12604 1 ap  (Proteintech)

Journal: iScience

Article Title: Differentiation-associated ISG expression of NK cells in chronic viral infection

doi: 10.1016/j.isci.2025.113216

Figure Lengend Snippet: Distinct ISG expression in NK cell clusters from healthy donors ScRNAseq analysis of isolated circulating CD3 − CD56 + NK cells from HCMV + /HD (HD1: 14752 cells, HD2: 14595 cells). (A) Unsupervised UMAP cluster analysis showed four distinct NK cell clusters C1-C4 (dark gray: C1, green: C2, light green: C3 and beige: C4). Each dot represents one CD3 − CD56 + NK cell. (B) Heatmap with centered averaged expression of NK cell lineage markers indicates four NK cell clusters as C1 = CD56 bright NK cells (CD56 bright ); C2 = CD56 dim conventional NK cells (cCD56 dim ); and two clusters with an adaptive phenotype, C3 and C4 = CD56 dim CD16 low adaptive NK cells (adCD56 dim I and adCD56 dim II). (C and D) ISG expression in CD56 bright , cCD56 dim , adCD56 dim I and adCD56dim II NK cells. (C) Violin plot with aggregated transcript counts and (D) heatmap with centered averaged expression. (E) Flow cytometric analysis of IFITM3, IRF1, IFIT2 and ISG20 protein expression in NK cells of HCMV + /HDs (n = 4–7). Representative flow cytometry histograms and bar graphs depicting the normalized MFI of IFITM3, IRF1, and IFIT2 and ISG20 expression in CD56 bright , cCD56 dim , and adCD56 dim NK cells. Protein marker expression was normalized to CD3 − CD56 + NK cells. Bar charts indicate the median value with IQR. Statistical analyses were performed (C) via Kruskal-Wallis test, p < 2.2 e−16 , and (E) via one-way repeated measures ANOVA (IFITM3, IRF1, ISG20) and Friedmann test (IFIT2), ∗: p < 0.05, ∗∗: p < 0.01, ∗∗∗∗: p < 0.0001. ScRNAseq, single cell RNA sequencing; HCMV + , human cytomegalovirus seropositive; HD, healthy donor; UMAP, uniform manifold approximation and projection; ISG, interferon-stimulated gene; MFI, median fluorescence intensity; IQR, interquartile range. See also and .

Article Snippet: Mouse monoclonal anti-human IFIT2 (clone F-12) , Santa Cruz Biotechnology , Cat#sc-390724 AF594.

Techniques: Expressing, Isolation, Flow Cytometry, Marker, RNA Sequencing, Fluorescence

Journal: iScience

Article Title: Differentiation-associated ISG expression of NK cells in chronic viral infection

doi: 10.1016/j.isci.2025.113216

Figure Lengend Snippet: Conserved ISG patterns in NK cells during chronic viral infection Comparative scRNAseq analysis of isolated circulating CD3 − CD56 + NK cells from patients suffering from HCMV + /cHBV or HCMV + /cHCV infection (HBV1: 9760 cells, HBV2: 9921 cells, HBV3: 10030 cells, HCV1: 9957 cells, HCV2: 9923 cells, HCV3: 9831 cells). (A) Unsupervised UMAP cluster analysis to identify NK cell clusters (dark gray: CD56 bright , green: cCD56 dim , light green: adCD56 dim I and beige: adCD56 dim II) in HCMV + /cHBV- and HCMV + /cHCV-infected patients. Each dot represents one CD3 − CD56 + NK cell. (B) Bar graph with frequencies of NK cell cluster distribution on RNA level in HCMV + /HDs, HCMV + /cHBV-, and HCMV + /cHCV-infected patients is depicted. (C) Centered average expression of ISGs in NK cell clusters from HCMV + /HD, HCMV + /cHBV-, and HCMV + /cHCV-infected patients is shown. (D) Normalized MFI of IFITM3, IRF1, IFIT2 and ISG20 on protein level in NK cell clusters obtained from HCMV + /cHBV- (n = 8–13)or HCMV + /cHCV-infected patient (n = 8–11)and from HCMV + /HDs (n = 4–7). (E) Normalized MFI of IFITM3, IRF1, IFIT2, and ISG20 on protein level in NK cells separated by subsets. Bar charts indicate the median value with IQR. MFI of ISG expression was normalized to CD3 − CD56 + NK cells. Statistical significance was assessed by: (D) ordinary one-way repeated measures ANOVA (IFITM3, ISG20) and Friedmann test (IRF1, IFIT2); and (E) RM one-way repeated measures ANOVA (IFITM3, ISG20), Kruskal-Wallis test (IRF1/CD56 bright /cCD56 dim , IFIT2/CD56 bright ) and Friedmann test (IRF1/adCD56 dim , IFIT2/cCD56 dim /adCD56 dim . p > 0.05, ∗: p < 0.05, ∗∗: p < 0.01, ∗∗∗: p < 0.001, ∗∗∗∗: p < 0.0001. cHBV, chronic hepatitis B virus infection; cHCV, chronic hepatitis C virus infection. n.s., not significant. See also .

Article Snippet: Mouse monoclonal anti-human IFIT2 (clone F-12) , Santa Cruz Biotechnology , Cat#sc-390724 AF594.

Techniques: Infection, Isolation, Expressing, Virus

Journal: iScience

Article Title: Differentiation-associated ISG expression of NK cells in chronic viral infection

doi: 10.1016/j.isci.2025.113216

Figure Lengend Snippet: ISG expression is associated with NK cell differentiation regulated by transcription factor networks (A) Merged unsupervised clustering of NK cell clusters (dark gray: CD56 bright , green: cCD56 dim , light green: adCD56 dim I and beige: adCD56 dim II) and trajectory inference analysis of NK cell clusters merged from HCMV + /HD, HCMV + /cHBV- and HCMV + /cHCV-infected patients. Each dot represents one CD3 − CD56 + NK cell. (B) IFITM3 expression profile with respect to pseudotime of the different NK cell clusters. The line indicates the fitting of the expression trend over pseudotime. (C) Statistical graphs depicting ISG expression in CD56 bright and cCD56 dim NK cells of HCMV + /HD ( n = 5), HCMV + /cHBV- ( n = 4) and HCMV + /cHCV-infected patients ( n = 3) ex vivo (white) and in vitro after differentiation at day 21 (light gray) measured by flow cytometry. (D) Binary heatmap represents the co-regulatory network analysis of the previously introduced four ISG sets and distinct transcription factors. White indicates non-regulation and gray indicates regulation. (E) Co-expression analysis of TCF1 and IFITM3 on protein level in CD56 bright , cCD56 dim , and adCD56 dim NK cells from HCMV + /HD ( n = 4), HCMV + /cHBV- ( n = 4) and HCMV + /cHCV-infected patients ( n = 5) is shown. (F and G) Representative flow cytometry dot plot and bar chart with percentage of (F) TCF1 expression and (G) co-expression analysis of TCF1 and IFITM3 of in vitro differentiated cCD56 dim NK cells (light gray) compared to ex vivo ISG expression in CD56 bright and cCD56 dim NK cells (white) from HCMV + /HD ( n = 5), HCMV + /cHBV- ( n = 4) and HCMV + /cHCV-infected patients ( n = 3). Bar charts indicate the median value with IQR. MFI of ISG expression was normalized to CD3 − CD56 + NK cells. Statistical significance was assessed by (D and G) one-way repeated measures ANOVA (IFITM3), Friedman test (IRF1, ISG20, IFIT2, and TCF1), and (F and H) simple linear regression. ∗: p < 0.05, ∗∗: p < 0.01, ∗∗∗: p < 0.001, ∗∗∗∗: p < 0.0001. See also .

Article Snippet: Mouse monoclonal anti-human IFIT2 (clone F-12) , Santa Cruz Biotechnology , Cat#sc-390724 AF594.

Techniques: Expressing, Cell Differentiation, Infection, Ex Vivo, In Vitro, Flow Cytometry

Journal: Journal of Virology

Article Title: IFIT3 RNA-binding activity promotes influenza A virus infection and translation efficiency

doi: 10.1128/jvi.00286-25

Figure Lengend Snippet: IFIT3 enhances influenza A virus replication. (A) IFIT3 RNA expression measured by RT-qPCR from A549 CRISPRa cells inoculated with non-targeting or IFIT3-targeting TRPPC viruses (MOI = 5). Data are normalized to the non-targeting control. (B) Influenza virus NP protein levels measured 8 hpi in A549 CRISPRa cells inoculated with non-targeting or IFIT3-targeting TRPPC viruses (MOI = 5). Data are normalized to the non-targeting control. (C) Single-cycle replication was measured in A549 CRISPRa cells infected with virus targeting IFIT3 or a non-targeting control (MOI = 0.05). Viral titers were determined 18 hpi. (D) Multicycle replication kinetics in A549 CRISPRa cells inoculated with virus targeting IFIT3 or a non-targeting control (MOI = 0.05). Titers were determined at the indicated time points by plaque assay. Data in panels A–D) are the mean of n = 3 ± SD. Significance was assessed by a two-way Student’s t -test. (E) Viral gene expression during single-cycle infection in WT, IFIT3 -/- , and IFIT2 -/- A549 cells using a WSN-based reporter virus measured 8 hpi. Data are the grand mean ± SEM of two independent biological replicates, each with three technical replicates. Significance was assessed by one-way ANOVA with Dunnett’s multiple comparisons test. * P < 0.5, ** P < 0.01, and **** P < 0.0001.

Article Snippet: Membranes were then probed with primary antibodies targeting V5 (Bethyl Laboratories, Inc., cat. no. A190-220A) at a 1:5,000 dilution, IFIT3 (Proteintech 15201-1-AP) at a 1:800 dilution, IFIT2 (Proteintech 12604-1-AP) at a 1:500 dilution, or GFP (Proteintech 50430-2-AP) at a 1:1,000 dilution.

Techniques: Virus, RNA Expression, Quantitative RT-PCR, Control, Infection, Plaque Assay, Gene Expression

Journal: Journal of Virology

Article Title: IFIT3 RNA-binding activity promotes influenza A virus infection and translation efficiency

doi: 10.1128/jvi.00286-25

Figure Lengend Snippet: IFIT2 and IFIT3 function independently while also exhibiting an additive pro-viral effect. (A) WT 293 cells were transfected with plasmids expressing IFIT2, IFIT3, both IFIT2 and IFIT3, or an empty vector control. Cells were subsequently inoculated with a WSN-based PASTN reporter virus (MOI = 0.01), and viral gene expression was measured 8 hours post-infection. (B) Viral spread was measured in WT, IFIT2 -/- , and IFIT3 -/- 293 cells following the same approach as in panel A, except infection was allowed to proceed for 24 hours before measuring reporter activity. (C) Representative western blot of cell lysate before infection demonstrating IFIT2 and IFIT3 expression levels. Data are mean ± SD of a representative biological replicate from a total of three. Significance was assessed by one-way ANOVA with Dunnett’s multiple comparisons test. * P < 0.5, *** P < 0.001, and **** P < 0.0001.

Article Snippet: Membranes were then probed with primary antibodies targeting V5 (Bethyl Laboratories, Inc., cat. no. A190-220A) at a 1:5,000 dilution, IFIT3 (Proteintech 15201-1-AP) at a 1:800 dilution, IFIT2 (Proteintech 12604-1-AP) at a 1:500 dilution, or GFP (Proteintech 50430-2-AP) at a 1:1,000 dilution.

Techniques: Transfection, Expressing, Plasmid Preparation, Control, Virus, Gene Expression, Infection, Activity Assay, Western Blot

Journal: Journal of Virology

Article Title: IFIT3 RNA-binding activity promotes influenza A virus infection and translation efficiency

doi: 10.1128/jvi.00286-25

Figure Lengend Snippet: IFIT3 directly binds RNA. (A) Electrophoretic mobility shift assays were performed using in vitro -transcribed RNAs derived from regions of the indicated viral genes in the presence or absence of recombinant IFIT3. RNAs were detected by Sybr gold staining (top), while proteins were visualized with Coomassie staining (bottom). (B) EMSAs were performed with increasing amounts of recombinant IFIT3 and the indicated RNAs, revealing dose-dependent RNA binding. RNA was detected by Sybr gold staining (top), while protein was visualized with Coomassie staining (bottom). (C) EMSAs were performed by forming complexes between NA1 RNA and increasing amounts of IFIT3. Where indicated, complexes were denatured by boiling before electrophoresis. (D) RNA-immunoprecipitations (RIPs) were performed on infected cell lysates. IFIT2 -/- A549 cells were infected with WSN (MOI = 0.02) for 24 hours, lysed, and lysates were immunoprecipitated with an antibody targeting IFIT3 or an IgG control. IFIT3-specific binding was quantified by RT-qPCR relative to an IgG control. A representative Western blot showing expression and specific capture of IFIT3 is shown below, in the input as well as pulldown with the IFIT3 antibody, but not the IgG antibody.

Article Snippet: Membranes were then probed with primary antibodies targeting V5 (Bethyl Laboratories, Inc., cat. no. A190-220A) at a 1:5,000 dilution, IFIT3 (Proteintech 15201-1-AP) at a 1:800 dilution, IFIT2 (Proteintech 12604-1-AP) at a 1:500 dilution, or GFP (Proteintech 50430-2-AP) at a 1:1,000 dilution.

Techniques: Electrophoretic Mobility Shift Assay, In Vitro, Derivative Assay, Recombinant, Staining, RNA Binding Assay, Electrophoresis, Infection, Immunoprecipitation, Control, Binding Assay, Quantitative RT-PCR, Western Blot, Expressing

Journal: Journal of Virology

Article Title: IFIT3 RNA-binding activity promotes influenza A virus infection and translation efficiency

doi: 10.1128/jvi.00286-25

Figure Lengend Snippet: Identification of a conserved RNA-binding surface on IFIT3. (A) A putative IFIT3 structure was modeled on the IFIT2 structure. Dimers (left) are depicted as pairs of ribbon and space-filling subunits, while monomers (right) are shown rotated to emphasize surfaces facing the dimer interface. The computed electrostatic surface potential is shown from blue (basic) to red (acidic). Residues identified by RBS-ID as cross-linking to RNA are indicated, and a conserved basic patch is outlined in black. (B) Protein alignment of a portion of IFIT3 from multiple influenza A virus hosts. Red arrows indicate sites identified via RBS-ID, and blue arrows indicate amino acids that were mutated based on sequence similarity to IFIT2 RNA-binding mutants.

Article Snippet: Membranes were then probed with primary antibodies targeting V5 (Bethyl Laboratories, Inc., cat. no. A190-220A) at a 1:5,000 dilution, IFIT3 (Proteintech 15201-1-AP) at a 1:800 dilution, IFIT2 (Proteintech 12604-1-AP) at a 1:500 dilution, or GFP (Proteintech 50430-2-AP) at a 1:1,000 dilution.

Techniques: RNA Binding Assay, Virus, Sequencing

Journal: Journal of Virology

Article Title: IFIT3 RNA-binding activity promotes influenza A virus infection and translation efficiency

doi: 10.1128/jvi.00286-25

Figure Lengend Snippet: RNA binding by IFIT3 is important for its pro-viral function. (A) Influenza NP expression assays were performed in IFIT3 -/- 293 cells co-transfected with WT or mutant IFIT3 or an empty-vector control. NP translation was measured with a luciferase reporter assay (left), and NP RNA levels were measured by qRT-PCR (right). Equivalent expression of WT and mutant IFIT3 was shown by Western blot. (B) IFIT3 (left) and IFIT2 (right) dimer formation was measured for WT proteins and RNA-binding mutants IFIT3 K249E (K/E) and IFIT2 R292E/K410E (DM2). WT and mutant proteins were expressed as GFP- and V5-tagged proteins in 293T cells. The GFP-tagged form was immunoprecipitated from cell lysates, and co-precipitating proteins were detected by blotting for V5-tagged versions. Co-precipitating proteins are shown in the top blot, while input proteins are detected in the middle and bottom blots. (C) IFIT2/IFIT3 heterodimer formation was measured using WT IFIT2 (Ι2) and IFIT3 (Ι3) proteins and RNA-binding mutants IFIT2 R292E/K410E (DM2) and IFIT3 K249E (K/E). Co-immunoprecipitations were performed as in panel B. (D) IFIT3 -/- 293 cells were transfected with plasmids expressing WT or mutant IFIT3 and subsequently inoculated with a WSN-based PASTN reporter virus (MOI = 0.01). Viral gene expression was measured 8 hours post-infection. IFIT3 expression was detected by western blot. (E) Viral spread was measured in IFIT3 -/- 293 cells following the same approach as in panel D, except infection was allowed to proceed for 24 hours before measuring reporter activity. Data are mean ± SD of three technical replicates from a representative biological replicate from a total of three biological replicates, except qRT-PCR, which is the grand mean of three independent biological replicates each measured in technical triplicate. Significance assessed by Student’s t -test for qRT-PCR or multiple-comparison testing with a one-way ANOVA with Dunnett’s multiple comparisons test. ns, not significant; * P < 0.5, ** P < 0.01, *** P < 0.001, and **** P < 0.0001.

Article Snippet: Membranes were then probed with primary antibodies targeting V5 (Bethyl Laboratories, Inc., cat. no. A190-220A) at a 1:5,000 dilution, IFIT3 (Proteintech 15201-1-AP) at a 1:800 dilution, IFIT2 (Proteintech 12604-1-AP) at a 1:500 dilution, or GFP (Proteintech 50430-2-AP) at a 1:1,000 dilution.

Techniques: RNA Binding Assay, Expressing, Transfection, Mutagenesis, Plasmid Preparation, Control, Luciferase, Reporter Assay, Quantitative RT-PCR, Western Blot, Immunoprecipitation, Virus, Gene Expression, Infection, Activity Assay, Comparison