Journal: npj Viruses

Article Title: STAT1 signaling controls cholesterol metabolism in epithelial cells and RSV-induced syncytia formation

doi: 10.1038/s44298-026-00173-w

Figure Lengend Snippet: A Sequence alignment of wild-type and STAT1 −/− to STAT1 gene of the human genome (GrCh38.p14). The red bar indicates the binding site of sgRNAs. The red star indicates the 17 bp deletion on the second allele. B Western blot of whole cell lysates of wild-type and STAT1 −/− HEp-2 cells targeting STAT1 (top) and β-Actin (bottom) as loading control. C Bright-field IncuCyte live-cell images of wild-type (left) and STAT1 −/− HEp-2 (right) at 72 hours (h) post-seeding. Images representative of three independent experiments (10 technical replicates). Scale bar indicated next to images. D Quantification of cell eccentricity for wild-type and STAT1 −/− HEp-2 cells at 72 h post-seeding based on live cell images. Eccentricity was assessed at approximately 60% confluence, which was reached at 72 h post-seeding for both cell types. Minimum area, minimum eccentricity, and Hole fill were set to 600 μm 2 , 0.3, and 1000 μm 2 , respectively. Mean ± SD from three independent experiments, with 10 replicates each, are shown. Statistical analysis: Unpaired t-test. E Quantitative analysis of IncuCyte live-cell images for cells per well, with images taken every 3 h for 4.5 days. Mean ± SD of three independent experiments (10 technical replicates) is shown, with non-linear regressions applied for wild-type (green) and STAT1 −/− (red) HEp-2 cells. Statistical analysis: Mann-Whitney test for comparison of ranks. F Volcano plot demonstrating the number of differentially expressed genes between wild-type and STAT1 −/− HEp-2 cells with thresholds p < 0.05 and |Log2 Fold change | ≥ 1. G Bar plot of Gene Set Enrichment Analysis of Gene Ontology. The plot shows significantly enriched biological processes in STAT1 −/− compared to wild-type HEp-2 cells.

Article Snippet: HEp-2 (ATCC, CCL-23) and STAT1 knockout (STAT1 −/− ) HEp-2 cells were cultured in Minimum Essential medium with Earl’s salts (Capricorn Scientific, MEM-A) supplemented with 10% fetal bovine serum (FBS, Gibco), 1% penicillin/streptomycin (P/S, Capricorn Scientific), 1% non-essential amino acids (Gibco), and 1% GlutaMAX (Gibco) at 37 °C, 5% CO 2 .

Techniques: Sequencing, Binding Assay, Western Blot, Control, MANN-WHITNEY, Comparison

Journal: npj Viruses

Article Title: STAT1 signaling controls cholesterol metabolism in epithelial cells and RSV-induced syncytia formation

doi: 10.1038/s44298-026-00173-w

Figure Lengend Snippet: A Viral replication kinetics of RSV-A-0594 (dashed lines) and rRSV-A-0594-eGFP (solid lines)-infected wild-type (green) and STAT1 −/− (red) HEp-2 cells (MOI 0.05). Mean ± SD of three independent experiments (four technical replicates) is shown. Statistical analysis: Two-way ANOVA with Fisher’s LSD test. B Fluorescence images of rRSV-A-0594-eGFP-infected (MOI 0.05) wild-type (top) and STAT1 −/− (bottom) Hep-2 cells at 48 hours post-infection (hpi). Images representative of three independent experiments (two technical replicates). Scale bar indicated next to images. C Graph showing the comparison of viral transcript numbers from RNA-Seq analysis between wild-type and STAT1 −/− HEp-2 cells from 0 to 72 hpi with thresholds set at p < 0.05 and |Log2 Fold change | ≥ 1. D Presatovir-based fusion inhibition assay on wild-type (green) and STAT1 −/− (red) HEp-2 cells at 72 hpi with rRSV-A-0594-eGFP (MOI 0.05). Fluorescence intensity (eGFP) relative to untreated, infected cells is shown. Solid lines indicate mean values, while dashed lines above and below each curve indicate the corresponding standard deviation (Mean ± SD). Horizontal dashed line and the right graph indicate Half Maximal Inhibitory Concentration (IC 50 ) of wild-type (green) and STAT1 −/− (red) HEp-2 based on non-linear regression fitted to graphs. Mean ± SD of three independent experiments (eight technical replicates) is shown. Statistical analysis for IC 50 comparison: Unpaired t-test with Welsh’s correction.

Article Snippet: HEp-2 (ATCC, CCL-23) and STAT1 knockout (STAT1 −/− ) HEp-2 cells were cultured in Minimum Essential medium with Earl’s salts (Capricorn Scientific, MEM-A) supplemented with 10% fetal bovine serum (FBS, Gibco), 1% penicillin/streptomycin (P/S, Capricorn Scientific), 1% non-essential amino acids (Gibco), and 1% GlutaMAX (Gibco) at 37 °C, 5% CO 2 .

Techniques: Infection, Fluorescence, Comparison, RNA Sequencing, Inhibition, Standard Deviation, Concentration Assay

Journal: npj Viruses

Article Title: STAT1 signaling controls cholesterol metabolism in epithelial cells and RSV-induced syncytia formation

doi: 10.1038/s44298-026-00173-w

Figure Lengend Snippet: A UpSet plot demonstrating differentially expressed genes that are shared and unique to infected wild-type and STAT1 −/− HEp-2 after comparison to respective uninfected controls with thresholds p < 0.05 and |Log2 Fold change | ≥ 1. B Cleveland Dot Plot of Gene Set Enrichment Analysis (GSEA) of Gene Ontology comparing processes enriched in both infected wild-type and STAT1 −/− HEp-2 cells based on Normalized Enrichment Scores (NES). C Quantification of total free cholesterol by luminescent enzymatic assay in uninfected wild-type (green) and STAT1 −/− (red) HEp-2 cells. Statistical analysis: Mann-Whitney test. D Quantification of total free cholesterol by luminescent enzymatic assay in wild-type and STAT1 −/− HEp-2 cells, which were uninfected (grey), treated with 5 mM methyl-beta-cyclodextrin (MβCD, orange), infected at MOI 0.05 (light red), infected at MOI (dark red), or treated with 100 ng/ml IFN-α2 and 50 ng/mL IFN-γ at 24 and 48 hpi. Only significant differences between untreated and treated cells within a cell type or within treatments between cell types are shown. Statistical analysis: two-way ANOVA with Fisher’s LSD test. Mean ± SD of four independent experiments (two technical replicates) shown for ( C , D ).

Article Snippet: HEp-2 (ATCC, CCL-23) and STAT1 knockout (STAT1 −/− ) HEp-2 cells were cultured in Minimum Essential medium with Earl’s salts (Capricorn Scientific, MEM-A) supplemented with 10% fetal bovine serum (FBS, Gibco), 1% penicillin/streptomycin (P/S, Capricorn Scientific), 1% non-essential amino acids (Gibco), and 1% GlutaMAX (Gibco) at 37 °C, 5% CO 2 .

Techniques: Infection, Comparison, Enzymatic Assay, MANN-WHITNEY

Journal: npj Viruses

Article Title: STAT1 signaling controls cholesterol metabolism in epithelial cells and RSV-induced syncytia formation

doi: 10.1038/s44298-026-00173-w

Figure Lengend Snippet: Representative fluorescence images of rRSV-A-0594-eGFP-infected wild-type ( A ) and STAT1 −/− ( B ) HEp-2 cells (MOI 0.05) at 48 hpi. Cells were left untreated (first row), treated with 50 μM Gemfibrozil (GFZ, second row), treated with 5 μM methyl-β-cyclodextrin (MβCD) and 50 μM GFZ (third row), or with 10 μM 25-hydroxycholesterol (25-HC, bottom panel). Free cholesterol in plasma membranes was visualized by Filipin III staining (0.05 mg/ml), and DNA was visualized by Propidium iodide (PI) staining (5 μg/ml). Images representative of three independent experiments (two technical replicates). Scale bar indicated next to images.

Article Snippet: HEp-2 (ATCC, CCL-23) and STAT1 knockout (STAT1 −/− ) HEp-2 cells were cultured in Minimum Essential medium with Earl’s salts (Capricorn Scientific, MEM-A) supplemented with 10% fetal bovine serum (FBS, Gibco), 1% penicillin/streptomycin (P/S, Capricorn Scientific), 1% non-essential amino acids (Gibco), and 1% GlutaMAX (Gibco) at 37 °C, 5% CO 2 .

Techniques: Fluorescence, Infection, Clinical Proteomics, Staining

Journal: npj Viruses

Article Title: STAT1 signaling controls cholesterol metabolism in epithelial cells and RSV-induced syncytia formation

doi: 10.1038/s44298-026-00173-w

Figure Lengend Snippet: A Fluorescence images from IncuCyte live-cell imaging of rRSV-A-0594-eGFP-infected wild-type (left) and STAT1 −/− (right) HEp-2 cells (MOI 0.05), which were left untreated (left column), treated with 50 μM Gemfibrozil (GFZ, middle column), or with 10 μM 25-hydroxycholesterol (25-HC, right column) at 12, 24, 36, and 48 hpi. Images representative of three independent (two technical replicates) experiments. Scale bar indicated next to images. B Quantification of syncytia size by IncuCyte live-cell imaging for green (fluorescent) area per image relative to green object count per image of rRSV-A-0594-eGFP-infected wild-type (solid lines) and STAT1 −/− (dashed lines) HEp-2 cells (MOI 0.05), either untreated (red), treated with 50 μM GFZ (blue), or 10 μM 25-HC (green). Images of cells were taken every hour for up to 48 hpi. Mean ± SEM of eight independent experiments (two technical replicates) is shown. Statistical analysis was conducted on the overall distribution of syncytia size during the course of infection using the Friedman test with Dunn’s multiple-comparison test.

Article Snippet: HEp-2 (ATCC, CCL-23) and STAT1 knockout (STAT1 −/− ) HEp-2 cells were cultured in Minimum Essential medium with Earl’s salts (Capricorn Scientific, MEM-A) supplemented with 10% fetal bovine serum (FBS, Gibco), 1% penicillin/streptomycin (P/S, Capricorn Scientific), 1% non-essential amino acids (Gibco), and 1% GlutaMAX (Gibco) at 37 °C, 5% CO 2 .

Techniques: Fluorescence, Live Cell Imaging, Infection, Comparison

Journal: npj Viruses

Article Title: STAT1 signaling controls cholesterol metabolism in epithelial cells and RSV-induced syncytia formation

doi: 10.1038/s44298-026-00173-w

Figure Lengend Snippet: Confocal microscopy images of RSV-A-0594-infected wild-type ( A ) and STAT1 −/− ( B ) HEp-2 cells (MOI 0.05) at 48 hpi. Infected cells were left untreated (top), treated with 50 μM Gemfibrozil (GFZ, middle), or 10 μM 25-hydroxycholesterol (25-HC, bottom). Cells were stained for RSV postfusion F protein on the cell surface. Intracellular actin was visualized by ActinRed staining and nuclei by NucBlue staining. Images representative of three independent experiments (two technical replicates). Scale bar indicated next to images.

Article Snippet: HEp-2 (ATCC, CCL-23) and STAT1 knockout (STAT1 −/− ) HEp-2 cells were cultured in Minimum Essential medium with Earl’s salts (Capricorn Scientific, MEM-A) supplemented with 10% fetal bovine serum (FBS, Gibco), 1% penicillin/streptomycin (P/S, Capricorn Scientific), 1% non-essential amino acids (Gibco), and 1% GlutaMAX (Gibco) at 37 °C, 5% CO 2 .

Techniques: Confocal Microscopy, Infection, Staining

Journal: npj Viruses

Article Title: STAT1 signaling controls cholesterol metabolism in epithelial cells and RSV-induced syncytia formation

doi: 10.1038/s44298-026-00173-w

Figure Lengend Snippet: Western blot of membrane fractions and total cell lysates in uninfected (‘Mock’) and rRSV-A-0594-eGFP-infected (MOI 0.5) wild-type ( A ) and STAT1 −/− HEp-2 ( B ) HEp-2 cells at 48 hpi. Infected cells were left untreated, treated with 50 μM Gemfibrozil (GFZ), treated with 5 μM methyl-β-cyclodextrin (MβCD) and 50 μM GFZ, or with 10 μM 25-hydroxycholesterol (25-HC). Membrane fractions were stained for prefusion F protein, postfusion F protein, and Na + -K + -ATPase (loading control). Total cell lysates were stained for total F protein (prefusion and postfusion conformation) and β-Actin (loading control). Images representative of three independent experiments.

Article Snippet: HEp-2 (ATCC, CCL-23) and STAT1 knockout (STAT1 −/− ) HEp-2 cells were cultured in Minimum Essential medium with Earl’s salts (Capricorn Scientific, MEM-A) supplemented with 10% fetal bovine serum (FBS, Gibco), 1% penicillin/streptomycin (P/S, Capricorn Scientific), 1% non-essential amino acids (Gibco), and 1% GlutaMAX (Gibco) at 37 °C, 5% CO 2 .

Techniques: Western Blot, Membrane, Infection, Staining, Control

Journal: Molecular Medicine Reports

Article Title: STAT1 accelerates cutaneous melanoma progression through TUBB4A expression regulation

doi: 10.3892/mmr.2026.13828

Figure Lengend Snippet: STAT1 knockdown significantly reduces melanoma cell viability and proliferation. STAT1 mRNA expression was analyzed in (A) A375 and (B) RPMI-7951 melanoma cells post-STAT1 knockdown using siRNA. (C) Western blotting was used to assess STAT1 protein levels in melanoma cell lines following siRNA-mediated knockdown. Cell viability was evaluated after STAT1 knockdown in (D) A375 and (E) RPMI-7951 cells using Cell Counting Kit-8 assays. (F) Colony formation assays were performed to assess the proliferative capacity of cells after STAT1 knockdown. (G) Quantification of colony formation assay results. **P<0.01 vs. si-NC group. siRNA, small interfering RNA; STAT1, signal transducer and activator of transcription 1; si-STAT1, siRNA targeting STAT1; si-NC, negative control siRNA.

Article Snippet: Melanoma cells (A375 and RPMI-7951) were seeded into 6-well plates (cat. no. 3516; Corning, Inc.) at a density of 500–1,000 cells per well and allowed to adhere for 24 h. Cells were then transfected with si-NC (cat. no. A09010) or si-STAT1 (cat. no. A09009; GenePharma), as well as transfected using a lentiviral vector for TUBB4A overexpression (Ov-TUBB4A; pReceiver-Lv105; GeneCopoeia, Inc.) or Ov-NC (pReceiver-Lv105 Empty Vector; GeneCopoeia, Inc.).

Techniques: Knockdown, Expressing, Western Blot, Cell Counting, Colony Assay, Small Interfering RNA, Negative Control

Journal: Molecular Medicine Reports

Article Title: STAT1 accelerates cutaneous melanoma progression through TUBB4A expression regulation

doi: 10.3892/mmr.2026.13828

Figure Lengend Snippet: STAT1 knockdown significantly promotes melanoma cell apoptosis and inhibits migration. (A and B) Apoptosis was assessed and quantified in A375 and RPMI-7951 cells following STAT1 knockdown using flow cytometry. (C) Transwell migration assays were conducted to evaluate cell migration following STAT1 knockdown. Magnification, ×200. (D) Quantification of migration capacity in A375 and RPMI-7951 cells following STAT1 knockdown. *P<0.05 vs. si-NC. STAT1, signal transducer and activator of transcription 1; si-STAT1, small interfering RNA targeting STAT1; si-NC, negative control small interfering RNA.

Article Snippet: Melanoma cells (A375 and RPMI-7951) were seeded into 6-well plates (cat. no. 3516; Corning, Inc.) at a density of 500–1,000 cells per well and allowed to adhere for 24 h. Cells were then transfected with si-NC (cat. no. A09010) or si-STAT1 (cat. no. A09009; GenePharma), as well as transfected using a lentiviral vector for TUBB4A overexpression (Ov-TUBB4A; pReceiver-Lv105; GeneCopoeia, Inc.) or Ov-NC (pReceiver-Lv105 Empty Vector; GeneCopoeia, Inc.).

Techniques: Knockdown, Migration, Flow Cytometry, Small Interfering RNA, Negative Control

Journal: Molecular Medicine Reports

Article Title: STAT1 accelerates cutaneous melanoma progression through TUBB4A expression regulation

doi: 10.3892/mmr.2026.13828

Figure Lengend Snippet: STAT1 regulates TUBB4A expression at the transcription level. (A) STAT1 mRNA levels were measured in A375 and RPMI-7951 cells after STAT1 knockdown via transfection with different siRNA sequences. (B) TUBB4A mRNA levels were measured in A375 and RPMI-7951 cells after STAT1 knockdown via transfection with different siRNA sequences. (C) Specific fragments of the TUBB4A promoter region were cloned into the luciferase reporter plasmids upstream of the firefly luciferase gene. (D) Transcriptional activity of various TUBB4A promoter fragments was analyzed by luciferase reporter assay in 293T cells, with the −1,783 and −1,771 fragments exhibiting the highest activity. (E) STAT1 siRNA-mediated knockdown significantly reduced STAT1 mRNA levels in A375 cells. (F) STAT1 knockdown significantly reduced the luciferase activity of the −1,783 fragment of the TUBB4A promoter, but not the −1,771 fragment. (G) Chromatin immunoprecipitation assays were performed in A375 and RPMI-7951 cells targeting the −1,783 binding site in the TUBB4A promoter region. Quantitative PCR provided evidence of STAT1 binding to this region. Genomic DNA input was set to 100%. **P<0.01 vs. si-NC; ## P<0.01 vs. PGL3; && P<0.01 vs. IgG. STAT1, signal transducer and activator of transcription 1; siRNA, small interfering RNA; si-NC, negative control siRNA; si-STAT1, siRNA targeting STAT1; si-STAT1-1, siRNA targeting STAT1 sequence 1; si-STAT1-2, siRNA targeting STAT1 sequence 2; TUBB4A, tubulin β4A; PGL3, promoter-gluc luciferase 3; LUC, firefly luciferase gene.

Article Snippet: Melanoma cells (A375 and RPMI-7951) were seeded into 6-well plates (cat. no. 3516; Corning, Inc.) at a density of 500–1,000 cells per well and allowed to adhere for 24 h. Cells were then transfected with si-NC (cat. no. A09010) or si-STAT1 (cat. no. A09009; GenePharma), as well as transfected using a lentiviral vector for TUBB4A overexpression (Ov-TUBB4A; pReceiver-Lv105; GeneCopoeia, Inc.) or Ov-NC (pReceiver-Lv105 Empty Vector; GeneCopoeia, Inc.).

Techniques: Expressing, Knockdown, Transfection, Clone Assay, Luciferase, Activity Assay, Reporter Assay, Chromatin Immunoprecipitation, Binding Assay, Real-time Polymerase Chain Reaction, Small Interfering RNA, Negative Control, Sequencing

Journal: Molecular Medicine Reports

Article Title: STAT1 accelerates cutaneous melanoma progression through TUBB4A expression regulation

doi: 10.3892/mmr.2026.13828

Figure Lengend Snippet: TUBB4A overexpression mitigates the effects of STAT1 knockdown on cell viability and proliferation. TUBB4A mRNA levels were measured in (A) A375 and (B) RPMI-7951 cells following TUBB4A overexpression mediated by a lentiviral vector. (C) TUBB4A protein expression was analyzed after its overexpression. Combined STAT1 knockdown and TUBB4A overexpression transfections were performed, followed by a western blot analysis of TUBB4A protein levels in (D) A375 and (E) RPMI-7951 cells. Cell viability was assessed via Cell Counting Kit-8 assays following combined STAT1 knockdown and TUBB4A overexpression in (F) A375 and (G) RPMI-7951 cells. (H) Colony formation assays were performed to assess the proliferative capacity of cells after STAT1 knockdown and TUBB4A overexpression. (I) Quantification of colony formation assay results. **P<0.01 vs. Ov-NC; ## P<0.01 vs. si-STAT1. TUBB4A, tubulin β4A; STAT1, signal transducer and activator of transcription 1; si-NC, negative control small interfering RNA; si-STAT1, small interfering RNA targeting STAT1; Ov-NC, negative control lentiviral overexpression vector; Ov-TUBB4A, lentiviral vector for TUBB4A overexpression.

Article Snippet: Melanoma cells (A375 and RPMI-7951) were seeded into 6-well plates (cat. no. 3516; Corning, Inc.) at a density of 500–1,000 cells per well and allowed to adhere for 24 h. Cells were then transfected with si-NC (cat. no. A09010) or si-STAT1 (cat. no. A09009; GenePharma), as well as transfected using a lentiviral vector for TUBB4A overexpression (Ov-TUBB4A; pReceiver-Lv105; GeneCopoeia, Inc.) or Ov-NC (pReceiver-Lv105 Empty Vector; GeneCopoeia, Inc.).

Techniques: Over Expression, Knockdown, Plasmid Preparation, Expressing, Transfection, Western Blot, Cell Counting, Colony Assay, Negative Control, Small Interfering RNA

Journal: Molecular Medicine Reports

Article Title: STAT1 accelerates cutaneous melanoma progression through TUBB4A expression regulation

doi: 10.3892/mmr.2026.13828

Figure Lengend Snippet: TUBB4A overexpression reverses the effects of STAT1 knockdown on apoptosis, migration and tumor growth. (A-D) Apoptosis and migration were evaluated in A375 and RPMI-7951 cells following STAT1 knockdown and TUBB4A overexpression. Magnification, ×200. (E) Representative images of isolated xenograft tumors in mice. Tumor volumes were measured in nude mice injected subcutaneously with 2×10 6 A375 cells that had been subject to STAT1 knockdown and TUBB4A overexpression. (F) Quantification of mouse tumor volumes showed that STAT1 knockdown significantly suppressed tumor growth, whereas TUBB4A overexpression reversed this inhibitory effect. ## P<0.01 vs. si-STAT1. TUBB4A, tubulin β4A; si-NC, negative control small interfering RNA; Ov-TUBB4A, lentiviral vector for TUBB4A overexpression.

Article Snippet: Melanoma cells (A375 and RPMI-7951) were seeded into 6-well plates (cat. no. 3516; Corning, Inc.) at a density of 500–1,000 cells per well and allowed to adhere for 24 h. Cells were then transfected with si-NC (cat. no. A09010) or si-STAT1 (cat. no. A09009; GenePharma), as well as transfected using a lentiviral vector for TUBB4A overexpression (Ov-TUBB4A; pReceiver-Lv105; GeneCopoeia, Inc.) or Ov-NC (pReceiver-Lv105 Empty Vector; GeneCopoeia, Inc.).

Techniques: Over Expression, Knockdown, Migration, Isolation, Injection, Negative Control, Small Interfering RNA, Plasmid Preparation

Journal: Cell Insight

Article Title: HTATSF1 regulates innate antiviral immune response by orchestrating TRAF3-IRF3 and TRAF6-NF-κB pathways

doi: 10.1016/j.cellin.2025.100294

Figure Lengend Snippet: HTATSF1 mediates viral nucleic acid- but not IFN-β-triggered signaling. (A) Effects of HTATSF1-deficiency on transcription of downstream genes induced by viral nucleic acid mimics. The control (g NC ) and HTATSF1-deficient (g HTATSF1 ) THP-1 cells (1 × 10 6 ) were untransfected or transfected with HT-DNA (2 μg/mL) or poly(I:C) (2 μg/mL) for the indicated times before RT-qPCR analysis to measure mRNA levels of the indicated genes. (B) Effects of HTATSF1-deficiency on transcription of downstream genes induced by cGAMP. The control (g NC ) and HTATSF1-deficient (g HTATSF1 ) THP-1 cells (1 × 10 6 ) were left untreated or treated with 2′3′-cGAMP (40 nM) for the indicated times before RT-qPCR analysis to measure mRNA levels of the indicated genes. (C) Effects of HTATSF1-deficiency on IFN-β-induced phosphorylation of STAT1. The control (g NC ) and HTATSF1-deficient (g HTATSF1 ) THP-1 cells (1 × 10 6 ) were left untreated or treated with IFN-β (100 ng/mL) for the indicated times before immunoblotting analysis with the indicated antibodies. (D) Effects of HTATSF1-deficiency on transcription of downstream genes induced by IFN-β. The control (g NC ) and HTATSF1-deficient (g HTATSF1 ) THP-1 cells (1 × 10 6 ) were left untreated or treated with IFN-β (100 ng/mL) for 2 h before RT-qPCR analysis to measure mRNA levels of the indicated genes. Data shown are mean ± SEM ( n = 3 technical replicates) from one representative experiment ( (A) , ( B) , (D) ), which were repeated for at least two times with similar results. n.s., not significant; ∗∗, P < 0.01 (unpaired t -test).

Article Snippet: Antibodies against HA (TA180128) (OriGene); Flag (F3165) and β-actin (A2228) (Sigma); p-TBK1 S172 (ab109272), TBK1 (ab40676), p-IRF3 S386 (ab76493), TAK1 (ab109526), TRAF6 (ab33915), p65 (ab7970), ubiquitin (ab7254), K48-linkage specific polyubiquitin (ab140601) and K63-linkage specific polyubiquitin (ab179434) (Abcam); IRF3 (sc-33641) and STAT1 (SC-417) (Santa Cruz Biotechnology); Myc (5605), p-IRF3 S396 (4947), p-STAT1 Y701 (9167), TRAF3 (4729), p-TAK1 T184/187 (4508), p-IκBα S32/36 (9246), IκBα (9242), p-IKKα/β S176/177 (2078), IKKβ (2370), and p-p65 S536 (3033) (Cell Signaling Technology), HTATSF1 (20805-1-AP) and HECTD3 (11487-1-AP) (Proteintech) were purchased from the indicated companies.

Techniques: Control, Transfection, Quantitative RT-PCR, Phospho-proteomics, Western Blot

Journal: Cell Insight

Article Title: HTATSF1 regulates innate antiviral immune response by orchestrating TRAF3-IRF3 and TRAF6-NF-κB pathways

doi: 10.1016/j.cellin.2025.100294

Figure Lengend Snippet: HTATSF1 is important for HSV-1- or SeV-triggered phosphorylation of downstream signaling components. (A) Effects of HTATSF1-deficiency on HSV-1- or SeV-induced phosphorylation of TBK1, IRF3, and STAT1 in THP-1 cells. The control (g NC ) and HTATSF1-deficient (g HTATSF1 ) THP-1 cells (1 × 10 6 ) were left uninfected or infected with HSV-1 (MOI = 1) or SeV (MOI = 1) for the indicated times before immunoblotting analysis with the indicated antibodies. (B) Effects of HTATSF1-deficiency on HSV-1- or SeV-induced phosphorylation of TBK1, IRF3, and STAT1 in BMDMs. Lyz2 -Cre; Htatsf1 fl/fl and Htatsf1 fl/fl BMDM cells (1 × 10 6 ) were left uninfected or infected with HSV-1 (MOI = 1) or SeV (MOI = 1) for the indicated times before immunoblotting analysis with the indicated antibodies.

Article Snippet: Antibodies against HA (TA180128) (OriGene); Flag (F3165) and β-actin (A2228) (Sigma); p-TBK1 S172 (ab109272), TBK1 (ab40676), p-IRF3 S386 (ab76493), TAK1 (ab109526), TRAF6 (ab33915), p65 (ab7970), ubiquitin (ab7254), K48-linkage specific polyubiquitin (ab140601) and K63-linkage specific polyubiquitin (ab179434) (Abcam); IRF3 (sc-33641) and STAT1 (SC-417) (Santa Cruz Biotechnology); Myc (5605), p-IRF3 S396 (4947), p-STAT1 Y701 (9167), TRAF3 (4729), p-TAK1 T184/187 (4508), p-IκBα S32/36 (9246), IκBα (9242), p-IKKα/β S176/177 (2078), IKKβ (2370), and p-p65 S536 (3033) (Cell Signaling Technology), HTATSF1 (20805-1-AP) and HECTD3 (11487-1-AP) (Proteintech) were purchased from the indicated companies.

Techniques: Phospho-proteomics, Control, Infection, Western Blot

Journal: Frontiers in Immunology

Article Title: Microglial histone H3K18 crotonylation promotes STAT1 expression and induces cognitive deficit in Alzheimer disease

doi: 10.3389/fimmu.2026.1744375

Figure Lengend Snippet: An enhanced H3K18cr upregulates microglia expression of pro-inflammation-related transcription factor STAT1. (A, B) Representative western blot images and corresponding quantitative analysis of STAT1, pY-STAT1, JAK1 and pY-JAK1 levels in BV2 cells treated with 0.1 mM crotonic acid for 6 hours. N = 3 per group. Data are presented as mean ± SEM. Two-tailed unpaired t-test, STAT1: t = 3.084, P < 0.05; pY-STAT1: t = 2.802, P < 0.05; pY-STAT1/STAT1: t = 1.553, P = 0.1955; JAK1: t = 0.8888, P = 0.4243. pY-JAK1: t = 0.7728, P = 0.4828. (C, D) Representative western blot images and quantitative analysis of H3, H3K18cr, STAT1 and pY-STAT1 levels in BV2 nuclear extracts following 0.1 mM crotonic acid treatment for 6 hours. N = 3 per group. Data are presented as mean ± SEM. Two-tailed unpaired t-test, STAT1: t = 2.934, P < 0.05; pY-STAT1: t = 3.168, P < 0.05; pY-STAT1/STAT1: t = 0.01308, P = 0.9902. (E, F) Representative western blot images and corresponding quantitative analysis of STAT1, pY-STAT1, JAK1 and pY-JAK1 levels in the hippocampus of mice from the Ctrl and CA. N = 3 per group. Data are presented as mean ± SEM. Two-tailed unpaired t-test, STAT1: t = 9.929, P < 0.001; pY-STAT1: t = 3.814, P < 0.05; pY-STAT1/STAT1: t = 0.5361, P = 0.6203; JAK1: t = 0.3434, P = 0.7486. pY-JAK1: t = 1.583, P = 0.1887. (G, H) Representative western blot images and corresponding quantitative analysis of STAT1 and pY-STAT1 levels in BV2 cells treated with 0.1 mM crotonic acid and 5.0 μM fludarabine. N = 3 per group. Data are presented as mean ± SEM. One-way ANOVA test followed by Tukey’s multiple comparisons test, STAT1: interaction F (2,6) = 19.02, P < 0.01; pY-STAT1: interaction F (2,6) = 50.90, P < 0.001. (I, J) Representative western blot images and corresponding quantitative analysis of STAT1 and pY-STAT1 levels in BV2 nuclear extracts following 0.1 mM crotonic acid and 5.0 μM fludarabine. N = 3 per group. Data are presented as mean ± SEM. One-way ANOVA test followed by Tukey’s multiple comparisons test, STAT1: interaction F (2,6) = 45.52, P < 0.001; pY-STAT1: interaction F (2,6) = 35.12, P < 0.001. (K) Quantitative of mRNA levels of TNF-α, IL-6 and IL-1β in BV2 cells. N = 3 mice per group. Data are presented as mean ± SEM. One-way ANOVA test followed by Tukey’s multiple comparisons test, IL-1β: interaction F (2,6) = 12.21, P < 0.01; IL-6: interaction F (2,6) = 59.86, P < 0.001; TNF-α: interaction F (2,6) = 30.37, P < 0.001.

Article Snippet: Similar as described previously , cells were seeded at a density of 10,000 cells/well in 96-well plates and treated with fresh complete medium containing STAT1 inhibitor (Fludarabine, MedChemExpress, USA) at final concentrations of 0 μM (vehicle control, medium only), 0.1 μM, 0.5 μM, 1.0 μM, 5.0 μM and 10.0 μM.

Techniques: Expressing, Western Blot, Two Tailed Test