Journal: Cell Research

Article Title: Depression compromises antiviral innate immunity via the AVP-AHI1-Tyk2 axis

doi: 10.1038/s41422-022-00689-9

Figure Lengend Snippet: a Western blot analysis of Tyr701 phosphorylation of STAT1 (p-STAT1) in Ahi1 +/+ and Ahi1 −/− mouse peritoneal macrophages treated with mIFNβ (1000 IU/mL). b Western blot analysis of IFNAR1 and IFNAR2 protein levels in Ahi1 +/+ and Ahi1 −/− mouse peritoneal macrophages. c Western blot analysis of the JAK-STAT signaling proteins (JAK1, Tyk2, STAT1, STAT2 and IRF9) in Ahi1 +/+ and Ahi1 −/− mouse peritoneal macrophages. d Western blot analysis of the JAK-STAT signaling proteins (JAK1, Tyk2, STAT1, STAT2 and IRF9) in RAW264.7 cells transfected with either control shRNAs (shCtrl) or two specific shRNAs against AHI1 (shAHI1: #1, #2). e RT-qPCR analysis of Tyk2 mRNA levels in RAW264.7 cells transfected with either shCtrl or two specific shAHI1 (#1, #2). f Western blot analysis of Tyk2 protein levels in Ahi1 +/+ and Ahi1 −/− mouse peritoneal macrophages treated with cycloheximide (CHX, 50 μg/mL) for durations. g Western blot analysis of Tyk2 protein levels in the spleen tissues from normal control mice (Normal, n = 5) or depression model mice ( n = 5). The ratio values indicate Tyk2/β-actin densitometric ratios analyzed by the ImageJ program. h Western blot analysis of Tyk2 and STAT1 protein levels in human PBMCs from healthy controls (Healthy, n = 5) and MDD patients ( n = 5). ns, not significant ( P > 0.05) (two-tailed unpaired Student’s t -test). Data are shown as means ± SD of three biological replicates ( e , f ), or are representative of three independent experiments ( a – d , f – h ).

Article Snippet: The antibodies with indicated dilutions were as follows: AHI1 (Santa Cruz, sc-515382, 1:500), JAK1 (Santa Cruz, sc-1677, 1:1000), Tyk2 (Cell Signaling Technology, 14193, 1:1000), STAT1 (Cell Signaling Technology, 9172, 1:1000), STAT2 (Cell Signaling Technology, D9J7L, 1:1000), IRF9 (Abcam, ab51639, 1:1000), IFNAR1 (Sino Biological, 13222-H08H, 1:1000), IFNAR2 (Santa Cruz, sc-137209, 1:1000), OTUD1 (Abcam, ab122481, 1:1000), Flag (Sigma, F7425, 1:5000), HA (Abcam, ab9110, 1:3000), Myc (Abmart, M20002H, 1:3000), pY701-STAT1 (Cell Signaling Technology, 9167, 1:1000), VSV-G (Santa Cruz, sc-66180, 1:1000), Tyk2 (Affinity, AF5223, 1:1000), β-Actin (Proteintech, 66009-1-Ig, 1:2000), Tubulin (Proteintech, 66031-1-Ig, 1:3000), Ubiquitin (Ub) (Santa Cruz, sc-8017, 1:500), K48-Ub (Cell Signaling Technology, 8081, 1:1000).

Techniques: Western Blot, Transfection, Quantitative RT-PCR, Two Tailed Test

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: 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