Journal: Molecular Neurodegeneration

Article Title: Protective paraspeckle hyper-assembly downstream of TDP-43 loss of function in amyotrophic lateral sclerosis

doi: 10.1186/s13024-018-0263-7

Figure Lengend Snippet: Endogenous dsRNA response and type I interferon promote paraspeckle hyper-assembly in stable cell lines. a and b Depletion of TDP-43, Dicer, Drosha, ADAR1 but not Ago2 or FUS causes intracellular build-up of dsRNA. dsRNA was detected by immunocytochemistry using J2 antibody. Representative images of all conditions are shown. Scale bars, 50 and 10 μm for general plane and close-up panels respectively. c Levels of Alu-containing RNA as analysed by qRT-PCR using specific primers recognising Alu elements (n = 4). *p < 0.05 (Mann-Whitney U -test). d and e Markers of activated cellular reponse to dsRNA are upregulated in TDP-43 depleted cells. Levels of phosphorylated PKR and eIF2α were analysed by Western blot ( d , representative blots are shown) and expression of IFNB1 and an IFN-stimulated gene CXCL10 - by qRT-PCR ( e , n = 6). *p < 0.05 (Mann-Whitney U -test). f IFNbeta treatment stimulates NEAT1 expression and paraspeckle formation. NEAT1 levels were measured by qRT-PCR (n = 6). **p < 0.01 (Mann-Whitney U -test). Staining for an IFN-inducible protein IFIT3 was used as a positive control. Scale bar, 10 μm. g Simultaneous IFNbeta knockdown partially reverses the effect of TDP-43 depletion on paraspeckles. * and #p < 0.05, ***p < 0.001 (one-way ANOVA with Holm-Sidak correction for multiple comparisons). Scale bar, 10 μm. In all panels, cells were harvested for analysis 48 h post-transfection. Paraspeckles in panels f and g were visualised by NEAT1_2 RNA-FISH

Article Snippet: The following commercial primary antibodies were used: TDP-43 (rabbit polyclonal, 10782–2-AP, Proteintech and mouse monoclonal, MAB7778-SP, R&D Biosystems); FUS (rabbit polyclonal, Proteintech, 11570–1-AP); p54nrb/NONO (rabbit polyclonal C-terminal, Sigma); PSF/SFPQ (rabbit monoclonal, ab177149, Abcam); Tuj (β-Tubulin III, mouse monoclonal, Sigma); dsRNA (mouse monoclonal, J2, Kerafast); cleaved caspase 3 (rabbit polyclonal, 9661, Cell Signaling); NF-κB p65 (rabbit monoclonal, D14E12, Cell Signaling); IFIT3 (rabbit polyclonal, Bethyl); p-eIF2α (rabbit monoclonal, ab32157, Abcam); p-PKR (rabbit polyclonal, Thr451, ThermoFisher); PKR (mouse monoclonal, MAB1980-SP, R&D Systems); eIF2α (rabbit monoclonal, D7D3, Cell Signaling); β-actin (mouse monoclonal, A5441, Sigma).

Techniques: Stable Transfection, Immunocytochemistry, Quantitative RT-PCR, MANN-WHITNEY, Western Blot, Expressing, Staining, Positive Control, Transfection

Journal: World journal of surgical oncology

Article Title: IFIT3 accelerates the progression of head and neck squamous cell carcinoma by targeting PD-L1 to activate PI3K/AKT signaling pathway.

doi: 10.1186/s12957-023-03274-5

Figure Lengend Snippet: Fig. 4 IFIT3 regulated the activation of EMT progress in HNSC cells. A The different expression genes between IFIT3 high groups and low groups. B KEGG pathway analysis of these different expression genes. C Enrichment analysis of pathways in cancer by GSEA. D Enrichment analysis of EMT by GSEA. E and F Protein levels of E-cadherin, N-cadherin, and vimentin were determined by Western blotting in IFIT3 knockdown and up-regulation in FaDu and Cal 27 cells

Article Snippet: In brief, paraffinization and rehydration were performed, following antigen retrieval, tissue microarray was treated with 3% hydrogen peroxide for the blocking of endogenous peroxidases, and then 1:100 diluted IFIT3 antibody (HPA059914, Atlas Antibodies), 1:100 diluted PD-L1 antibody (CST, USA), and 1:100 diluted ki67 antibody (Affinity, China) were used to incubate tissue sections overnight at 4 °C.

Techniques: Activation Assay, Expressing, Western Blot, Knockdown

Journal: World journal of surgical oncology

Article Title: IFIT3 accelerates the progression of head and neck squamous cell carcinoma by targeting PD-L1 to activate PI3K/AKT signaling pathway.

doi: 10.1186/s12957-023-03274-5

Figure Lengend Snippet: Fig. 5 IFIT3 regulated the activation of PD-L1 and PI3K/AKT signaling pathway in HNSC cells. A PD-L1 mRNA level was positively correlated with IFIT3 in TCGA HNSC. B Enrichment analysis of PI3K/AKT pathway in cancer by GSEA. C and D CD44, PD-L1, PI3K, p-PI3K, AKT, and p-AKT protein levels were measured by Western blotting

Article Snippet: In brief, paraffinization and rehydration were performed, following antigen retrieval, tissue microarray was treated with 3% hydrogen peroxide for the blocking of endogenous peroxidases, and then 1:100 diluted IFIT3 antibody (HPA059914, Atlas Antibodies), 1:100 diluted PD-L1 antibody (CST, USA), and 1:100 diluted ki67 antibody (Affinity, China) were used to incubate tissue sections overnight at 4 °C.

Techniques: Activation Assay, Western Blot

Journal: World journal of surgical oncology

Article Title: IFIT3 accelerates the progression of head and neck squamous cell carcinoma by targeting PD-L1 to activate PI3K/AKT signaling pathway.

doi: 10.1186/s12957-023-03274-5

Figure Lengend Snippet: Fig. 6 IFIT3 interacts with PD-L1 in HNSC. A and B IHC analysis showed PD-L1 expression was positively correlated with IFIT3. C IFIT3 protein interacting with PD-L1was examined in FaDu and Cal27 cells by co‑ip. D Co-localization based on IF assays, IFIT3 interacted with PD-L1 in the cytoplasm. Scale bar, 20 μm. E Sphere formation experiment was used to detect the effect of PD-L1 on sphere formation ability caused by IFIT3. Scale bar, 200 μm

Article Snippet: In brief, paraffinization and rehydration were performed, following antigen retrieval, tissue microarray was treated with 3% hydrogen peroxide for the blocking of endogenous peroxidases, and then 1:100 diluted IFIT3 antibody (HPA059914, Atlas Antibodies), 1:100 diluted PD-L1 antibody (CST, USA), and 1:100 diluted ki67 antibody (Affinity, China) were used to incubate tissue sections overnight at 4 °C.

Techniques: Expressing

Journal: World journal of surgical oncology

Article Title: IFIT3 accelerates the progression of head and neck squamous cell carcinoma by targeting PD-L1 to activate PI3K/AKT signaling pathway.

doi: 10.1186/s12957-023-03274-5

Figure Lengend Snippet: Fig. 8 IFIT3 promoted tumorigenicity and the metastasis of HNSC in nude mice. A–D Xenograft tumor volume and weight of the xenograft tumors were measured after mice were sacrificed, IFIT3 knockdown suppressed tumor growth (A, C). IFIT3 upregulation enhanced tumor growth (B, D). E IHC analysis of IFT3 and PD-L1 expression in xenograft tumor tissues. F IFIT3 knockdown suppressed numbers of lung metastatic nodules stained with H&E. G. IFIT3 over-expression promoted numbers of lung metastatic nodules stained with H&E

Article Snippet: In brief, paraffinization and rehydration were performed, following antigen retrieval, tissue microarray was treated with 3% hydrogen peroxide for the blocking of endogenous peroxidases, and then 1:100 diluted IFIT3 antibody (HPA059914, Atlas Antibodies), 1:100 diluted PD-L1 antibody (CST, USA), and 1:100 diluted ki67 antibody (Affinity, China) were used to incubate tissue sections overnight at 4 °C.

Techniques: Knockdown, Expressing, Staining, Over Expression

Journal: Neuron

Article Title: The Parabrachial Nucleus Directly Channels Spinal Nociceptive Signals to the Intralaminar Thalamic Nuclei, but Not the Amygdala.

doi: 10.1016/j.neuron.2020.06.017

Figure Lengend Snippet: Figure 3. Inhibition of the Ipsilateral Spinoparabrachial Pathway Impaired Formalin-Evoked Pain-Related Behaviors (A) Top left: schematic for virus injection in spinal cord and cannula implantation above PBN. Top right: distribution of hM4Di-positive fibers and track of cannula in the PBN. Yellow line, outer and injection cannula. Scale bar, 200 mm. Bottom: timeline of the experiment. (B) Raster plot showing formalin-evoked behaviors after CNO injection into the ipsilateral PBN (0.5 mg/mouse). (C and D) Graph showing formalin-evoked licking (C) and flinching (D) behaviors after CNO injection (n = 6–7 mice, unpaired Student’s t test). (E and F) Duration of formalin-evoked licking (E) and flinching (F) behaviors after CNO injection (n = 6–7 mice, two-way ANOVA). (G) Schematic depicting viral injection into the right dorsal spinal cord and cannula implantation above the contralateral PBN of wild-type mice. (H) Raster plot showing formalin-evoked licking behavior after CNO injection into the contralateral PBN (0.5 mg/mouse). (I and J) Duration of formalin-evoked licking (I) and flinching (J) behaviors after CNO injection into the contralateral PBN (n = 6 mice, two-way ANOVA). (K) Schematic showing viral injection into the right dorsal spinal cord and ipsilateral PBN of wild-type mice. (L) Raster plot showing formalin-evoked licking behavior after CNO injection (1 mg/kg, intraperitoneally [i.p.]). (M and N) Duration of formalin-evoked licking (M) and flinching (N) behaviors after CNO injection (n = 5–9 mice, two-way ANOVA) *p < 0.05, **p < 0.01. Error bars represent SEMs. See also Figure S3.

Article Snippet: To manipulate the activity of the spinoparabrachial pathway, AAV2/9-hSyn-hChR2(H134R)-mCherry (titer: 4.22 3 1012 v.g./mL, Taitool) or AAV2/8-hSyn-hM4Di-mCherry (titer: 5.53 1012 v.g./mL, Addgene) virus was injected into the right superficial dorsal spinal cord, except for the activation of spinoparabrachial pathway arising from left spinal cord, in which virus was injected into the left side of superficial dorsal spinal cord.

Techniques: Inhibition, Virus, Injection

Journal: Neuron

Article Title: The Parabrachial Nucleus Directly Channels Spinal Nociceptive Signals to the Intralaminar Thalamic Nuclei, but Not the Amygdala.

doi: 10.1016/j.neuron.2020.06.017

Figure Lengend Snippet: Figure 4. Functional Roles of Tacr1+ PBN Neurons in Processing Nociceptive Information (A) Schematic for labeling PBNSC neurons in H2B-GFP mice. (B) Distribution pattern of GFP+ (green) and Tacr1+ (red) neurons in the PBN. Scale bar, 50 mm. (C) Magnified view of the boxed area in (B). Scale bar, 50 mm. (D) Percentage of different cell types among GFP+ neurons (n = 3–4 mice). (E) Schematic showing viral injection into the right PBN of Tacr1-flpo mice. (F) Distribution pattern of hM4Di+ (DsRed) neurons in the right PBN. Scale bar, 200 mm. (G and H) Duration of formalin-evoked licking (G) and flinching (H) behaviors after CNO injection (1 mg/kg, i.p.) (n = 7–8 mice, two-way ANOVA). (I) Schematic showing viral injection into right PBN of Tacr1-flpo mice. (J) Distribution pattern of hM3Dq+ (DsRed) neurons in the right PBN. Scale bar, 200 mm. (K) Raster plot showing CNO-evoked licking behavior on right hind paw after CNO injection (2 mg/kg, i.p.). (L) Duration of licking behavior on right hind paw after saline or CNO injection (i.p.) (n = 6–10 mice, two-way ANOVA). (M) Schematic showing viral injection and optical fiber implantation in Tacr1-flpo mice. (N) Top: schematic showing recording of Tacr1+ PBN neurons in response to mechanical stimulation. Bottom: raw traces of fluorescent GCaMP6s signal of Tacr1+ PBN neurons in response to pinch or brush stimulation. Blue bars indicate pinch (5 s) or brush (3 s) stimulation.

Article Snippet: To manipulate the activity of the spinoparabrachial pathway, AAV2/9-hSyn-hChR2(H134R)-mCherry (titer: 4.22 3 1012 v.g./mL, Taitool) or AAV2/8-hSyn-hM4Di-mCherry (titer: 5.53 1012 v.g./mL, Addgene) virus was injected into the right superficial dorsal spinal cord, except for the activation of spinoparabrachial pathway arising from left spinal cord, in which virus was injected into the left side of superficial dorsal spinal cord.

Techniques: Functional Assay, Labeling, Injection, Saline

Journal: iScience

Article Title: Glucocorticoid receptor-mediated Nr1d1 chromatin circadian misalignment in stress-induced irritable bowel syndrome

doi: 10.1016/j.isci.2023.107137

Figure Lengend Snippet:

Article Snippet: IFIT3 Rabbit pAb , ABclonal , A3924.

Techniques: Recombinant, Reporter Assay, Knock-Out, Software

Journal: Nature Communications

Article Title: A basally active cGAS-STING pathway limits SARS-CoV-2 replication in a subset of ACE2 positive airway cell models

doi: 10.1038/s41467-024-52803-7

Figure Lengend Snippet: a WT and STING KO SCC25, H596, OE21, and Detroit 562 cells were subjected to RNA-seq analysis. Heatmap shows differentially expressed genes (absolute log 2 fold change of > 2 and an adjusted q value of < 0.05 from n = 4 independent replicates for each cell type) in the IFN and inflammatory pathways in any given cell line between the WT vs. STING KO variant. b–g RT-qPCR analysis of IFIT2, MX1 and IFIT3 in uninfected SCC25, H596, OE21 and Detroit 562 cells treated with increasing concentrations of SN-011 ( b – d ) and H-151 ( e – g ) for 48 h. Data show the mean from n = 2 independent biological replicates, error bars show SEM ( h , i ) WT and STING KO SCC25 and STING KO cells were pre-treated with 20 μM SN-011 ( h ) or H-151 ( i ) for 24 h were infected with WT SARS-CoV-2 at MOI: 1 i.u./cell for 72 h. Data show the copies of cell-associated SARS-CoV-2 N RNA in compound-treated cells normalized relative to mock-treated samples. Data show the mean from n = 3 independent biological replicates, error bars show SEM. Significance was assessed by multiple unpaired two-tailed t tests and p- values provided in the figure. Source data are provided as a Source Data file.

Article Snippet: After washing with PBST, samples were incubated in a goat anti-mouse secondary antibody conjugated to Alexa Fluor Plus 488 (Invitrogen, Cat# A-11029, 1:1000) at room temperature for 1 h. IFIT3, IFIT2, ISG15 and STING were detected by incubation with a primary rabbit polyclonal IFIT3 antibody (Novus Biologicals NBP2-32500, 1:500), rabbit polyclonal IFIT2 antibody (Novus Biologicals NBP2-15180SS, 1:500), rabbit polyclonal ISG15 antibody (Proteintech 15981-1-AP, 1:250) and rabbit polyclonal STING antibody (Proteintech 19851-1-AP, 1:200) respectively as described above followed by incubation with a goat anti-rabbit fluorescent secondary antibody (Invitrogen Goat anti-Rabbit Alexa Fluor Plus 568TM, Cat# A-11036, 1:1000 or Invitrogen Goat anti-Rabbit Alexa FluorTM Plus 647, Catalog # A32733TR, 1:1000).

Techniques: RNA Sequencing, Variant Assay, Quantitative RT-PCR, Infection, Two Tailed Test

Journal: Nature Communications

Article Title: A basally active cGAS-STING pathway limits SARS-CoV-2 replication in a subset of ACE2 positive airway cell models

doi: 10.1038/s41467-024-52803-7

Figure Lengend Snippet: a Relative expression of ISGs including ISG15, IFIT1, IFIT2, IFIT3, and IRF7 in SCC25 cGAS KO, STING KO or NT control cells infected with SARS-CoV-2 at a MOI of 2 i.u./cell. Cells were collected at 72 hpi, and ISG expression was analyzed by RT-qPCR. Data show the fold induction of ISGs in infected over uninfected cells from n = 3 biological replicates, errors bars show the SEM. b , c SCC25 STING KO or NT control cells were infected with SARS-CoV-2 at an MOI of 2 i.u./cell. Immunofluorescence detection for IFIT3 expression (in red) and SARS-CoV-2 nucleocapsid (N) viral protein (in green), and cellular nuclei (DAPI, in blue) at 72 hpi from a representative experiment ( n = 2) are shown. Images were collected with an epifluorescence microscope, 4X objective ( b ), or Zeiss LSM 880 Airyscan confocal microscope equipped with a × 63/1.4 objective ( c ) as detailed in Methods. Scale bars = 250 μm ( b ) or 10 μm ( c ). d , e SCC25 STING KO cells were infected with SARS-CoV-2-mNG at MOI:1 i.u./cell, and cells were single-cell sorted at 72 hpi to isolate infected (mNG positive) and bystander cells as detailed in Methods. Expression of the indicated ISGs ( d ) and inflammatory genes ( e ) were analyzed by RT-qPCR in these sorted cell populations. Data show the mean from two independent replicates, error bars show the SEM. Source data are provided as a Source Data file.

Article Snippet: After washing with PBST, samples were incubated in a goat anti-mouse secondary antibody conjugated to Alexa Fluor Plus 488 (Invitrogen, Cat# A-11029, 1:1000) at room temperature for 1 h. IFIT3, IFIT2, ISG15 and STING were detected by incubation with a primary rabbit polyclonal IFIT3 antibody (Novus Biologicals NBP2-32500, 1:500), rabbit polyclonal IFIT2 antibody (Novus Biologicals NBP2-15180SS, 1:500), rabbit polyclonal ISG15 antibody (Proteintech 15981-1-AP, 1:250) and rabbit polyclonal STING antibody (Proteintech 19851-1-AP, 1:200) respectively as described above followed by incubation with a goat anti-rabbit fluorescent secondary antibody (Invitrogen Goat anti-Rabbit Alexa Fluor Plus 568TM, Cat# A-11036, 1:1000 or Invitrogen Goat anti-Rabbit Alexa FluorTM Plus 647, Catalog # A32733TR, 1:1000).

Techniques: Expressing, Control, Infection, Quantitative RT-PCR, Immunofluorescence, Microscopy