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

Journal: The Journal of Neuroscience

Article Title: Interferon-α Causes Neuronal Dysfunction in Encephalitis

doi: 10.1523/JNEUROSCI.5595-08.2009

Figure Lengend Snippet: Working Memory Correct (WMC) and Repeat Reference Memory (RRM) errors by trial for HIVE and control saline treated mice. HIVE mice that received IFNα NAb made less WMC (p=0.0017) and RRM (p=0.013) errors as memory load increased during trials than HIVE mice that received no treatment or control antibodies (A,B). HIVE mice with IFNα NAb were able to maintain performance similar to control mice without treatment as memory load increased in WMC and RRM errors (C, D). HIVE mice without treatment made significantly more WMC (p=0.016) and RRM (p=0.0002) errors as the memory load increased compared to control and HIVE IFNα NAb mice (C, D).

Article Snippet: Control and HIVE mice received i.p. injections of either 300 μl of saline (control treatment), 100 μg/kg of isotype matched control rabbit polyclonal antibody (control antibody; Vector; Burlingame, CA) or 100 μg/kg of rabbit polyclonal anti-mouse IFNα NAb (PBL, Piscataway, NJ).

Techniques: Control, Saline

Journal: The Journal of Neuroscience

Article Title: Interferon-α Causes Neuronal Dysfunction in Encephalitis

doi: 10.1523/JNEUROSCI.5595-08.2009

Figure Lengend Snippet: Densitometry analysis of microgliosis, astrogliosis, and dendritic arborization in all mice. Densitometry analysis of microgliosis in all control and HIV mouse groups (A). Mice treated with IFNα NAb had significantly less microgliosis compared to other HIVE mouse groups (p=0.0061). Densitometry analysis of astrogliosis in all control and HIV mouse groups (B). There was no statistical difference in astrogliosis between HIV groups. Densitometry analysis of dendritic arborization (MAP2 staining) in all control and HIV mouse groups (C). HIVE mice treated with IFNα NAb did not have a significant loss of dendritic arborization compared to control mice.

Article Snippet: Control and HIVE mice received i.p. injections of either 300 μl of saline (control treatment), 100 μg/kg of isotype matched control rabbit polyclonal antibody (control antibody; Vector; Burlingame, CA) or 100 μg/kg of rabbit polyclonal anti-mouse IFNα NAb (PBL, Piscataway, NJ).

Techniques: Control, Staining

Journal: The Journal of Neuroscience

Article Title: Interferon-α Causes Neuronal Dysfunction in Encephalitis

doi: 10.1523/JNEUROSCI.5595-08.2009

Figure Lengend Snippet: Real Time RT-PCR analysis of IFNα4 and interferon stimulated gene product 15 (ISG15) mRNA in control, HIVE control, HIVE control Ab, and HIVE IFNα NAb mice (A, B). All three treatments of HIV infected mice expressed higher levels of IFNα4 compared to control mice, but there was no significant difference between the three groups. HIV infected mice treated with IFNα NAb produced significantly less ISG15 compared to other HIV infected mice (p=0.0216)(B).

Article Snippet: Control and HIVE mice received i.p. injections of either 300 μl of saline (control treatment), 100 μg/kg of isotype matched control rabbit polyclonal antibody (control antibody; Vector; Burlingame, CA) or 100 μg/kg of rabbit polyclonal anti-mouse IFNα NAb (PBL, Piscataway, NJ).

Techniques: Quantitative RT-PCR, Control, Infection, Produced

Journal: The Journal of Neuroscience

Article Title: Interferon-α Causes Neuronal Dysfunction in Encephalitis

doi: 10.1523/JNEUROSCI.5595-08.2009

Figure Lengend Snippet: Immunohistochemistry staining for PKR in mouse brain tissue. PKR staining in control mouse (A). PKR staining in HIV mouse injected with saline (B). PKR staining in HIV mouse injected with control Ab (C). PKR staining in HIV mouse injected with IFNα NAb (D). Densitometry analysis of PKR staining in untreated control mice and all groups of HIVE mice (E). HIVE mice treated with IFNα NAb had significantly less PKR present in brain tissue compared to other HIVE mice (p<0.0001).

Article Snippet: Control and HIVE mice received i.p. injections of either 300 μl of saline (control treatment), 100 μg/kg of isotype matched control rabbit polyclonal antibody (control antibody; Vector; Burlingame, CA) or 100 μg/kg of rabbit polyclonal anti-mouse IFNα NAb (PBL, Piscataway, NJ).

Techniques: Immunohistochemistry, Staining, Control, Injection, Saline

Journal: The Journal of Neuroscience

Article Title: Interferon-α Causes Neuronal Dysfunction in Encephalitis

doi: 10.1523/JNEUROSCI.5595-08.2009

Figure Lengend Snippet: Immunofluorescence stain of dendritic arbors (MAP2) during a dose response of changes in dendritic arborization to increasing doses IFNα (A). Increasing the dose of IFNα in the neuron culture decreased dendritic arborization of neurons. Dendrite length decreases as the amount of IFNα increases in the neuron cultures (n=100 neurons/dose) (B). 200 IU of IFNα is enough to significantly decreases the length of dendrites (p=0.0003). Dendritic branches decrease as the amount of IFNα increases in the neuron cultures (n=100 neurons/dose) (C). 200 IU of IFNα is enough to decrease the branching of dendrites (p=0.0004). Dendritic length and branching measurements of neuron cultures treated with 300 IU of IFNα and a blocking treatment of IFNα nAb or an NMDA antagonist (MK801 or AP5) (D,E). NMDA antagonists protected against loss of dendritic length (p<0.0001)(D), however, were not able to prevent loss of dendritic branching in the neuron cultures (E).

Article Snippet: Control and HIVE mice received i.p. injections of either 300 μl of saline (control treatment), 100 μg/kg of isotype matched control rabbit polyclonal antibody (control antibody; Vector; Burlingame, CA) or 100 μg/kg of rabbit polyclonal anti-mouse IFNα NAb (PBL, Piscataway, NJ).

Techniques: Immunofluorescence, Staining, Blocking Assay

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: Mycobacterium tuberculosis inhibits autocrine type I interferon signaling to increase intracellular survival.

doi: 10.4049/jimmunol.1801303

Figure Lengend Snippet: (A) Ifn-β−/− BMDMs were infected with Mtb H37Rv at MOI of 3 for 4h and flow cytometry was conducted at 4hpi to measure surface receptor expression levels of IFNAR1 and IFNAR2. (B and C) Ifn-β−/− BMDMs were infected as described in the presence of 300pg/ml IFN-β. Cell lysates were collected at 20 min post infection and immunoblotted for pJAK1 (Y1022/1023), total JAK1, pTYK2 (Y1054/1055), and total TYK2. (D and E) Ifn-β−/− BMDMs were infected as described in the presence of 300pg/ml IFN-γ. Cell lysates were collected at 20 min post infection and immunoblotted for pJAK1 (Y1022/1023), total JAK1, pTYK2 (Y1054/1055), and total TYK2. (F) Ifn-β BMDMs were treated with DMSO or increasing amounts of a JAK inhibitor for 1 hour, then stimulated with 200pg/ml IFN-β for 4 hours. Cell lysates were then collected and immunoblotted for pSTAT1 (Y701), total STAT1, and β-actin. Densitometry was performed using ImageJ software and phosphorylated protein bands were normalized to loading control for each condition. Densitometric ratios are relative to the UI +IFNβ or the UI +IFNγ conditions. Data and densities shown represent one representative experiment out of three.

Article Snippet: After infection, BMDMs were blocked with 5% FCS and rat anti-mouse CD16/CD32 Fc Block (BD Biosciences, 553141) for 15 min followed by incubation with either PE-conjugated mouse anti-IFNAR1 (Biolegend, 127311) or PE-conjugated goat anti-IFNAR2 (R&D Systems, FAB1083P) for 30 min on ice.

Techniques: Infection, Flow Cytometry, Expressing, Software

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: Type I IFN is siloed in endosomes

doi: 10.1073/pnas.1921324117

Figure Lengend Snippet: Persistence of IFN-I signaling in ISG15- and USP18-deficient cells. ( A ) Control, ISG15-, or USP18-deficient fibroblasts were given 0.197 nM (1,000 u/mL) IFNα2b for 12 h, washed, and rested for 36 h in all experiments. Relative units of MX1 mRNA was quantified by qPCR. ( B ) Following A , EU was added for 24 h, RNA containing EU isolated (nascent RNA), and qPCR performed. ( C and D ) Western blotting or IF for phospho-STAT1, phospho-STAT2, and total STAT2 as per A . Band densitometry of pSTAT1 relative to tubulin for C . (Magnification in D : 40×.) ( E ) Cells were incubated with 10 μM Cerdulatinib (Cerd) or vehicle (dimethyl sulfoxide [DMSO]) for 4 h following A , and Western blotting was performed. ( F and G ) Cells were primed and rested with antibodies present, and ( F ) qPCR or ( G ) Western blotting was performed. ( H ) qPCR for IFNB mRNA after prime-rest protocol or polyinosinic:polycytidylic acid (poly I:C). ( I ) Cells were prime-rested as per A , and supernatants were placed on naïve control cells for 8 h (NT) or with anti-IFNα. Control cells given 0.197 nM IFN-I (8 h) served as a positive control, and RSAD2 mRNA was quantified. SEM represented, unpaired Student t tests: * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001. ns = not significant.

Article Snippet: For IFN-I blocking, anti-human IFNα antibody (PBL 31110–1) was used at 0.2 μg/mL, with sheep serum (Millipore S3772, 1:3 for equivalent concentration) as vehicle.

Techniques: Control, Isolation, Western Blot, Incubation, Positive Control

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: Type I IFN is siloed in endosomes

doi: 10.1073/pnas.1921324117

Figure Lengend Snippet: IFN-I is retained intracellularly. ( A ) HeLa cells expressing Rab5-mNeonGreen (green) incubated with 5 nM DY647 IFNα2 (magenta) for 17 h, washed, rested for 25 h, and visualized by LLSM ( Top ). Untreated cells show background autofluorescence ( Bottom ). Z projections are of five sections (0.52-µm total thickness). Box plot shows correlation coefficients for colocalization of IFNα2 and Rab5 ( n = 10 cells). ( B ) Control, ISG15-, and USP18-deficient cells were given DY647 IFNα2 for 17 h and imaged, or rested for 24 h and IFN-I vesicles quantified. ( C ) EM of Control or IFNAR2-deficient cells given 0.5 nM of IFN-I biotin for 15 min. Yellow arrows denote positive immunoreactivity. ( D ) Prime-rested cells evaluated by SiMoA, P = 0.0093. ( E ) EM of control cells given 0.5 nM of IFN-I biotin for 12 h, washed and rested in media containing 0.2 μg/mL anti-IFNa or vehicle (sheep serum) for 36 h. Representative example of ( 1 ) negative control without IFN-I biotin; ( 2 ) IFN-I biotin 12 h, 36 h rest; ( 3 ) IFN-I biotin 12 h, anti-IFNα during 36 h rest; and ( 4 ) IFN-I biotin 12 h, sheep serum during 36 h rest. Asterisk denotes unlabeled endosome; arrow denotes positive immunoreactivity within endosome; n = 50 cells per condition. ( F ) Cells were primed-rested in DMSO or 100 μM PitStop2, and qPCR was performed. Cells given IFN-I with DMSO or PitStop2 for 6 h served as controls. SEM represented, unpaired Student t tests: * P < 0.05, **** P < 0.0001.

Article Snippet: For IFN-I blocking, anti-human IFNα antibody (PBL 31110–1) was used at 0.2 μg/mL, with sheep serum (Millipore S3772, 1:3 for equivalent concentration) as vehicle.

Techniques: Expressing, Incubation, Control, Negative Control