: Cells were lysed with RIPA buffer and the lysates were collected by centrifugation at 12,000 rpm for 10 min. Proteins were labelled with biotin and loaded on protein microarray slides (Full Moon BioSystems, Sunnyvale, CA, USA) with 6 replicates according to the manufacturer's instruction.

Techniques: De-Phosphorylation Assay, High Throughput Screening Assay, Microarray, Western Blot, Infection, Stable Transfection, Expressing, Real-time Polymerase Chain Reaction