Journal: bioRxiv

Article Title: FUNGAL DYSBIOSIS CORRELATES WITH THE DEVELOPMENT OF TUMOUR-INDUCED CACHEXIA IN MICE

doi: 10.1101/2020.06.29.171397

Figure Lengend Snippet: Equimolar samples of DNA extracted from stool samples obtained from each mouse were consolidated into two pools, representing SC and CC groups. Samples from these pools were then used in qPCR experiments, with primers previously described in the literature as specific to Saccharomyceae, Kazachstania and Malassezia genera, as well as to the species Saccharomyces cereviseae, Malassezia dermatis, Malassezia japonica, Rhizopus oryzeae and Penicillium citrinum . The Ct values obtained for each of these taxa were normalized by the Ct obtained after amplifying the pooled DNAs with the primer pair used to amplify the fungal ITS1 amplicons . Next, the normalized Ct s were used to calculate the relative prevalence of each genus/species in the CC pool, in relation to the SC pool (CC/CR). The relative quantification values for each taxon ( Fold Change ) are shown in the graph, as the mean ± standard deviation of three independent experiments (each performed in triplicate). The species Rhizopus oryzae was detected only in the DNA pool from SC animals, so its SC/CC ratio is represented in the graph as negative, to infinity (-INF). The species P enicillium citrinum was not detected (N/D) in any of the experiments, with either DNA pool.

Article Snippet: Average sizes of the generated fragments were evaluated after electrophoresis in a Bioanalyzer 2100 (Agilent), using the High Sensitivity DNA Chip and the libraries were quantified by qPCR, in an ABI Prism 7500 Fast Sequence Detection System (Applied Biosystems), with the aid of the universal quantification kit NEBNext (New England Biolabs), according to the manufacturer’s instructions.

Techniques: Quantitative Proteomics, Standard Deviation

Journal: International journal of cancer

Article Title: The intracellular juxtamembrane domain of discoidin domain receptor 2 (DDR2) is essential for receptor activation and DDR2-mediated cancer progression.

doi: 10.1002/ijc.28901

Figure Lengend Snippet: Figure 1. The IJM region is necessary for collagen-induced DDR2 activation. (a) Overall topology and alignment of the transmembrane (TM) domain and the intracellular juxtamembrane region (IJM) of DDR1a and DDR2. The IJM was divided into three regions: JM1, JM2 and JM3. (b) Schematic diagram of various DDR2 constructs used in our study. (c) HEK293T cells transiently transfected with plasmids encoding the full-length DDR2 and F-DJM1-JM2 mutant were stimulated by Type I collagen for 60 min. Tyrosine phosphorylation of the F-DJM1-JM2 mutant was inhibited compared to that of the full-length DDR2. (d) HEK293T cells were transfected with plasmids encoding full-length DDR2, F- DJM1 and F-DJM2 and were stimulated by collagen. The F-DJM2 mutant showed a significant decrease in tyrosine phosphorylation. **p < 0.01, Student’s t-test.

Article Snippet: The antibodies used in our study were as follows: mouse anti-myc (sc-40; Santa Cruz Biotechnology, Santa Cruz, CA), rabbit anti-HA (sc-805; Santa Cruz Biotechnology), rabbit anti-DDR1 (sc-532; Santa Cruz Biotechnol- ogy), goat anti-DDR2 (sc-7555; Santa Cruz Biotechnology), anti-human DDR2 (AF2538; R&D Systems, Minneapolis, MN), anti-phosphotyrosine (clone 4G10; Upstate Biotechnology, Lake Placid, NY) and peroxidase- and fluoresceinconjugated goat anti-mouse IgG (Jackson ImmunoResearch Laboratories, West Grove, PA).

Techniques: Activation Assay, Construct, Transfection, Mutagenesis, Phospho-proteomics

Journal: International journal of cancer

Article Title: The intracellular juxtamembrane domain of discoidin domain receptor 2 (DDR2) is essential for receptor activation and DDR2-mediated cancer progression.

doi: 10.1002/ijc.28901

Figure Lengend Snippet: Figure 2. DDR2 dimerizes via the JM2 of the IJM region. (a and b) HEK293T cells were transiently cotransfected with plasmids encoding TM- JM1-JM2-myc and TM-JM1-JM2-HA. Immunoprecipitation and Western blot analysis showed that the cytoplasmic domains of DDR2 bind to each other via the intact TM-JM1-JM2 domain and form homodimers. Asterisks indicate the expected size of TM-JM1-JM2. (c) HEK293T cells were transfected with plasmids encoding F-DJM1 and F-DJM2 mutants. A crosslinking assay showed that dimers of F-DJM1 were not changed compared to full-length DDR2, whereas dimers were significantly decreased for F-DJM2.

Article Snippet: The antibodies used in our study were as follows: mouse anti-myc (sc-40; Santa Cruz Biotechnology, Santa Cruz, CA), rabbit anti-HA (sc-805; Santa Cruz Biotechnology), rabbit anti-DDR1 (sc-532; Santa Cruz Biotechnol- ogy), goat anti-DDR2 (sc-7555; Santa Cruz Biotechnology), anti-human DDR2 (AF2538; R&D Systems, Minneapolis, MN), anti-phosphotyrosine (clone 4G10; Upstate Biotechnology, Lake Placid, NY) and peroxidase- and fluoresceinconjugated goat anti-mouse IgG (Jackson ImmunoResearch Laboratories, West Grove, PA).

Techniques: Immunoprecipitation, Western Blot, Transfection

Journal: International journal of cancer

Article Title: The intracellular juxtamembrane domain of discoidin domain receptor 2 (DDR2) is essential for receptor activation and DDR2-mediated cancer progression.

doi: 10.1002/ijc.28901

Figure Lengend Snippet: Figure 3. JM2 has a dominant-negative effect on DDR2 activation. (a and b) HEK293T cells were cotransfected with plasmids encoding full- length DDR2-myc and TM-JM1-JM2-HA. Immunoprecipitation and Western blotting showed that the full-length DDR2 and TM-JM1-JM2 bind to each other to form heterodimers. The immunoprecipitates obtained with anti-IgG antibodies were used as a negative control. (c) H1299 cells transfected with plasmids encoding full-length DDR2 and TM-JM1-JM2 and HeLa cells were lysed, and the whole cell lysates (W) were separated into the plasma membrane (P) and cytosol (C) fractions. EGFR and a-tubulin were used as positive controls for the plasma mem- brane and cytosol fractions, respectively. Endogenous full-length DDR2 (HeLa cells), forced-expressed full-length DDR2 and TM-JM1-JM2 pro- teins were appropriately localized in the plasma membrane. (d) HEK293T cells were transfected with plasmids encoding a C-terminally myc- tagged full-length DDR2 and TM-JM1-JM2. Only under the permeabilized condition, full-length DDR2-myc and TM-JM1-JM2-myc were visual- ized, indicating that the C-termini of these proteins were located in the cytosol and not extracellular space. Full-length DDR2 was used as a positive control. Bar, 50 mm. (e) HEK293T cells were cotransfected with plasmids encoding full-length DDR2 (500 ng) and an increasing amount of TM-JM1-JM2 (100, 300 and 500 ng) as indicated and then stimulated with collagen. Tyrosine phosphorylation gradually decreased with an increasing amount of TM-JM1-JM2. (f) HeLa cells were transiently transfected with a plasmid encoding TM-JM1-JM2 and were then stimulated. Tyrosine phosphorylation was significantly decreased in endogenous DDR2. **p < 0.01, Student’s t-test. [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]

Article Snippet: The antibodies used in our study were as follows: mouse anti-myc (sc-40; Santa Cruz Biotechnology, Santa Cruz, CA), rabbit anti-HA (sc-805; Santa Cruz Biotechnology), rabbit anti-DDR1 (sc-532; Santa Cruz Biotechnol- ogy), goat anti-DDR2 (sc-7555; Santa Cruz Biotechnology), anti-human DDR2 (AF2538; R&D Systems, Minneapolis, MN), anti-phosphotyrosine (clone 4G10; Upstate Biotechnology, Lake Placid, NY) and peroxidase- and fluoresceinconjugated goat anti-mouse IgG (Jackson ImmunoResearch Laboratories, West Grove, PA).

Techniques: Dominant Negative Mutation, Activation Assay, Immunoprecipitation, Western Blot, Negative Control, Transfection, Clinical Proteomics, Membrane, Positive Control, Phospho-proteomics, Plasmid Preparation

Journal: International journal of cancer

Article Title: The intracellular juxtamembrane domain of discoidin domain receptor 2 (DDR2) is essential for receptor activation and DDR2-mediated cancer progression.

doi: 10.1002/ijc.28901

Figure Lengend Snippet: Figure 4. JM2 regulates the collagen-binding affinity of DDR2. (a and b) HEK293T cells transiently transfected with various DDR2 constructs were harvested, and protein expression was verified by Western blot- ting (a). Collagen-binding affinities were reduced in F-DJM2, F-DJM1- JM2 and extra mutants but not the F-DJM1 mutant in a dose- dependent manner (b). Nontransfected (NC) and TM-JM1-JM2 samples were used as negative controls. **p< 0.01, Student’s t-test.

Article Snippet: The antibodies used in our study were as follows: mouse anti-myc (sc-40; Santa Cruz Biotechnology, Santa Cruz, CA), rabbit anti-HA (sc-805; Santa Cruz Biotechnology), rabbit anti-DDR1 (sc-532; Santa Cruz Biotechnol- ogy), goat anti-DDR2 (sc-7555; Santa Cruz Biotechnology), anti-human DDR2 (AF2538; R&D Systems, Minneapolis, MN), anti-phosphotyrosine (clone 4G10; Upstate Biotechnology, Lake Placid, NY) and peroxidase- and fluoresceinconjugated goat anti-mouse IgG (Jackson ImmunoResearch Laboratories, West Grove, PA).

Techniques: Binding Assay, Transfection, Construct, Expressing, Western Blot, Mutagenesis

Journal: International journal of cancer

Article Title: The intracellular juxtamembrane domain of discoidin domain receptor 2 (DDR2) is essential for receptor activation and DDR2-mediated cancer progression.

doi: 10.1002/ijc.28901

Figure Lengend Snippet: Figure 5. Colony formation and proliferation of tumor cells are suppressed by overexpression of JM2. (a and b) Formalin-fixed tissue micro- array slides were used in immunohistochemistry experiments. DDR2 was overexpressed in bladder, testis, lung, kidney, prostate and stom- ach cancers. Bar, 50 mm. (c) Stable TM-JM1-JM2–expressing H1299 cells were stimulated by collagen and then harvested. Immunoprecipitation and Western blot analysis showed that tyrosine phosphorylation of DDR2 was decreased by TM-JM1-JM2 overexpres- sion (labeled JM1/2), but phosphorylation of DDR1 was unaffected. (d) A colony-forming assay of H1299 cells showed that the number and projected area of colonies were decreased by TM-JM1-JM2 overexpression. Bar, 100 mm. (e) Proliferation of H1299 cells was assessed by cell counting (left) and an MTT assay (right). Cell proliferation was inhibited by TM-JM1-JM2 overexpression. **p < 0.01, Student’s t-test; control, nontransfected cells; Mock, empty vector stably transfected cells; JM1/2, TM-JM1-JM2 stably transfected cells. [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]

Article Snippet: The antibodies used in our study were as follows: mouse anti-myc (sc-40; Santa Cruz Biotechnology, Santa Cruz, CA), rabbit anti-HA (sc-805; Santa Cruz Biotechnology), rabbit anti-DDR1 (sc-532; Santa Cruz Biotechnol- ogy), goat anti-DDR2 (sc-7555; Santa Cruz Biotechnology), anti-human DDR2 (AF2538; R&D Systems, Minneapolis, MN), anti-phosphotyrosine (clone 4G10; Upstate Biotechnology, Lake Placid, NY) and peroxidase- and fluoresceinconjugated goat anti-mouse IgG (Jackson ImmunoResearch Laboratories, West Grove, PA).

Techniques: Over Expression, Microarray, Immunohistochemistry, Expressing, Immunoprecipitation, Western Blot, Phospho-proteomics, Labeling, Cell Counting, MTT Assay, Control, Plasmid Preparation, Stable Transfection, Transfection

Journal: PLoS ONE

Article Title: Expression of MicroRNA-15b and the Glycosyltransferase GCNT3 Correlates with Antitumor Efficacy of Rosemary Diterpenes in Colon and Pancreatic Cancer

doi: 10.1371/journal.pone.0098556

Figure Lengend Snippet: ( A ) Venn diagram representing overlap of the genes significantly modulated (p<0.05 and fold change (FC) >±50%) by RE-2 at 30, 60 and 100 µg/mL after 48 h treatment in SW620 cells according to microarray data. ( B ) Up-regulation of GCNT3 gene expression in SW620 colon cancer cells by five RE's with different composition (RE-1, RE-2, RE-3, RE-4 and RE-5) at the same concentration (90 µg/mL) during 48 h. Bars represent the mean ± SEM of two independent experiments, each performed with biological triplicates and technical duplicates. ANOVA with Bonferroni's post hoc test was applied to test statistically significant differences among RE's. ( C ) Up-regulation of GCNT3 gene expression in SW620 colon cancer cells by 48 h treatment with RE-4 at 60 µg/mL and carnosic acid, carnosol, and the combination of both compounds at the concentrations present in this RE. Bars represent the mean ± SEM of two independent experiments, each performed with biological triplicates and technical duplicates. Student's t test was applied to determine the statistically significant differences between treated and control cells. ( D ) Down-regulation of miR-15b gene expression in SW620 colon cancer cells by 48 h treatment with RE-4 at 60 µg/mL and carnosic acid, carnosol, and the combination of both compounds at the concentrations present in this RE. Student's t test was used to test statistically significant differences between treated and control cells. ( E ) Modulation of miR-15b and miR-939 (as a negative control) in plasma of colon cancer xenograft nude mice after orally intake of RE-5 (1 mg/mL in drinking water), in comparison with the control group of mice. Bars represent mean ± SEM (n = 8). Student's t test was used to assess statistically significant differences between treated and control groups. * p≤0.05, ** p≤0.01, *** p≤0.001. RE: supercritical rosemary extract, Cc+Cl: Carnosic acid and carnosol.

Article Snippet: For gene expression analysis of GCNT3, we used the specific Taqman gene expression assay Hs01921181_s1 (Applied Biosystems). qRT-PCR was performed in the 7900HT Real-Time PCR System (Applied Biosystems), according to the manufacturer's protocol.

Techniques: Microarray, Gene Expression, Concentration Assay, Control, Negative Control, Clinical Proteomics, Comparison

Journal: PLoS ONE

Article Title: Expression of MicroRNA-15b and the Glycosyltransferase GCNT3 Correlates with Antitumor Efficacy of Rosemary Diterpenes in Colon and Pancreatic Cancer

doi: 10.1371/journal.pone.0098556

Figure Lengend Snippet: Modulation of gene and miRNA expression by RE-2 in colon cancer cells.

Article Snippet: For gene expression analysis of GCNT3, we used the specific Taqman gene expression assay Hs01921181_s1 (Applied Biosystems). qRT-PCR was performed in the 7900HT Real-Time PCR System (Applied Biosystems), according to the manufacturer's protocol.

Techniques: Expressing

Journal: Molecular psychiatry

Article Title: Ovarian steroids regulate gene expression related to DNA repair and neurodegenerative diseases in serotonin neurons of macaques

doi: 10.1038/mp.2014.178

Figure Lengend Snippet: Gene names, symbol, AB assay ID, context sequence, and NCBI gene reference information of genes included on the Taqman qRT-PCR card. The microarray signal intensity of a representative probe set is also shown.

Article Snippet: 10. PARP2* , poly(ADP-ribosyl)transferase-like 2 , capable of catalyzing a poly(ADP-ribosyl)ation reaction , XM_001088314.2 , Rh02857777_m1 , 13 , 172 , 39.

Techniques: Sequencing, Microarray, Activity Assay, Transduction, Translocation Assay, Ubiquitin Proteomics, Conjugation Assay, Activation Assay, Membrane

Journal: Nature communications

Article Title: TGF-β3-expressing CD4+CD25(-)LAG3+ regulatory T cells control humoral immune responses.

doi: 10.1038/ncomms7329

Figure Lengend Snippet: Figure 2 | LAG3 þ Treg regulate B-cell functions through Fas. (a–d) Treatment of MRL/lpr mice with adoptive transfer of T-cell subsets. Ten-week-old MRL/lpr mice were injected i.v. with LAG3 þ Treg (n ¼ 9), CD4 þCD25 LAG3 T cells (LAG3 T; n ¼ 9), CD4 þCD25 þ Treg (CD25 þ Treg; n ¼ 9) or CD4 þCD25 CD45RBhigh T cells (naive T; n ¼ 8) from MRL/ þ mice (1 105 cells each). The mice of LAG3 þ Treg x3 group (n ¼ 8) were injected with LAG3 þ Treg (1 105 cells) at 10 weeks of age followed by a twice weekly injection of the same amount of LAG3 þ Treg. The control group received PBS (n ¼ 13). (a) Proteinuria progression. *Po0.05 versus control group (Mann–Whitney U-test). (b) Quantification of serum anti-ds DNA antibodies. *Po0.05 (Bonferroni post-test). (c) Haematoxylin and eosine (H&E) staining (upper panels) and IgG immunofluorescent staining (lower panels) of kidney sections. Scale bars, 50 mm. (d) Glomerular scores. *Po0.05 (Mann–Whitney U-test). (e) LAG3 þ Treg-mediated suppression of in vitro NP-specific antibody responses. B cells and Th cells purified from NP-OVA/alum-pre-immunized B6 mice and OT-II mice, respectively, were incubated with or without LAG3 þ Treg from non-immunized OT-II mice in the presence or absence of anti-FasL blocking antibody, and supernatants were analysed for anti-NP-BSA antibodies using ELISA. See also Supplementary Fig. 1e (n ¼ 6 per group). *Po0.05 (Bonferroni post-test). (f) NP-specific antibody responses of Rag1KO mice injected with B6 B cells and OT-II Th cells with or without LAG3 þ Treg from WT, B6/lpr or B6/gld mice, as outlined in Fig. 1e (n ¼ 6 per group). Anti- FasL blocking antibody (200 mg per mouse) was injected i.v. weekly. *Po0.05 (Bonferroni post-test). (g–i) Flow cytometry plots (g) and quantification of splenic CD4 þCD25 CXCR5 þPD-1 þ TFH (h) and B220 þGL-7 þFas þ GCB (i) from the same mice as in f. Statistical significances in h,i were analysed by Bonferroni post-test (*Po0.05). The experiments in e,f were repeated three times. The means±s.d. are indicated.

Article Snippet: B cells undergoing apoptosis on day 3 and total IgG production in the culture supernatants on day 7 were determined using the Annexin V Apoptosis Detection Kit (BD Pharmingen) and a mouse IgG ELISA Quantitation Set (Bethyl Laboratories), respectively, according to the manufacturer’s protocol.

Techniques: Adoptive Transfer Assay, Injection, Control, MANN-WHITNEY, Staining, In Vitro, Incubation, Blocking Assay, Enzyme-linked Immunosorbent Assay, Flow Cytometry

Journal: Nature communications

Article Title: TGF-β3-expressing CD4+CD25(-)LAG3+ regulatory T cells control humoral immune responses.

doi: 10.1038/ncomms7329

Figure Lengend Snippet: Figure 3 | LAG3 þ Treg suppress B-cell activation through TGF-b3. (a) Microarray comparisons of the gene expression profiles between B6 CD25 þ Treg and B6 LAG3 þ Treg. Normalized expression values from B6 CD4 þCD25 CD45RBhigh naive Tcells are depicted according to the colour scale shown. (b) Tgfb3 mRNA expression in sorted T-cell subsets taken from the spleens of B6 mice (n ¼ 3 per group). (c–e) TGF-b1, 2 and 3 protein levels in the culture supernatants of the indicated T-cell subsets from B6 mice determined using ELISA. Cells were seeded at 1 105 cells per well (n ¼ 4 per group). (f) CFSE- labelled B cells were stimulated with or without anti-IgM mAb in the presence or absence of rTGF-b3 (n ¼ 3 per group). (g) Viability of anti-IgM-stimulated B cells in the presence or absence of rTGF-b3 (1 ng ml 1) was assessed by 7-AAD (n ¼ 3 per group). (h) The effects of TGF-b3 on total IgG production in the culture supernatants of anti-CD40/IL-4-stimulated B cells, determined as in Fig. 1d (n ¼ 3 per group). *Po0.05 (unpaired two-tailed Student’s t-test). (i–k) STAT6 (i), Syk (j) and NF-kB p65 (k) phosphorylation in stimulated B cells with or without rTGF-b3, calculated as the ratio of phosphorylated to total protein levels (n ¼ 3 per group). See also Supplementary Fig. 10. *Po0.05 (unpaired two-tailed Student’s t-test). (l) TGF-b3 protein levels in the culture supernatants of freshly isolated B6 LAG3 þ Treg or, naive B6 CD4 þ Tcells cultured under Th0, Th1, Th2 or Th17 conditions determined using ELISA. Cells were seeded at 3 105 cells per well (n ¼ 3 per group). The means±s.d. are indicated.

Article Snippet: B cells undergoing apoptosis on day 3 and total IgG production in the culture supernatants on day 7 were determined using the Annexin V Apoptosis Detection Kit (BD Pharmingen) and a mouse IgG ELISA Quantitation Set (Bethyl Laboratories), respectively, according to the manufacturer’s protocol.

Techniques: Activation Assay, Microarray, Gene Expression, Expressing, Enzyme-linked Immunosorbent Assay, Two Tailed Test, Phospho-proteomics, Isolation, Cell Culture

Journal: Nature communications

Article Title: TGF-β3-expressing CD4+CD25(-)LAG3+ regulatory T cells control humoral immune responses.

doi: 10.1038/ncomms7329

Figure Lengend Snippet: Figure 5 | PD-1 expression on B cells is important for the suppressive activity of LAG3 þ Treg. (a) Resistance to TGF-b3-mediated B-cell suppression in PD-1-deficient (PD-1KO) mice. CFSE-labelled B cells from B6, PD-1KO, B6/lpr or B6/gld mice were stimulated in vitro for 72 h with anti-IgM and anti-CD40 in the presence or absence of rTGF-b3 (1 ng ml 1). Histograms are gated on B220 þ B cells. (b) Bcl-xL and Bcl-2a1 mRNA levels in anti-IgM-stimulated B cells from WT or PD-1KO mice in the presence or absence of rTGF-b3 (1 ng ml 1; n ¼ 3 per group). *Po0.05 (unpaired two-tailed Student’s t-test). (c) Blockade of LAG3 þ Treg-mediated suppression of in vitro NP-specific antibody responses by anti-PD-L1 blocking mAb. B cells and Th cells purified from NP-OVA/alum-pre-immunized B6 mice and OT-II mice, respectively, were incubated with or without LAG3 þ Treg from non-immunized OT-II mice in the presence or absence of anti-PD-L1 blocking mAb, and supernatants were analysed for anti-NP-BSA antibodies by ELISA (n ¼ 6 per group). *Po0.05 (Bonferroni post-test). (d) NP-specific antibody responses of Rag1KO mice injected with B6 B cells and OT-II Th cells from B6 or PD-1KO mice with or without LAG3 þ Treg from B6 mice. Anti-PD-L1 blocking antibody (200 mg per mouse) was injected i.v. every 3 days. Anti-NP-BSA antibody levels were determined as in Fig. 1e (n ¼ 6 per group). *Po0.05 (Bonferroni post-test). (e) PD-1 expression on B cells. B cells from B6 mice were stimulated in vitro for 72 h with or without anti-IgM and anti-CD40 antibodies in the presence or absence of rTGF-b3 (1 ng ml 1). Histograms are gated on B220 þ B cells. Data are representative of three independent experiments. The means±s.d. are indicated.

Article Snippet: B cells undergoing apoptosis on day 3 and total IgG production in the culture supernatants on day 7 were determined using the Annexin V Apoptosis Detection Kit (BD Pharmingen) and a mouse IgG ELISA Quantitation Set (Bethyl Laboratories), respectively, according to the manufacturer’s protocol.

Techniques: Expressing, Activity Assay, In Vitro, Two Tailed Test, Blocking Assay, Incubation, Enzyme-linked Immunosorbent Assay, Injection

Journal: Nature communications

Article Title: TGF-β3-expressing CD4+CD25(-)LAG3+ regulatory T cells control humoral immune responses.

doi: 10.1038/ncomms7329

Figure Lengend Snippet: Figure 6 | IL-27 induces TGF-b3-producing Egr2 þ Treg from naive T cells. (a) IL-27-mediated induction of Egr2 and LAG-3 on CD4 þ T cells. Freshly isolated naive CD4 þ T cells were stimulated with anti-CD3/CD28 mAb in the presence or absence of IL-27. Cells were stained for Egr2 and LAG-3 expression on day 5. (b) Quantitative RT–PCR analysis of Tgfb3 mRNA expression in naive WT CD4 þ Tcells activated as in a, assessed on day 3. *Po0.05 (unpaired two-tailed Student’s t-test). (c) ELISA for TGF-b3 in culture supernatants of activated naive WT, Egr2 CKO, STAT1 KO, or Stat3fl/flCD4-Cre þ

Article Snippet: B cells undergoing apoptosis on day 3 and total IgG production in the culture supernatants on day 7 were determined using the Annexin V Apoptosis Detection Kit (BD Pharmingen) and a mouse IgG ELISA Quantitation Set (Bethyl Laboratories), respectively, according to the manufacturer’s protocol.

Techniques: Isolation, Staining, Expressing, Quantitative RT-PCR, Two Tailed Test, Enzyme-linked Immunosorbent Assay

Journal: Journal of cancer science & therapy

Article Title: Blocking PD1/PDL1 Interactions Together with MLN4924 Therapy is a Potential Strategy for Glioma Treatment.

doi: 10.4172/1948-5956.1000543

Figure Lengend Snippet: Figure 1: PDL1 expression in clinical brain tumor samples, glioma cell lines and CSF samples from the immunocompetent glioma mice model. A) Graph (left) represents PDL1 mRNA expression in brain tumors and melanomas (cBioPortal (http://www.cbioportal.org)). Note that the average PDL1 mRNA level in GBM is similar to the average PDL1 mRNA level in melanomas. The PDL1 values in GBM and low-grade gliomas which exceed the average PDL1 mRNA level in melanomas are highlighted in the red box. Kaplan-Meier plot (right) illustrates survival rates of glioma patients with high and low PDL1 expression levels; 9% versus 24% of 2 year survival for high and low PDL1 expression; the difference is significant, P=0.02 (data has been obtained from the Human Pathology Atlas). B) Immunohistochemical detection of PDL1 in the tissue microarray of normal and brain tumor samples. The images were taken at 40x magnification. C) Western blot illustrates PDL1 and Actin protein levels in control and brain tumor clinical samples. Graph represents PDL1 to Actin ratios for corresponding protein samples; 0.87 ± 0.32 (n=7) versus 0.19 ± 0.1 (n=7) for tumor and normal samples, respectively, the difference is significant, P=0.003. Two samples (marked by light grey color) of patients with hemorrhage have been excluded from the average. D) Western blots illustrate PDL1 protein levels in established and PDGx glioma cell lines (left) and in the extracellular media collected from these cell lines (right). The protein content of the collected media was concentrated six folds before an analysis (see method). The graph (right) provides PDL1 concentrations in CSF samples from control and tumor-bearing mice (see method). The immunocompetent glioma mice model with GL261 cells was utilized for this experiment. E) Images illustrate interactions of tumor neuro spheres from XD456 (a) and from two parental U251-IDH1-R132H cell lines with different PDL1 expression levels (b) with primary T-cells. Note that tumor neuro spheres with high PDL1 levels keep their integrity after 48 hours of interaction with primary T-cells (illustrated in the insert). The graph represents the average percent of neuro spheres after 48 hours of interaction with T-cells, 89 ± 7% (n=4) and 14 ± 5% (n=4) for cell lines with high and low PDL1 levels, respectively. The difference is significant, P=0.0002. Primary T-cells were loaded with calcein, AM (green) before experiments for visualization.

Article Snippet: The PDL1 level in CSF samples was analyzed by using the Mouse PDL1 ELISA Kit (Boster, Pleasanton, CA, USA).

Techniques: Expressing, Immunohistochemical staining, Microarray, Western Blot, Control

Journal: Journal of cancer science & therapy

Article Title: Blocking PD1/PDL1 Interactions Together with MLN4924 Therapy is a Potential Strategy for Glioma Treatment.

doi: 10.4172/1948-5956.1000543

Figure Lengend Snippet: Figure 2: HIF1A accumulation evokes PDL1 up-regulation in glioma cell lines. A) HIF1A accumulations induced by CoCl2 treatment of U251 and U87 cell lines evoke PDL1 up-regulation in protein and mRNA levels. Western blot illustrates HIF1A and PDL1 levels in nuclear and cytoplasmic fractions, respectively, in control and after treatment with CoCl2 (85uM). Lamin A/C and alpha Tubulin were utilized to verify nuclear and cytoplasmic fractions, respectively. Graphs illustrate normalized PDL1/ GAPDH mRNA ratios after cell treatment with CoCl2 (85uM) at different time points. In each experiment, data has been normalized to the corresponding ratios in untreated cells, results are presented as mean ± S.D. B) Transcription initiation complexes encoded by pAC154-dual-dCas9VP160 plasmids, guided by sgRNAs to the HIF1A binding domains in the first intron of the PDL1 gene, evoke PDL1 overexpression. Western blot illustrates PDL1 and Actin protein levels in U251 cells after transfection with plasmids encoding control (scrambled sequence) sgRNA or HIF1A sgRNAs. Note that transcription initiation complexes guided by sgRNAs to the HIF1A binding domains (A), (B), and (A) with (B) simultaneously increased PDL1 expression by 1.5, 3.9 and 1.8 folds, respectively, compared to the control (PDL1 expression in the presence of transcription initiation complexes guided by scrambled sgRNA). In each experiment, PDL1 expression was normalized to the Actin expression.

Article Snippet: The PDL1 level in CSF samples was analyzed by using the Mouse PDL1 ELISA Kit (Boster, Pleasanton, CA, USA).

Techniques: Western Blot, Control, Binding Assay, Over Expression, Transfection, Sequencing, Expressing

Journal: Journal of cancer science & therapy

Article Title: Blocking PD1/PDL1 Interactions Together with MLN4924 Therapy is a Potential Strategy for Glioma Treatment.

doi: 10.4172/1948-5956.1000543

Figure Lengend Snippet: Figure 3: MLN4924 treatment induces up-regulation of HIF1A and PDL1 in glioma cell lines. A) The inhibitory dose- response curves for MLN4924 in established, PDGx and PDGX-stem glioma cell lines. The IC50s are 0.3 ± 0.2 uM (n=4), 2.7 ± 1 uM (n=6), 3 ± 2 uM (n=3), 3 ± 1 uM (n=4), 2.9 ± 0.5 uM (n=4), 0.8 ± 0.2 uM (n =4), 0.2 ± 0.1uM (n=4) for LN221, U251, U87, XD456, JX10, XD456-stem, X14P-stem cell lines, respectively, after treatment with MLN4924 for 5 days. B) Western blots illustrate HIF1A and PDL1 protein levels in nuclear and cytoplasmic fractions in the control and after treatment with MLN4924 (1 uM, 5 days). LaminA/C and alpha Tubulin were utilized to verify nuclear and cytoplasmic fractions, respectively. C) The graph illustrates normalized PDL1/18S mRNA ratios after treatment with MLN4924 (1uM, 5 days) for different cell lines. Note the significant enhancement of the PDL1/18 mRNA ratio for all cell lines after MLN4924 treatment: 8 ± 3, 25 ± 5, 5 ± 1, 8 ± 3, 4.5 ± fold increase compared to the corresponding control values for U251, Ln229, U87, XD456, JX6 cell lines, respectively, P<0.05, n=3.

Article Snippet: The PDL1 level in CSF samples was analyzed by using the Mouse PDL1 ELISA Kit (Boster, Pleasanton, CA, USA).

Techniques: Western Blot, Control

Journal: Journal of cancer science & therapy

Article Title: Blocking PD1/PDL1 Interactions Together with MLN4924 Therapy is a Potential Strategy for Glioma Treatment.

doi: 10.4172/1948-5956.1000543

Figure Lengend Snippet: Figure 4: Glioma cells treated with MLN4924 decrease T-cell proliferation via utilization of PD1/PDL1 signaling pathway. A) The graph illustrates an inhibition of T-cell proliferation after T-cell encounter with U251 cells (1); with U251 cells in the presence of an inhibitor of PD1/PDL1 interaction (2); with U251 cells treated with MLN4924 (1uM, for 4 days) (3); with U251 cells treated with MLN4924 (1uM, for 4 days) and in the presence of an inhibitor of PD1/PDL1 interaction (4). After MLN4924 treatment, glioma cells were washed and placed in the media with/and without an inhibitor of PD1/PDL1 interaction (4 uM). Note, that glioma cells treated with MLN4924 induce a stronger decrease of T-cell proliferation compared to untreated cells (55 ± 8% (n=4) versus 30 ± 8% (n=4), respectively, the difference is significant with P=0.0005). The reduction of T-cell proliferation induced by glioma cells treated with MLN4924 is inhibited in the presence of an inhibitor of PD1/PDL1 interaction (P=0.0009, n=4). B) The graph illustrates an inhibition of T-cell proliferation after T-cell encounter with XD456 cells (1); with UXD456 cells in the presence of an inhibitor of PD1/PDL1 interaction (2); with XD456 cells treated with MLN4924 (1uM, for 4 days) (3); with XD456 cells treated with MLN4924 (1uM, for 4 days) and in the presence of an inhibitor of PD1/PDL1 interaction (4). After MLN4924 treatment, glioma cells were washed and placed in media with/and without an inhibitor of PD1/PDL1 interaction. Note, that glioma cells treated with MLN4924 induce a stronger decrease of T-cell proliferation compared to untreated cells (74 ± 8% (n=4) versus 41 ± 7% (n=4), respectively, the difference is significant with P=0.0003). The reduction of T-cell proliferation induced by glioma cells treated with MLN4924 is inhibited in the presence of an inhibitor of PD1/PDL1 interaction (P=0.007, n=4).

Article Snippet: The PDL1 level in CSF samples was analyzed by using the Mouse PDL1 ELISA Kit (Boster, Pleasanton, CA, USA).

Techniques: Inhibition

Journal: Cancers

Article Title: Cancer-Associated Fibroblasts Promote Tumor Aggressiveness in Head and Neck Cancer through Chemokine Ligand 11 and C-C Motif Chemokine Receptor 3 Signaling Circuit

doi: 10.3390/cancers14133141

Figure Lengend Snippet: Characterization of CAFs and NFs obtained from clinical surgical tissues from patients with head and neck cancer (HNC) ( a ) Morphological comparisons between CAFs and NFs from a representative HNC case showed that CAFs (right panel) consisted of more cytoplasmic protrusions than NFs (left panel). Photographs were captured at 40× magnification. ( b ) Quantitative PCR (left panel) of the culture medium showed a significantly higher expression of vimentin and α-SMA in CAFs than in NFs. Western blot analysis (right panel) also demonstrated that levels of vimentin and α-SMA were significantly higher in CAFs than in NFs. ( c ) Flow cytometric analysis of the cell surface markers, CD10 and GPR77, showed a marked increase in their expression in CAFs compared to NFs. ( d ) A heat map of the gene microarray of NFs and CAFs showed that there were several differences in the expression profile of the secreted genes. The arrow indicates a marked discrepancy of the relative mRNA levels of CCL11 in CAF compared with NF. ( e ) The RT-PCR (left panel) and ELISA (right panel) analysis showed an increased expression of CCL11 in CAFs compared with that in NFs. ( f ) Western blot analysis showed that the protein level of CCL11 was higher in CAFs than in NFs in cell lysates. ( g ) Western blot analysis showed a higher CCL11 expression in CAF-CM compared to NF-CM. The asterisk indicated a significant difference (*: p < 0.05; **: p < 0.01) between experimental and control groups.

Article Snippet: CAFs and NFs supernatant CCL11concentrations were measured using the CCL11 (human) ELISA kit (R&D system) following the manufacturer’s protocol.

Techniques: Real-time Polymerase Chain Reaction, Expressing, Western Blot, Microarray, Reverse Transcription Polymerase Chain Reaction, Enzyme-linked Immunosorbent Assay, Control

Journal: Cancers

Article Title: Cancer-Associated Fibroblasts Promote Tumor Aggressiveness in Head and Neck Cancer through Chemokine Ligand 11 and C-C Motif Chemokine Receptor 3 Signaling Circuit

doi: 10.3390/cancers14133141

Figure Lengend Snippet: CCL11 produced by CAFs causes increased migration and invasion, and the EMT of HNC cells. ( a ) Comparative analysis of the migration and invasion of HNC cells associated with CCL11. Four test groups were classified for comparative analysis of migration and invasive abilities. FaDu and NPC204 cells cultured with medium containing CAF-induced CCL11 presented greater abilities of migration and invasion, with a statistically significant difference, than three other groups: NF, NF with CCL11, and CAFs treated with CCL11 antibody. The asterisk indicates a significant difference (*: p < 0.05; **: p < 0.01) ( b ) Comparative photographs of the infiltrating behavior of FaDu and NPC204 cells in an organotypic culture in four groups seeded onto a mixture layer containing NFs or CAFs with CCL11 or CCL11 antibody. The arrow(s) indicate infiltration buds from the HNC cells seeded above. ( c ) Representative blots of the EMT-associated markers in FaDu and NPC204 cells, as observed upon Western blotting analysis in five groups, showed that treatment with CAF-conditioned medium or the application of rCCL11 decreased the expression of epithelial-type markers (E-cadherin), and increased the expression of mesenchymal-type markers (fibronectin) and EMT regulators (Snail and Twist). In addition, increased expression of invasion-related MMP2 and MMP9 was also seen in those two groups, compared with other groups. The asterisk indicates a significant difference ( p < 0.05) between experimental and control groups. Results are expressed as mean ± SD.

Article Snippet: CAFs and NFs supernatant CCL11concentrations were measured using the CCL11 (human) ELISA kit (R&D system) following the manufacturer’s protocol.

Techniques: Produced, Migration, Cell Culture, Western Blot, Expressing, Control

Journal: Cancers

Article Title: Cancer-Associated Fibroblasts Promote Tumor Aggressiveness in Head and Neck Cancer through Chemokine Ligand 11 and C-C Motif Chemokine Receptor 3 Signaling Circuit

doi: 10.3390/cancers14133141

Figure Lengend Snippet: Comparative analysis of induction of CSC properties and drug resistance in HNC cells associated with CCL11. ( a ) Four groups were classified for comparative analysis of the ability of sphere formation. Increased ability of sphere formation in two test groups of HNC cells exposed to a CAF medium and the group with treatment of rCCL11 was noted. ( b ) Flow cytometric analysis showed a significant increase in CD44 and CD44/CD24, as well as in CD133 in HNC cells exposed to rCCL11, compared to control HNC cells ( p < 0.05). ( c ) Flow cytometric analysis showed a marked increase in ALDH-1 activity in HNC cells exposed to rCCL11 compared to control HNC cells. ( d ) Western blot analysis showed that CSC-representative markers, Oct-4, Nanog, and Sox-2, were also overexpressed in addition to the increased expression of two important drug resistance genes, ABCG-2 and MDR-1 , in HNC cells exposed to rCCL11. ( e ) Treatment with Cisplatin at 24 h showed a significant increase in chemoresistance in both FaDu and NPC204 cells exposed to rCCL11 compared with control HNC cells. The asterisk indicates a significant difference (*: p < 0.05; **: p < 0.01) between experimental and control groups.

Article Snippet: CAFs and NFs supernatant CCL11concentrations were measured using the CCL11 (human) ELISA kit (R&D system) following the manufacturer’s protocol.

Techniques: Control, Activity Assay, Western Blot, Expressing

Journal: Cancers

Article Title: Cancer-Associated Fibroblasts Promote Tumor Aggressiveness in Head and Neck Cancer through Chemokine Ligand 11 and C-C Motif Chemokine Receptor 3 Signaling Circuit

doi: 10.3390/cancers14133141

Figure Lengend Snippet: CCL11 and CCR3 expression with associated signal pathway in HNC cell lines and their correlation to clinical outcomes in 104 HNC patients. ( a ) Confocal microscopic images showed CCL11 (green) localized to both cell and nuclear membranes, while CCR3 (red) localized only to the cell membrane in FaDu cells; CCL11 and CCR3 co-localized at the cell membrane (yellow). In NPC204 cells, CCL11 (green) and CCR3 (red) were found to co-localize at protrusions polarized to the cells (yellow). ( b ) Using the crisp technique, higher expression of CCR3, MMP2, and MMP3 was found in over-expressed CCL11 cloned-FaDu and NPC204 cells. Cloned CCL11-overexpressed cells were abolished by adding eotaxin siRNA or CCR3 antibody, which reversed the expression of CCR3 and invasion-related MMP2 and MMP9. ( c ) Higher phosphorylation levels of p38 MAPK and ERK were found in cloned CCL11-overexpressed FaDu and NPC204 cells and were reversed by treatment of the p38 MAPK inhibitor (SB203580) and ERK inhibitor (FR180204), respectively. The phosphorylation level of JNK was kept in low condition before and after treatment of the JNK inhibitor (SP600125). ( d ) Photomicrographs of immunohistochemical staining from tissue microarray showing CCL11 and CCR3 expression in three different representative groups of HNC patients (magnification, ×200). ( e ) Kaplan–Meier survival analysis of patients showed that overexpression of CCL11 and CCR3 were statistically associated with poor overall survival).

Article Snippet: CAFs and NFs supernatant CCL11concentrations were measured using the CCL11 (human) ELISA kit (R&D system) following the manufacturer’s protocol.

Techniques: Expressing, Membrane, Clone Assay, Phospho-proteomics, Immunohistochemical staining, Staining, Microarray, Over Expression

Journal: Cancers

Article Title: Cancer-Associated Fibroblasts Promote Tumor Aggressiveness in Head and Neck Cancer through Chemokine Ligand 11 and C-C Motif Chemokine Receptor 3 Signaling Circuit

doi: 10.3390/cancers14133141

Figure Lengend Snippet: The diagrammatic illustration demonstrates the major mechanism that CAFs secreting CCL11 promotes HNC cell migration and invasion and induces properties of drug resistance and stemness, shown as follows. CAFs in TME secret CCL11 binding to the CCR3 receptors on HNC cells via the paracrine effect. The signal induces overexpression of transcriptional factors, such as Snail and Twist, which regulate EMT and are also responsible for self-induction of CCL11 in an autocrine fashion. As a result, CCL11, via paracrine or autocrine signaling when targeting CCR3 receptors, play a functional role in the induction of EMT and CSC properties, for further tumor progression.

Article Snippet: CAFs and NFs supernatant CCL11concentrations were measured using the CCL11 (human) ELISA kit (R&D system) following the manufacturer’s protocol.

Techniques: Migration, Binding Assay, Over Expression, Functional Assay

Journal: PLoS ONE

Article Title: A FOXM1 Dependent Mesenchymal-Epithelial Transition in Retinal Pigment Epithelium Cells

doi: 10.1371/journal.pone.0130379

Figure Lengend Snippet: A. Quantification of change in cell density (number of DAPI positive nuclei per cm 2 imaged area) upon FOXM1 overexpression or knockdown, 72h post transfection. Data is normalized to appropriate controls (Empty vector for pFOXM1 and non-targeting siRNA for siFOXM1). Bars represent Mean + SD (n = 4). P<0.0001 (Student’s t-test). B. Heatmap showing changes in gene expression of a panel of representative markers over a timecourse of RPE culture where cells are seeded at high (100000 cells/cm 2 ) or low (8000 cells/cm 2 ) density. C. Plot showing differential expression of BMP7 and Wnt5B transcripts extrapolated from the microarray data. The shaded area represents 95% confidence intervals around the point estimates (circles) of the difference between the mean high density expression vs the mean low density expression.

Article Snippet: Where required, media was supplemented with recombinant human Wnt5B (500ng/ml; R&D Systems), BMP-4/7 (75ng/ml; R&D Systems), Thiostrepton (Sigma), LDN-193189 (10μM; Stemgent), WAY-262611 (10μM; Enzo Lifesciences).

Techniques: Over Expression, Knockdown, Transfection, Plasmid Preparation, Gene Expression, Quantitative Proteomics, Microarray, Expressing

Journal: PLoS ONE

Article Title: A FOXM1 Dependent Mesenchymal-Epithelial Transition in Retinal Pigment Epithelium Cells

doi: 10.1371/journal.pone.0130379

Figure Lengend Snippet: A. Immunocytochemistry for PMEL17 where cells are seeded at either low density (16000 cells/cm 2 ) in the presence or absence of BMP4/7 (top left) or at high density (25000 cells/cm 2 ) in the presence or absence of Wnt5B (bottom left) and cultured for a period of 14 days. Also shown is the expression of BEST1 under the same conditions (top and bottom right). ACTB and B2M are used as housekeeping genes. Bars represent Mean + SD (n = 3). B. Quantification of immunocytochemistry for % CRALBP at Day 21 where cells are either treated with media alone (Control) or media supplemented with 10μM LDN-193189 added at Day 2,4,6,8,11,14 or 18. * indicates significant difference between control and compound treatment (One way ANOVA with Dunnett’s multiple comparisons). C. Quantification of immunocytochemistry for % CRALBP at Day 28 where cells are either treated with media alone (Control) or media supplemented with 10μM WAY-262611 added at Day 2,7,14 or 21. * indicates significant difference between control and compound treatment (One way ANOVA with Dunnett’s multiple comparisons). D. qPCR based measurement of BMP7 and Wnt5B transcript expression at Day 10 post siFOXM1 transfection (relative to transfection with non-targeting siRNA used as a control). GAPDH , HPRT1 and IPO8 were used as housekeeping genes. Bars represent Mean + SD (n = 3). P<0.05 (Student’s t-test).

Article Snippet: Where required, media was supplemented with recombinant human Wnt5B (500ng/ml; R&D Systems), BMP-4/7 (75ng/ml; R&D Systems), Thiostrepton (Sigma), LDN-193189 (10μM; Stemgent), WAY-262611 (10μM; Enzo Lifesciences).

Techniques: Immunocytochemistry, Cell Culture, Expressing, Control, Transfection

Journal: PLoS ONE

Article Title: A FOXM1 Dependent Mesenchymal-Epithelial Transition in Retinal Pigment Epithelium Cells

doi: 10.1371/journal.pone.0130379

Figure Lengend Snippet: RPE first acquire a mesenchymal morphology upon dissociation and culture followed by proliferation and mesenchymal-epithelial transition to re-uptake an epithelial phenotype. Proliferation of RPE is directly regulated by FOXM1 which also affects expression of BMP7 and Wnt5B by an unknown mechanism. Both these activities are required for successful MET and epithelialization.

Article Snippet: Where required, media was supplemented with recombinant human Wnt5B (500ng/ml; R&D Systems), BMP-4/7 (75ng/ml; R&D Systems), Thiostrepton (Sigma), LDN-193189 (10μM; Stemgent), WAY-262611 (10μM; Enzo Lifesciences).

Techniques: Expressing

Journal: Laboratory investigation; a journal of technical methods and pathology

Article Title: Activin A balance regulates epithelial invasiveness and tumorigenesis

doi: 10.1038/labinvest.2014.97

Figure Lengend Snippet: (a) Immunohistochemistry staining with antibody against phosphorylated Smad2 (pSmad2) and TGFβ receptor II (TβRII) showed increased nuclear signal for pSmad2 in the invasive ECdnT organotypic cultures. Scale bar 50 micron. (b) Analysis of immunohistochemistry staining for TβRII and pSmad2 in 83 ESCC cases in a tissue microarray shows no significant correlation. Fisher’s exact test, two tailed p= 0.3182. (c) Five paired normal adjacent and ESCC tissues (GSE17531) were analyzed for INHBA mRNA expression, which identified upregulation of INHBA in four ESCC samples. (d) Waterfall plot of a publically available dataset (GSE23400) represented upregulation of INHBA in the ESCC (grey bars) samples vs. normal (black bars).

Article Snippet: The following treatments were added to the organotypic cultures at the time of epithelial seeding and renewed with every media change: Five ng/ml recombinant human TGFβ1, 10ng/ml Activin A, 100 ng/ml Follistatin and 600 ng/ml neutralizing antibody against Activin A (all from R&D Systems, Minneapolis, MN), or 1 μM A83-01 (Tocris, Bristol, UK) and 1 μM GM6001 (Millipore EMD, Billerica, MA).

Techniques: Immunohistochemistry, Staining, Microarray, Two Tailed Test, Expressing

Journal: Laboratory investigation; a journal of technical methods and pathology

Article Title: Activin A balance regulates epithelial invasiveness and tumorigenesis

doi: 10.1038/labinvest.2014.97

Figure Lengend Snippet: (a) Esophageal epithelial cells expressing wild-type full-length E-cadherin (E), dominant-negative mutant E-cadherin (EC) or dominant-negative mutant E-cadherin and TGFβ receptor II (ECdnT) were grown in organotypic cultures with either fetal esophageal fibroblasts (FEF) or cancer-associated fibroblasts (CAF) embedded in the underlying matrix. Immunofluorescence staining with antibody against αSMA (green) and podoplanin (red) showed similar expression pattern in the cultures. Scale bar is 50 micron. (b) Activin A concentration in conditioned media from organotypic cultures is higher in invasive cultures as measured using indirect ELISA. * p=0.003, ** p= 0.005, *** p=0.03 (c) Stimulation of epithelial cells with Act A in monolayer plastic culture demonstrated phosphorylation of Smad. Neutralizing antibody against Activin (nAb) prevented the induction of pSmad2 by Act A. Following stimulation with Act A or with conditioned media from organotypic culture increased expression of vimentin was detected after 48 hours by Western Blot. The increase was reversed in the presence of neutralizing antibody (nAb). (d) Inhibition with the Act A antagonist, Follistatin, or a pan-TGFβ inhibitor A83-01 suppressed MMP-9 secretion in E, EC and ECdnT cells as measured by gelatin zymography. Upper bands reflect pro-MMP, lower bands activated, cleaved MMP (arrow).

Article Snippet: The following treatments were added to the organotypic cultures at the time of epithelial seeding and renewed with every media change: Five ng/ml recombinant human TGFβ1, 10ng/ml Activin A, 100 ng/ml Follistatin and 600 ng/ml neutralizing antibody against Activin A (all from R&D Systems, Minneapolis, MN), or 1 μM A83-01 (Tocris, Bristol, UK) and 1 μM GM6001 (Millipore EMD, Billerica, MA).

Techniques: Expressing, Dominant Negative Mutation, Immunofluorescence, Staining, Concentration Assay, Indirect ELISA, Phospho-proteomics, Western Blot, Inhibition, Zymography

Journal: Laboratory investigation; a journal of technical methods and pathology

Article Title: Activin A balance regulates epithelial invasiveness and tumorigenesis

doi: 10.1038/labinvest.2014.97

Figure Lengend Snippet: (a) Esophageal epithelial cells expressing wild-type full-length E-cadherin (E), dominant-negative mutant E-cadherin (EC) or dominant-negative mutant E-cadherin and TGFβ receptor II (ECdnT) were grown in organotypic cultures in the presence of recombinant Activin A (Act A), its antagonist Follistatin or a neutralizing antibody against Activin A (nAb); H&E staining. Stimulation with Act A inhibited invasion of E and EC cells, but failed to suppress ECdnT cell invasion. Follistatin increased cell invasion in all cell types, while the neutralizing antibody prevented invasion of E and EC cells, without an effect on ECdnT cells. (b) Immunohistochemistry staining with ki67-antibody showed no differences in cell proliferation. Scale bars are 50 micron. (c) Indirect ELISA with antibody against Act A measured increased levels after addition of recombinant Act A in fibroblasts (FEF) and ECdnT. Untreated ECdnT cells (Control) secreted higher baseline levels of Act A than FEF, which were reduced by Follistatin. (d) TGFβ1 concentration was increased in response to stimulation with Act A and overall baseline secretion was higher in control ECdnT cells than fibroblasts as measured by indirect ELISA. Follistatin inhibited TGFβ1 secretion.

Article Snippet: The following treatments were added to the organotypic cultures at the time of epithelial seeding and renewed with every media change: Five ng/ml recombinant human TGFβ1, 10ng/ml Activin A, 100 ng/ml Follistatin and 600 ng/ml neutralizing antibody against Activin A (all from R&D Systems, Minneapolis, MN), or 1 μM A83-01 (Tocris, Bristol, UK) and 1 μM GM6001 (Millipore EMD, Billerica, MA).

Techniques: Expressing, Dominant Negative Mutation, Recombinant, Staining, Immunohistochemistry, Indirect ELISA, Control, Concentration Assay

Journal: Laboratory investigation; a journal of technical methods and pathology

Article Title: Activin A balance regulates epithelial invasiveness and tumorigenesis

doi: 10.1038/labinvest.2014.97

Figure Lengend Snippet: (a) Separating the cellular matrix and epithelium of the organotypic cultures growing ECdnT cells through a collagen I layer, dashed lines, prevented cell invasion in the absence (control) and presence of Act A (+Act A). When the cellular matrix of the organotypic culture was treated with puromycin to kill the embedded fibroblasts before the ECdnT cells were seeded, epithelial formation occurred but invasion was inhibited with and without Act A stimulation. (b) Treatment of ECdnT organotypic cultures with a pan-MMP inhibitor, GM6001, suppressed cell invasion, which was not restored in the presence of Act A. Untreated (no tx) control ECdnT cells in organotypic culture invaded into the underlying matrix. TGFβ1 treatment inhibited epithelial cell invasion. Scale bars are 50 micron. (c) Immunohistochemistry showed nuclear localization of phosphorylated Smad (pSmad2, red) in control and Act A stimulated conditions. Collagen IV, red, was disrupted in invasive cultures after Act A treatment. Loss of the fibroblasts (FEF), labeled green with antibody against vimentin (no staining in the lower panels), had no effect on the nuclear localization of pSmad2. The collagen IV layer was not disrupted in non-invasive cultures in the absence of FEFs.

Article Snippet: The following treatments were added to the organotypic cultures at the time of epithelial seeding and renewed with every media change: Five ng/ml recombinant human TGFβ1, 10ng/ml Activin A, 100 ng/ml Follistatin and 600 ng/ml neutralizing antibody against Activin A (all from R&D Systems, Minneapolis, MN), or 1 μM A83-01 (Tocris, Bristol, UK) and 1 μM GM6001 (Millipore EMD, Billerica, MA).

Techniques: Control, Immunohistochemistry, Labeling, Staining