Journal: Biomolecules

Article Title: Antiviral Functions of Type I and Type III Interferons in the Olfactory Epithelium

doi: 10.3390/biom13121762

Figure Lengend Snippet: Upregulation of type I and III interferon transcript levels. Relative expression of Ifna2, Ifna4, Ifnb2, and Ifnl2/3 in the olfactory mucosa using qRT-PCR ( A – D ). Relative expression of Ifnar1 and Ifnlr1 in the olfactory mucosa at 24 h PI ( E ). Biological triplicates were included ( A – E ). Immunostaining of IFNAR1 (green in ( F )) and IFNLR1 (green in ( G )) with OMP (red in ( F , G )) in the OE. Bar = 15 μm.

Article Snippet: The antibodies used were: chicken anti-OMP (custom, 1:1000), goat anti-GFP (Rockland (Baltimore, MD, USA), 2.2 µg/mL), rabbit anti-IFNAR1 (Sino Biological (Beijing, China), 1:20), goat anti-IFNLR1 (Novus Biologicals (Centennial, CO, USA), 10 μg/mL), and rabbit anti-pSTAT1 (Abcam, 3.0 µg/mL).

Techniques: Expressing, Quantitative RT-PCR, Immunostaining

Journal: Biomolecules

Article Title: Antiviral Functions of Type I and Type III Interferons in the Olfactory Epithelium

doi: 10.3390/biom13121762

Figure Lengend Snippet: Interferon signaling is required for suppressing VSV replication in the olfactory mucosa. The expression levels of the viral genes, VSV-GFP, VSV-M, and VSV-N, in olfactory mucosae at 24 h PI were measured in Ifnar1 −/− ( A ), Ifnlr1 −/− ( B ), Ifnar1 −/− ; Ifnlr1 −/− ( C ), and Stat1 −/− ( D ) and compared to wildtype littermates. Student t -test, * p < 0.05.

Article Snippet: The antibodies used were: chicken anti-OMP (custom, 1:1000), goat anti-GFP (Rockland (Baltimore, MD, USA), 2.2 µg/mL), rabbit anti-IFNAR1 (Sino Biological (Beijing, China), 1:20), goat anti-IFNLR1 (Novus Biologicals (Centennial, CO, USA), 10 μg/mL), and rabbit anti-pSTAT1 (Abcam, 3.0 µg/mL).

Techniques: Expressing

Journal: The Journal of Reproduction and Development

Article Title: Pregnancy-associated changes of peroxisome proliferator-activated receptor delta (PPARD) and cytochrome P450 family 21 subfamily A member 2 (CYP21A2) expression in the bovine corpus luteum

doi: 10.1262/jrd.2019-132

Figure Lengend Snippet: Primers used in real-time PCR

Article Snippet: The 5 µm-thick sections from the paraffin-embedded luteal tissue were incubated at room temperature (20˚C) with rabbit polyclonal anti-human PPARD antibody (#ARP38765_T100, Aviva Systems Biology, San Diego, CA, USA; 1:50), rabbit polyclonal anti-human CYP21A2 antibody (#ARP60144_P050, Aviva Systems Biology; 1:25), rabbit polyclonal anti-human IFNαR1 antibody (#bs-4116R, Bioss, Woburn, MA, USA; 1:50) or rabbit polyclonal anti-human IFNαR2 antibody (#bs-7022R, Bioss; 1:50) for 12 h. The signals were detected using anti-rabbit IgG-Biotin conjugate (Sigma-Aldrich, LLC, St Louis, MO, USA; 1:100) for 1 h, and then counterstained with hematoxylin.

Techniques: Sequencing

Journal: The Journal of Reproduction and Development

Article Title: Pregnancy-associated changes of peroxisome proliferator-activated receptor delta (PPARD) and cytochrome P450 family 21 subfamily A member 2 (CYP21A2) expression in the bovine corpus luteum

doi: 10.1262/jrd.2019-132

Figure Lengend Snippet: Changes in the relative amount of mRNA for IFNαR1 and IFNαR2 in the bovine corpus luteum (CL) on day 18 of pregnant cows (P18) and on day 15 of non-pregnant cows (NP15). Data represent means ± SEM for four cows per stage and have been expressed as relative ratios of the mRNAs to 18S ribosomal RNA ( RN18S1 ). Localization of IFNαR1 and IFNαR2 in bovine CL obtained on P18 and NP15. Intensive immunoreactivity was observed in the luteal cells (black arrowheads), endothelial cells (red arrowheads), and immune cells (yellow arrowheads) obtained from the CL of both pregnant and nonpregnant cows. No positive immunoreactivity was observed in the negative control. Scale bar = 50 µm.

Article Snippet: The 5 µm-thick sections from the paraffin-embedded luteal tissue were incubated at room temperature (20˚C) with rabbit polyclonal anti-human PPARD antibody (#ARP38765_T100, Aviva Systems Biology, San Diego, CA, USA; 1:50), rabbit polyclonal anti-human CYP21A2 antibody (#ARP60144_P050, Aviva Systems Biology; 1:25), rabbit polyclonal anti-human IFNαR1 antibody (#bs-4116R, Bioss, Woburn, MA, USA; 1:50) or rabbit polyclonal anti-human IFNαR2 antibody (#bs-7022R, Bioss; 1:50) for 12 h. The signals were detected using anti-rabbit IgG-Biotin conjugate (Sigma-Aldrich, LLC, St Louis, MO, USA; 1:100) for 1 h, and then counterstained with hematoxylin.

Techniques: Negative Control

Journal: Oncogene

Article Title: Evaluating the therapeutic potential of ADAR1 inhibition for triple-negative breast cancer

doi: 10.1038/s41388-020-01515-5

Figure Lengend Snippet: A) Core ISG Score in TNBC and Non-TNBC breast cancer samples. Data were extracted from TCGA database. B) Core ISG Score in ER-positive, ERBB2(HER2)-positive and TNBC cell lines. Data were extracted from CCLE database. C) ADAR1-dependency score positively correlates with Core ISG Score in breast cancer cell lines. Upper panel: Core ISG Score in breast cancer cell lines. Lower panel: ADAR1-dependency scores. D) Immunoblots showing protein levels of IFNAR1, PKR, p-PKR (T446), p-eIF2α (S51) and GAPDH (loading control) in MDA-MB231 cells. IFNAR1 was knocked down in ShADAR1-treated MDA-MB231 cells to determine if IFNAR1 loss reverses ADAR1-knockdown phenotype. Images are representative, N=3. F) FF assay showing that IFNAR1 loss partially rescued ADAR1-knockdown phenotype in MDA-MB231 cells. Images are representative, N=3. G) Quantification of FF in F) . Relative plate occupancy was determined using ImageJ software and normalized to ShNT-ShNT. Data are represented as mean ± SD. N=3.

Article Snippet: Primary antibodies used in this study include ADAR1 (Santa Cruz, Dallas, TX USA, sc-73408; Bethyl Laboratories, Montgomery, TX USA, A303–883A; Abcam, Cambridge, MA USA, ab126745), cleaved PARP (Cell Signaling, #9541), PKR (Cell Signaling, #3072), PKR Thr-446-P (Abcam, ab32036), IFNAR1 (Bethyl, A304–290A), ISG15 (Santa Cruz, sc-166755), GAPDH (Bethyl, A300–641A), β-Tubulin (Abcam, ab6046), EIF2S1/eIF2α Ser-51-P (Abcam, 32157), EIF2S1 (Abcam, ab5369), ATF4 (Cell Signaling, #11815).

Techniques: Western Blot, Control, Knockdown, Software

Journal: Science Advances

Article Title: LATS1 is a central signal transmitter for achieving full type-I interferon activity

doi: 10.1126/sciadv.abj3887

Figure Lengend Snippet: ( A and B ) Immunoblotting (IB) analysis of Ser 909 phosphorylation of LATS1 (pS-LATS1) in 2fTGH cells treated with IFN-α (1000 IU/ml) (A) or in MEFs treated with mouse IFN-β (mIFNβ; 1000 IU/ml) (B). ( C to E ) IB analysis of pS-LATS1 in 2fTGH treated with IFN-γ (3000 IU/ml) (C) or IFN-λ (100 ng/ml) (D) or in Ifnar1 +/+ and Ifnar1 −/− MEFs treated with mIFNβ (1000 IU/ml) (E). ( F ) IB analysis of pS-LATS1 in 2fTGH treated with tumor necrosis factor–α (TNFα) (20 ng/ml) or interleukin-6 (IL-6) (100 ng/ml). ( G and H ) IP or IB analysis of pYAP (G) and YAP (H) in HEK293T treated with IFN-α as indicated. ( I ) IP-IB analysis of pYAP in HEK293T transfected with control short hairpin RNAs (shRNAs; shCON) or shLATS1 or shLATS2 and then treated with IFN-α (1000 IU/ml, 1 hour). ( J ) IB analysis of YAP in Lats1/2 +/+ or Lats1/2 −/− MEFs treated with mIFNβ (1000 IU/ml). ( K ) RT-qPCR analysis of C yr61 and C tgf in Lats1/2 +/+ or Lats1/2 −/− MEFs treated with mIFNβ (1000 IU/ml, 4 hours). ( L ) Cell counting kit 8 assay for analyzing the proliferation of Lats1/2 +/+ and Lats1/2 −/− MEFs treated with mIFNβ (500 IU/ml, 48 hours). ( M ) Cell numbers were counted in Lats1/2 +/+ and Lats1/2 −/− MEFs treated with mIFNβ (500 IU/ml) for 1, 2, and 3 days. IFN-mediated inhibition rate of cell proliferation was calculated. Data are representative of three independent experiments (A to J) or are shown as means and SD of three biological replicates (K to M). N.S, not significant ( P > 0.05). * P < 0.05, ** P < 0.01, and *** P < 0.001 (two-tailed unpaired Student’s t test).

Article Snippet: The antibodies with the indicated dilutions were as follows: anti-pY701 (STAT1) (Cell Signaling Technology, 9167; 1:1000), anti-pYAP (Cell Signaling Technology, 13008; 1:1000), anti-pJAK1 (Cell Signaling Technology, 3331S; 1:1000), anti-pTyk2 (Cell Signaling Technology, 9321; 1:1000), anti-Flag (Sigma-Aldrich, F7425; 1:5000), anti-HA (Abcam, ab9110; 1:3000), anti-JAK1 (Santa Cruz Biotechnology, sc-1677; 1:1000), anti-STAT1 (Cell Signaling Technology, 9172; 1:1000), anti-Tyk2 (Cell Signaling Technology, 14193; 1:1000), anti-pS909-LATS1 (Cell Signaling Technology, 9157; 1:1000), anti-Myc (Abmart, M20002H; 1:2000), anti–VSV-G (Abcam, ab1874; 1:2000), anti–β-actin (Proteintech, 66009-1-Ig; 1:1000), anti-tubulin (Proteintech, 66031-1-Ig; 1:3000), anti-LATS1 (Cell Signaling Technology, 3477; 1:1000), anti-Lamb1 (Proteintech, 12987-1-AP; 1:1000), anti-pS727 (STAT1) (Cell Signaling Technology, 8826; 1:1000), anti-YAP (Cell Signaling Technology, 14074S; 1:1000), anti-IFNAR1 (Sino Biological, 13222-T20; 1:1000), anti–IFN-ɑ/βRβ (IFNAR2, D-6) (Santa Cruz Biotechnology, sc-271105; 1:500), anti-STAT1 (Santa Cruz Biotechnology, sc-464; 1:1000), anti-STAT3 (Cell Signaling Technology, 9139S; 1:1000), anti-pSTAT3 (Cell Signaling Technology, 9145S; 1:1000), anti–Pan-pY (Cell Signaling Technology, 8954S; 1:1000), anti-CDK8 (Santa Cruz Biotechnology, sc-13155; 1:1000), anti-p65 (Cell Signaling Technology, 8242S; 1:1000), anti-p-p65 (Cell Signaling Technology, 3033S; 1:1000), and anti-LATS2 (Affinity, AF7940; 1:1000).

Techniques: Western Blot, Transfection, Control, Quantitative RT-PCR, Cell Counting, Inhibition, Two Tailed Test

Journal: Science Advances

Article Title: LATS1 is a central signal transmitter for achieving full type-I interferon activity

doi: 10.1126/sciadv.abj3887

Figure Lengend Snippet: ( A ) IP-IB analysis of endogenous LATS1-IFNAR1 or LATS1-IFNAR2 interaction in mouse primary splenocytes. ( B ) IP-IB analysis of pan-tyrosine phosphorylation of LATS1 (Pan-pY) in HEK293T treated with IFN-α (1000 IU/ml). ( C ) IP-IB analysis of pan-pY-LATS1 in HEK293T transfected with shCON or shRNAs against JAK1 or Tyk2 (shJAK1 or shTyk2) and then treated with IFN-α (1000 IU/ml, 30 min). The first lane represents an IgG control for IP. ( D ) IP-IB analysis of pan-pY-LATS1 in HEK293T cotransfected with Flag-LATS1 and increasing amounts of HA-Tyk2. ( E and F ) IP-IB analysis of pan-pY-LATS1 in HEK293T cotransfected with HA-Tyk2 and Flag-LATS1 mutants (E) or in Lats1/2 −/− MEFs transfected with LATS1 [wild type (WT) or Y200F/Y277F] and HA-Tyk2 (F). ( G ) Flag-LATS1 proteins were immunoprecipitated from HEK293T cotransfected with Flag-LATS1 and HA-Tyk2 by Flag agarose. The red “y” represents Y200/277. m/z is the mass/charge ratio. The “b”s and “y”s indicate mass spectrometry–identified fragment ions from the N termini (b) and C termini (y) of the peptides after fragmentation. The presented diagrams provided the tandem mass spectra of the identified peptides. ( H ) In vitro kinase assay using Flag-LATS1 pulled down from HEK293T transfected with Flag-LATS1 (WT or YF/YF) and recombinant Tyk2. ( I ) IP-IB analysis of LATS1-pan-pY (pY-LATS1) and pS909 (pS-LATS1) in Lats1/2 −/− MEFs transfected with LATS1 (WT or Y200F/Y277F) and treated with IFN-α (1000 IU/ml, 30 min). ( J ) IP-IB analysis of pS909-LATS1 in HEK293T transfected with Flag-LATS1 (WT or its phosphomimetic mutant: Y200E, Y277E, or Y200E/Y277E). Data are representative of three independent experiments (A to F and H to J).

Article Snippet: The antibodies with the indicated dilutions were as follows: anti-pY701 (STAT1) (Cell Signaling Technology, 9167; 1:1000), anti-pYAP (Cell Signaling Technology, 13008; 1:1000), anti-pJAK1 (Cell Signaling Technology, 3331S; 1:1000), anti-pTyk2 (Cell Signaling Technology, 9321; 1:1000), anti-Flag (Sigma-Aldrich, F7425; 1:5000), anti-HA (Abcam, ab9110; 1:3000), anti-JAK1 (Santa Cruz Biotechnology, sc-1677; 1:1000), anti-STAT1 (Cell Signaling Technology, 9172; 1:1000), anti-Tyk2 (Cell Signaling Technology, 14193; 1:1000), anti-pS909-LATS1 (Cell Signaling Technology, 9157; 1:1000), anti-Myc (Abmart, M20002H; 1:2000), anti–VSV-G (Abcam, ab1874; 1:2000), anti–β-actin (Proteintech, 66009-1-Ig; 1:1000), anti-tubulin (Proteintech, 66031-1-Ig; 1:3000), anti-LATS1 (Cell Signaling Technology, 3477; 1:1000), anti-Lamb1 (Proteintech, 12987-1-AP; 1:1000), anti-pS727 (STAT1) (Cell Signaling Technology, 8826; 1:1000), anti-YAP (Cell Signaling Technology, 14074S; 1:1000), anti-IFNAR1 (Sino Biological, 13222-T20; 1:1000), anti–IFN-ɑ/βRβ (IFNAR2, D-6) (Santa Cruz Biotechnology, sc-271105; 1:500), anti-STAT1 (Santa Cruz Biotechnology, sc-464; 1:1000), anti-STAT3 (Cell Signaling Technology, 9139S; 1:1000), anti-pSTAT3 (Cell Signaling Technology, 9145S; 1:1000), anti–Pan-pY (Cell Signaling Technology, 8954S; 1:1000), anti-CDK8 (Santa Cruz Biotechnology, sc-13155; 1:1000), anti-p65 (Cell Signaling Technology, 8242S; 1:1000), anti-p-p65 (Cell Signaling Technology, 3033S; 1:1000), and anti-LATS2 (Affinity, AF7940; 1:1000).

Techniques: Transfection, Control, Immunoprecipitation, Mass Spectrometry, In Vitro, Kinase Assay, Recombinant, Mutagenesis

Journal: medRxiv

Article Title: Polymorphism in IFNAR contributes to glucocorticoid response and outcome in ARDS and COVID-19

doi: 10.1101/2022.03.10.22272123

Figure Lengend Snippet: A genetic sample was available from 202/296 (104/144 in the IFN beta-1a treatment arm and 98/152 in the placebo arm) of the study subjects. Number of the subjects at risk is indicated on the x-axis. ( A) The rs9984273 minor allele C (CC or CT) was significantly associated with survival in patients compared to those who were homozygous with the major allele (TT). ( B) A similar association was not observed in the placebo arm. ( C) Further non-adjusted sub-group analyses encompassing the entire Phase III study population (both active and placebo arms) revealed that the use of glucocorticoids had the strongest association with altering the treatment effect of INF beta-1a. (D) The logarithmic change in serum IFN gamma and IL-6 levels according to the genetic background of rs9984273 in patients of the INTEREST trial with ARDS of pulmonary origin. Patients homozygous with the major allele (TT) compared to the minor allele (CT or CC) of rs9984273 show higher serum IFN-gamma and IL-6 levels when given glucocorticoids.

Article Snippet: The first stage antibodies were anti-alpha chain of the IFN alpha/beta receptor (St John’s Laboratory STJ112765) that was used 1:2000 and 1:5000 and anti-Stat1 (1:400, 9175S), anti-pStat1(1:100, 9167S) and anti-Stat2 (1:200, 72604S) all from Cell Signalling.

Techniques:

Journal: medRxiv

Article Title: Polymorphism in IFNAR contributes to glucocorticoid response and outcome in ARDS and COVID-19

doi: 10.1101/2022.03.10.22272123

Figure Lengend Snippet: (A ) STAT1 expression in the lung after 4-day culture in the presence of IFN beta with or without hydrocortisone (HC). ( B) pSTAT1 expression in the same specimens as in A. ( C) Example photomicrographs showing higher STAT2 expression in a TT patient than in a CT patient and the effect of HC on its nuclear translocation. Most STAT2 remains in the cytoplasm of the CT patients, whereas nuclear expression is prominent in the TT patient. Indicated insets are shown in the bottom row. Arrows. ( D ) Combined results of all patients noting that two CT samples are excluded in the data as the patients were already under glucocorticoid treatment at the time of sample acquisition. Ns, not significant; *P<0.05; **P<0.01; and ***P<0.001

Article Snippet: The first stage antibodies were anti-alpha chain of the IFN alpha/beta receptor (St John’s Laboratory STJ112765) that was used 1:2000 and 1:5000 and anti-Stat1 (1:400, 9175S), anti-pStat1(1:100, 9167S) and anti-Stat2 (1:200, 72604S) all from Cell Signalling.

Techniques: Expressing, Translocation Assay

Journal: International Journal of Molecular Sciences

Article Title: UL34 Deletion Restricts Human Cytomegalovirus Capsid Formation and Maturation

doi: 10.3390/ijms23105773

Figure Lengend Snippet: Characterisation of UL34 as an augmenting viral protein with leaky late expression kinetics. ( A ) Intracellular HCMV genome copies at 120 HPI, relative to genome copies at 12 HPI. MOI = 3, n = 3, bars = SD. ( B ) Western blot analysis of UL34 expression in MRC5 cells treated with 100 μg/mL phosphonoacetic acid (PAA) and subsequently infected with either AD169-GFP WT or AD169 HA-UL34 HCMV (5 DPI, MOI = 3). Membranes were probed with primary antibodies against HCMV viral proteins, HA, or β-actin loading control. ( C ) Immuno-fluorescence analysis of host GM130 and viral UL99 in WT MRC5 cells infected with WT or ΔUL34 AD169-GFP virus. 4 DPI, MOI = 0.1, scale bars = 20 μm. ( D ) Growth kinetics of ΔUL34 AD169-GFP virus, as measured by IE1 fluorescent focus assay in cell culture supernatants from WT and UL34-complementing MRC5 cells. MOI = 3, n = 3, bars = SD. ( E ) Spread of ΔUL34 AD169-GFP virus in WT and UL34-complementing MRC5 cells, as quantified by fixing, staining, and counting IE1 positive cells at indicated time points. MOI = 0.01, n = 3, bars = SD.

Article Snippet: Membranes blocking was performed in 5% ( w / v ) skim milk in 1X TBST (150 mM NaCl, 50 mM Tris pH 7.4, 0.1% ( v / v ) Tween 20) at 4 °C for 1 h, membranes were incubated with primary antibodies: anti-UL99 (Clone 10B4 [ ]), anti-UL83 (Clone 8F5 [ ]), anti-MCP (custom ordered rabbit polyclonal, Cusabio, Houston, TX, USA), anti-HCMV gB (ab6499, Abcam, Cambridge, UK), anti-IE1 (Clone 1B12 [ ]), anti-UL26 [ ], anti-HA (ab130275, Abcam, Cambridge, UK), mouse anti-lamin A/C (4777T, Cell Signaling Technology, Danvers, MA, USA), rabbit phospho-lamin A/C (ser-22) (13448T, Cell Signaling Technology, Danvers, MA, USA) and anti-β-actin (A2228, Sigma, Burlington, MA, USA).

Techniques: Expressing, Western Blot, Infection, Fluorescence, Cell Culture, Staining