Journal: Thorax

Article Title: Double-stranded RNA induces disproportionate expression of thymic stromal lymphopoietin versus interferon-beta in bronchial epithelial cells from donors with asthma.

doi: 10.1136/thx.2009.125930

Figure Lengend Snippet: Figure 4 Effect of inhibitors on the double-stranded RNA (dsRNA)-induced thymic stromal lymphopoietin (TSLP) response. Bronchial epithelial cells (BECs) were preincubated for 2 h with (A) chloroquine (Chlq) or (B) protein kinase inhibitor (PKRi) before stimulation with dsRNA. (C) Cycloheximide (CHX) was added 1 h before dsRNA and (D) type I interferon (IFN) receptor antibody (R Ab) was added at the same time as dsRNA. Cells were harvested at 3 h and TSLP gene expression was determined by RTqPCR. Data are presented as percentage stimulation in relation to TSLP mRNA expression for cells stimulated with dsRNA. The results are shown as mean6SEM from at least four independent experiments. *p<0.05 vs dsRNA-treated cells (Wilcoxon signed rank test). In (D), data from BECs from healthy subjects and those with asthma are combined owing to the small numbers of BECs tested.

Article Snippet: A neutralising antibody for the type I IFN receptor (Interferonsource-PBL, Biomedical Laboratories, Piscataway, New Jersey, USA) and its negative control mouse IgG2a isotype control (eBioscience, Hatfield, UK) were added at a concentration of 12 mg/ml at the time of addition of dsRNA.

Techniques: Gene Expression, Expressing

Journal: British Journal of Cancer

Article Title: Inhibition of interferon-signalling halts cancer-associated fibroblast-dependent protection of breast cancer cells from chemotherapy

doi: 10.1038/s41416-020-01226-4

Figure Lengend Snippet: a MDA-MB-231-GFP/luc cells were cultured alone (0%) or with CAFs (20%) and were treated with 10 nM epirubicin. Epithelial cells were then collected by FACS and RNA was prepared. Three separate biological repeats were performed giving three pairs of samples. Gene expression was assessed using Affymetrix Clariom D microarrays, and comparisons were made between 0% and 20% groups using hierarchical clustering. b MDA-MB-231-GFP/luc or MDA-MB-468-GFP cells were cultured on their own (0%) or in combination with CAFs (20%), with or without 10 nM epirubicin. Epithelial cells were collected by FACS and RNA was prepared. Relative expression of interferon response genes OAS1, MX1 and miR-155 was determined using qPCR. c MDA-MB-157 cells were cultured on their own (0%) or in combination with CAF-GFP cells (20%), with or without 10 nM epirubicin. Epithelial cells were collected by FACS and RNA was prepared. Relative expression of interferon response genes OAS1, MX1 and miR-155 was determined using qPCR. b, c Data represent the mean of technical triplicates (±SD) from one biological experiment, apart from miR-155 analysis in MDA-MB-231 cells, which is from three biological experiments (±SE) and is analysed using two-tailed Mann–Whitney U tests (selected significant difference shown).

Article Snippet: Epirubicin hydrochloride (Sigma; St Louis, USA); recombinant IFNα and IFNγ (Peprotech; Rocky Hill, USA); mouse anti-human IFN Type I R2 antibody (#MMHAR-2; PBL Assay Science; Piscataway, USA); goat anti-human IFN Type II R1 antibody, mouse IgG2A control, goat IgG control (#AF673, #MAB00, #AB-108-C 3; R&D Systems; Minneapolis, USA); rabbit anti-IFNβ1 and anti-claudin-3 antibodies (#PA5-20390, #PA5-16867; ThermoFisher; Waltham, USA); rabbit monoclonal MX1 antibody (#D3W7I; CST; MA, USA).

Techniques: Cell Culture, Gene Expression, Expressing, Two Tailed Test, MANN-WHITNEY

Journal: British Journal of Cancer

Article Title: Inhibition of interferon-signalling halts cancer-associated fibroblast-dependent protection of breast cancer cells from chemotherapy

doi: 10.1038/s41416-020-01226-4

Figure Lengend Snippet: TMAs of tissue from 109 TNBC resections were assembled and expression of IFNβ1 in fibroblasts, and MX1 and claudin-3 in tumour cells was determined using immunohistochemistry. a Representative images of immunohistochemistry, showing tissue scored ‘3’ for IFNβ in fibroblasts (left), ‘3’ for MX1, and ‘positive’ for claudin-3. b The cohort was split into groups with high or low expression of IFNβ1 in fibroblasts (left) or MX1 in tumour cells (right) using ROC analyses. Cumulative disease-free survival in the groups was compared using Kaplan-Meier analyses and log rank tests. c The cohort was split into claudin-low or claudin-high groups, based on expression levels of claudin-3 (positive or negative). The claudin-low group ( n = 49) were analysed as in b .

Article Snippet: Epirubicin hydrochloride (Sigma; St Louis, USA); recombinant IFNα and IFNγ (Peprotech; Rocky Hill, USA); mouse anti-human IFN Type I R2 antibody (#MMHAR-2; PBL Assay Science; Piscataway, USA); goat anti-human IFN Type II R1 antibody, mouse IgG2A control, goat IgG control (#AF673, #MAB00, #AB-108-C 3; R&D Systems; Minneapolis, USA); rabbit anti-IFNβ1 and anti-claudin-3 antibodies (#PA5-20390, #PA5-16867; ThermoFisher; Waltham, USA); rabbit monoclonal MX1 antibody (#D3W7I; CST; MA, USA).

Techniques: Expressing, Immunohistochemistry

Journal: British Journal of Cancer

Article Title: Inhibition of interferon-signalling halts cancer-associated fibroblast-dependent protection of breast cancer cells from chemotherapy

doi: 10.1038/s41416-020-01226-4

Figure Lengend Snippet: a, b MDA-MB-231-GFP/luc (left) or MDA-MB-157 (right) cells were cultured alone, or with breast CAFs or CAF-GFP cells, respectively. Cultures were treated with type I (1 μg/ml) or type II (5 μg/ml) interferon-signalling blocking antibodies or appropriate isotype controls for 24 h. Cultures were then treated with 10 nM (MDA-MB-231 cells) or 25 nM (MDA-MB-157 cells) epirubicin or vehicle control, and were redosed with antibodies for a further 24 h. Epithelial cells were then collected by FACS. a Clonogenic survival was determined. Data are presented as colony counts or relative survival after epirubicin (colony counts relative to untreated). b Relative expression of the marker of IFN-signalling activity MX1 was determined. c MDA-MB-231-GFP/luc cells were cultured alone, or with primary breast CAFs and were treated with antibodies and epirubicin/control exactly as above. Clonogenic survival was determined (left): data are presented as colony counts or relative survival after epirubicin (colony counts relative to untreated). Relative expression of the marker of IFN-signalling activity MX1 was also determined (right). a , b, c Data represent means (±SE) of three independent experimental repeats. Two-tailed Mann–Whitney U tests were carried out and selected differences are shown (ns not significant).

Article Snippet: Epirubicin hydrochloride (Sigma; St Louis, USA); recombinant IFNα and IFNγ (Peprotech; Rocky Hill, USA); mouse anti-human IFN Type I R2 antibody (#MMHAR-2; PBL Assay Science; Piscataway, USA); goat anti-human IFN Type II R1 antibody, mouse IgG2A control, goat IgG control (#AF673, #MAB00, #AB-108-C 3; R&D Systems; Minneapolis, USA); rabbit anti-IFNβ1 and anti-claudin-3 antibodies (#PA5-20390, #PA5-16867; ThermoFisher; Waltham, USA); rabbit monoclonal MX1 antibody (#D3W7I; CST; MA, USA).

Techniques: Cell Culture, Blocking Assay, Control, Expressing, Marker, Activity Assay, Two Tailed Test, MANN-WHITNEY