Journal: Experimental Biology and Medicine

Article Title: Annona atemoya leaf extract ameliorates cognitive impairment in amyloid-β injected Alzheimer’s disease-like mouse model

doi: 10.1177/1535370219886269

Figure Lengend Snippet: Molecular mechanisms responsible for anti-AD effects of AAL extract. (a) HT22 cells were exposed to H2O2 in the absence or presence of AAL extract (50 μg/mL) for 6 h. Antibody microarray assay was performed using the phospho-specific antibody microarray slide (Full Moon BioSystems). For data acquisition, GenePix 4100A scanner (Axon Instrument, USA) was used. The normalization data were analyzed using Genowiz 4.0™ (Ocimum Biosolutions). The phosphorylation ratio was calculated and represented as fold changes of indicated phosphoproteins after H2O2 treatment normalized to total protein expression (upper panel). Total protein quantification is shown (lower panel). (b) HT22 cells were exposed to H2O2 with or without various concentrations of AAL extract (0, 12.5, 25, or 50 μg/mL) for 6 h. Cell lysates were prepared from HT22 cells and equal amounts of protein were subjected to Western blotting using anti-phospho-CaMK2 β/ν/δ, GRK2, EGFR, Myc, FER, caveolin-1, NFκB p65, and MLRN 2 antibodies to validate the Ab microarray. (c) Protein extracts were prepared from hippocampal tissues in an Aβ-induced AD mouse model. Vehicle or various concentrations of AAL extract (50, 100, or 200 mg/kg) were administered to Aβ mice for 23 days. Western blotting was performed for anti-phospho-EGFR and phospho-GRK2. The validity of the two phospho-antibodies was determined using pre-stained protein marker (Bio-Rad). GAPDH was used as an internal control. Shown blots are representative results from three independent experiments.

Article Snippet: Molecular mechanisms responsible for anti-AD effects of AAL extract. (a) HT22 cells were exposed to H 2 O 2 in the absence or presence of AAL extract (50 μg/mL) for 6 h. Antibody microarray assay was performed using the phospho-specific antibody microarray slide (Full Moon BioSystems).

Techniques: Microarray, Phospho-proteomics, Expressing, Western Blot, Staining, Marker, Control

Journal: Acta Neuropathologica

Article Title: Activin receptors regulate the oligodendrocyte lineage in health and disease

doi: 10.1007/s00401-018-1813-3

Figure Lengend Snippet: Activin receptor signaling regulates oligodendrocyte differentiation. a Mean number of oligodendrocyte lineage cells (Olig2+) per field ± s.e.m. in corpus callosum of P16 Acvr1b fl/fl ( n = 3 mice), PDGFRa-Cre; Acvr1b fl/+ ( n = 4 mice) and PDGFRa-Cre; Acvr1b fl/fl mice ( n = 7 mice). b Mean proportion of oligodendrocyte lineage cells (Olig2+) which are mature oligodendrocytes (CC1+) versus immature cells (CC1−), per field ± s.e.m. in corpus callosum of P16 Acvr1b fl/fl ( n = 4 mice) and PDGFRa-Cre; Acvr1b fl/fl mice ( n = 7 mice). Multiple t tests with false discovery rate of 1%, *** P = 0.000026. c Images of differentiating oligodendrocytes (cytoplasmic Olig1+ and nuclear Olig2+) in corpus callosum of P16 Acvr1b fl/fl and PDGFRa-Cre; Acvr1b fl/fl mice. Scale bar 25 μm. d Mean number of cytoplasmic Olig1 and Olig2 double positive cells per field ± s.e.m. in corpus callosum of P16 Acvr1b fl/fl ( n = 3 mice), PDGFRa-Cre; Acvr1b fl/+ ( n = 4 mice) and PDGFRa-Cre; Acvr1b fl/fl mice ( n = 7 mice). Two-tailed Student’s t test, ** P = 0.0047, 0.0026, respectively. e Images of maturing oligodendrocytes (MAG+ MBP−) at P1 in corpus callosum of Acvr1b fl/fl and PDGFRa-Cre; Acvr1b fl/fl mice. Scale bar 25 μm. f Mean number of MAG+ cultured oligodendrocytes per field in vehicle control-treated or activin-A-treated conditions (10 ng ml −1 ) in vitro. n = 3 biological replicates. Two-tailed paired Student’s t test, * P = 0.0484. g Images of cultured OPCs treated with vehicle or 10 ng ml −1 activin-A and immunostained for MAG (green), counterstained with Hoechst (blue). Scale bar 25 μm. h Data-mining of microarray of human fetal brain at 9 and 12 gestational weeks (gw) represented as fold change in expression (normalized to 9 gw), showing paralleled expression changes between activin-A ( INHBA ) and oligodendrocyte differentiation-associated genes ( MAG , MOG ) in development

Article Snippet: Samples were applied to TGF-β phospho-antibody microarray slides (Full Moon Biosystems) which have 176 immobilized antibodies against phosphorylated and unphosphorylated specific residues in proteins associated with the 5 TGFβ signaling pathways, with 6 technical replicates per antibody.

Techniques: Two Tailed Test, Cell Culture, Control, In Vitro, Microarray, Expressing

Journal: bioRxiv

Article Title: Oncogenic hijacking of a developmental transcription factor evokes therapeutic vulnerability for ROS-induction in Ewing sarcoma

doi: 10.1101/578666

Figure Lengend Snippet: a) Western blot analysis 96h after Dox-induced shRNA-mediated SOX6 knockdown in RDES and TC-32 EwS cells. GAPDH served as loading control. b) Top: Volcano plot of microarray data showing differentially expressed genes (DEGs) after shRNA-mediated SOX6 knockdown compared to a non-targeting shCtrl. A summary of two EwS cell lines is shown. Bottom: Representative enrichment plots from GSEA of transcriptome profiles of RDES and TC-32 EwS cells 96h after induction of shRNA-mediated SOX6 silencing. c) Left: Quantification of the sphere index after 12 days of Dox-treatment in RDES and TC-32 cells. Horizontal bars represent means and whiskers the SEM, n =3. P values determined via two-sided Mann-Whitney test. Right: Representative micrographs of RDES/TR/shSOX6_3 spheres. Scale bar=1 mm. d) Analysis of tumor growth of xenografted RDES and TC-32 cells containing either Dox-inducible specific shRNAs against SOX6 (shSOX6_2/shSOX6_3) or a non-targeting control shRNA (shCtrl). When tumors were palpable (arrow), mice were randomized and henceforth treated with Dox (+) or vehicle (–). Data are represented as means and SEM, n ≥3 mice per condition. P values determined via two-sided Mann-Whitney test. e) Representative micrographs of xenografts from ( d ) showing IHC stains for SOX6, cleaved caspase 3 and Ki67. Scale bar=20µm. f) Quantification of the relative number of mitoses per high-power field (HPF) of xenografts shown in ( d ). Horizontal bars represent means and whiskers the SEM, n ≥3. P values determined via two-sided Mann-Whitney test. g) Quantification of the relative number of cells positive for cleaved caspase 3 of xenografts shown in ( d ). Horizontal bars represent means and whiskers the SEM, n ≥3. *** P <0.001, ** P <0.01, * P <0.05.

Article Snippet: Slides were incubated with the polyclonal cleaved caspase 3 primary antibody (rabbit, 1:100; 9661, Cell Signaling, Frankfurt am Main, Germany) for 60 min at RT followed by ImmPRESS Reagent Kit.

Techniques: Western Blot, shRNA, Microarray, MANN-WHITNEY

Journal: bioRxiv

Article Title: Oncogenic hijacking of a developmental transcription factor evokes therapeutic vulnerability for ROS-induction in Ewing sarcoma

doi: 10.1101/578666

Figure Lengend Snippet: a) Analysis of publicly available matched gene expression and drug-response data of up to 22 EwS cell lines per drug. Highlighted in dark grey, top 7 drugs with P <0.02; pink = Elesclomol. b) LN_IC50 (µM) of the top 7 drugs including Federatinib (JAK-2 inhibitor), PHA-793887 (CDK2/5/7 inhibitor), Rucaparib (PARP inhibitor), Serdemetan (p53 activator), Imatinib (tyrosine kinase inhibitor) and Olaparib (PARP1/2 inhibitor) with P <0.02. Horizontal bars represent means and whiskers SEM, n ≥18 EwS cell lines. c ) Quantification of relative viability of indicated cell lines by a Resazurin assay after treatment with Elesclomol at indicated concentrations for 72h. Modeled dose-response curves and calculated IC50 values (nM) are displayed for SOX6-high expressing EwS cells (black and grey) and the SOX6-low expressing osteosarcoma cell line SAOS-2 and the mesenchymal cell line MSC-52 (dark and light green), n ≥3. d ) Analysis of relative SOX6 expression in indicated cell lines by qRT-PCR. Horizontal bars represent means and whiskers SEM, n ≥3. P values determined via two-sided Mann-Whitney test. e ) Analysis of cell viability of indicated cell lines by a Resazurin assay. Horizontal bars represent means and whiskers SEM, n ≥5. P values determined via two-sided Mann-Whitney test. f ) Quantification of relative Elesclomol IC50 values by a Resazurin assay in indicated cell lines after 72h of Elesclomol treatment and concomitant addition of Dox. Horizontal bars represent means and whiskers SEM, n =7. P values determined via two-sided Mann-Whitney test. g ) Quantification of relative Annexin V positivity of indicated EwS cells 48h after treatment with Elesclomol (10 nM). Horizontal bars represent means and whiskers SEM, n =10. P values determined via unpaired two-sided t-test with Welch’s correction. h ) Analysis of tumor growth of TC-32 EwS cells in NSG mice treated once per day (day 0-4 and day 7-9) with Elesclomol (intravenously, 5 mg/kg). Data represent means and SEM, n =5 mice per condition. P values determined via two-sided Mann-Whitney test. i ) Left: Quantification of the average number of cleaved caspase 3 positive cells per 3 HPF in TC-32 xenografts shown in ( h ). Horizontal bars represent means and whiskers SEM, n =5 per condition. P values determined via two-sided Mann-Whitney test. Right: representative micrographs. Scale bar=100 µm. j ) Left: Quantification of necrotic area in TC-32 xenografts shown in ( h ). Horizontal bars represent means and whiskers SEM, n =5 per condition. P values determined via two-sided Mann-Whitney test. Right: Representative micrographs. Scale bar = 900 µm. *** P <0.001, ** P <0.01, * P <0.05.

Article Snippet: Slides were incubated with the polyclonal cleaved caspase 3 primary antibody (rabbit, 1:100; 9661, Cell Signaling, Frankfurt am Main, Germany) for 60 min at RT followed by ImmPRESS Reagent Kit.

Techniques: Expressing, Resazurin Assay, Quantitative RT-PCR, MANN-WHITNEY

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