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: Cell

Article Title: Heterogeneous Tumor-Immune Microenvironments among Differentially Growing Metastases in an Ovarian Cancer Patient

doi: 10.1016/j.cell.2017.07.025

Figure Lengend Snippet: Immune Infiltration Status Shows Heterogeneous Microenvironments across Tumor Samples (A) Tumor purity and immune component estimated by analyzing Affymetrix-based transcriptomics ( A) ( Yoshihara et al., 2013 ). (B) Fractions of immune cell subsets in tumor samples inferred from gene-expression data using CIBERSORT ( Newman et al., 2015 ). Width of bars is proportional to the −log 10 p value of the deconvolution ( B). CIBERSORT empirical p value, ∗ p < 0.05. (C) Representative images of hematoxylin and eosin staining of tumor samples and immunofluorescence staining for DAPI, cytotoxic T cells (CD8 + ), helper T cells (CD4 + FOXP3 − ), T cells (CD3 + ), T-regs (CD4 + FOXP3 + ), macrophages (CD68 + ), and immune-checkpoint PD-L1. Complete slides are shown in Figure S3 . (D) Image-based cell quantification of whole slides ( C).

Article Snippet: Next, slides were incubated with anti-FoxP3 (Abcam, cat#ab20034, 5 μg/ml) for 4 hr, followed by 60 min incubation with biotinylated horse anti-mouse IgG (Vector Laboratories, cat# MKB-22258) at 1:200 dilution.

Techniques: Expressing, Staining, Immunofluorescence

Journal: Cell

Article Title: Heterogeneous Tumor-Immune Microenvironments among Differentially Growing Metastases in an Ovarian Cancer Patient

doi: 10.1016/j.cell.2017.07.025

Figure Lengend Snippet: Complete Slide Hematoxylin and Eosin and Immunofluorescent Staining, Related to Figure 3 and Hematoxylin and eosin staining of tumor samples. Immunofluorescence staining for cytotoxic T cells (CD8 + ), helper T cells (CD4 + FOXP3 − ), and regulatory T cells (CD4 + FOXP3 + ).

Article Snippet: Next, slides were incubated with anti-FoxP3 (Abcam, cat#ab20034, 5 μg/ml) for 4 hr, followed by 60 min incubation with biotinylated horse anti-mouse IgG (Vector Laboratories, cat# MKB-22258) at 1:200 dilution.

Techniques: Staining, Immunofluorescence

Journal: Cell

Article Title: Heterogeneous Tumor-Immune Microenvironments among Differentially Growing Metastases in an Ovarian Cancer Patient

doi: 10.1016/j.cell.2017.07.025

Figure Lengend Snippet:

Article Snippet: Next, slides were incubated with anti-FoxP3 (Abcam, cat#ab20034, 5 μg/ml) for 4 hr, followed by 60 min incubation with biotinylated horse anti-mouse IgG (Vector Laboratories, cat# MKB-22258) at 1:200 dilution.

Techniques: Plasmid Preparation, Staining, Recombinant, Sequencing, Microarray, Software, Expressing