primary antibodies against stat1  (Cell Signaling Technology Inc)

Journal: Cell Reports Medicine

Article Title: Therapeutic stress triggers tumor STAT1 acetylation to disarm immunotherapy

doi: 10.1016/j.xcrm.2025.102448

Figure Lengend Snippet: STAT1 Lys637 acetylation correlates with poor response to ICB therapy (A) Schematic of a syngeneic murine oral cancer model receiving anti-PD1 injection. The murine oral squamous cell carcinoma cell line MOC-L2-1 with Stat1 knockdown (shmStat1) and reconstituted with human STAT1 (hSTAT1(WT) or hSTAT1(K637Q) or hSTAT1(K637R)) was inoculated subcutaneously into C57BL/6J mice until tumors reached a volume of 100 mm 3 . Five doses of anti-PD1 or isotype IgG were administered to tumor-bearing mice. n = 9–10 per group. (B) Tumor growth inhibition (TGI, %) calculated as the relative change in tumor volume between day 0 and day 38 in different groups. Data presented as mean ± SEM. ∗∗∗ p < 0.001. (C) Tumor weight in the mouse experiments. Data presented as mean ± SEM. ∗∗ p < 0.01; ∗∗∗ p < 0.001; ns, not significant. (D) Kaplan-Meier overall survival curves for HNSCC patients ( n = 63) stratified by H-score cutoff of 166 with median follow-up of 8.0 months (range 0.5–45.1). (E) Kaplan-Meier overall survival curves for GC patients ( n = 46) stratified by H-score cutoff of 166 with median follow-up of 9.7 months (range 1.97–60.2). (F) Kaplan-Meier overall survival curves for hepatocellular carcinoma (HCC) patients ( n = 39) stratified by H-score cutoff of 166 with median follow-up of 15.5 months (range 3.1–81.1). (G) Comparison of STAT1 Lys637 acetylation levels between HNSCC responders ( n = 27) and non-responders ( n = 36) to ICB treatment. Statistical analyses were performed using an unpaired Student’s t test. ∗∗ p < 0.01. (H) Comparison of STAT1 K637 acetylation levels between HNSCC disease control patients ( n = 42) and those with progressive disease ( n = 21) following ICB therapy. Statistical analyses were performed using an unpaired Student’s t test. ∗∗∗ p < 0.001. See also , , , , and .

Article Snippet: Primary antibodies against STAT1 (Cell Signaling Technology, Danvers, MA, USA) were used, and Hoechst 33342 (Thermo Fisher Scientific, Waltham, MA, USA) was used for nuclear staining.

Techniques: Injection, Knockdown, Inhibition, Comparison, Control

Journal: Cell Reports Medicine

Article Title: Therapeutic stress triggers tumor STAT1 acetylation to disarm immunotherapy

doi: 10.1016/j.xcrm.2025.102448

Figure Lengend Snippet: Impaired IFN-γ response and reduced STAT1 protein in cetuximab-resistant HNSCC (A) RT-qPCR of IFN-γ response-associated gene expression, including tumor immunology-related genes (upper), antiviral-related genes (middle), and antigen processing and presentation genes (lower) in OECM-1-WT and OECM-1-Ctx R cells. n = 3 (each with two technical replicates). The cells were then treated with IFN-γ (100 ng/mL) for 24 h. Data are presented as mean ± SD. Statistical significance was determined using unpaired Student’s t test. ∗ p < 0.05; ∗∗ p < 0.01; ∗∗∗ p < 0.001; ns, not significant. (B) RT-qPCR of IFN-γ signaling-associated components in OECM-1-WT/CAL-27-WT and OECM-1-Ctx R /CAL-27-Ctx R cells. n = 3 (each with two technical replicates). Data are presented as mean ± SD. Statistical significance was determined using unpaired Student’s t test. ∗ p < 0.05; ∗∗∗ p < 0.001; ns, not significant. (C) Representative western blot analysis of IFN-γ signaling-related proteins in OECM-1-WT/OECM-1-Ctx R and CAL-27-WT/CAL-27-Ctx R cells. GAPDH was the loading control. The experiments were performed in triplicate. (D) Heatmap showing STAT1 and STAT3 protein levels from mass spectrometry in OECM-1 cells after cetuximab treatment (500 μg/mL) across different passages. (E) Representative western blot analysis of STAT family in OECM-1-WT/OECM-1-Ctx R and CAL-27-WT/CAL-27-Ctx R cells. α-tubulin was used as the loading control. The experiments were performed in triplicate. (F) Representative western blot analysis of STAT1 protein levels in OECM-1 cells across different passages of cetuximab treatment (500 μg/mL). GAPDH was used as a loading control. The experiments were performed in triplicate. (G) Left: Schematic of the mouse experiment. Murine oral squamous cell carcinoma MOC-L2-1 cells were transduced with a doxycycline (DOX)-inducible vector for the knockdown of Stat1 (shStat1) or a scramble control (shScr) and were then inoculated subcutaneously into C57BL/6 mice. Doxycycline administration was initiated on day 18 to induce vector expression in syngeneic tumors. Mice were treated with either isotype IgG or murine anti-PD1 (200 μg) for 8 doses at specified time points. Right: Tumor growth curves are presented as mean ± SD. n = 3 per group. Statistical significance was determined using unpaired Student’s t test. ∗∗ p < 0.01. (H) Upper: Histogram showing weights of shScr and shStat1 MOC-L2-1 tumors. n = 3 per group. Statistical significance was determined using unpaired Student’s t test. ∗ p < 0.05. Lower: Representative images of tumors. See also .

Article Snippet: Primary antibodies against STAT1 (Cell Signaling Technology, Danvers, MA, USA) were used, and Hoechst 33342 (Thermo Fisher Scientific, Waltham, MA, USA) was used for nuclear staining.

Techniques: Quantitative RT-PCR, Gene Expression, Western Blot, Control, Mass Spectrometry, Transduction, Plasmid Preparation, Knockdown, Expressing

Journal: Cell Reports Medicine

Article Title: Therapeutic stress triggers tumor STAT1 acetylation to disarm immunotherapy

doi: 10.1016/j.xcrm.2025.102448

Figure Lengend Snippet: Tyrosine 701 phosphorylation promotes STAT1 degradation in cetuximab-resistant HNSCC (A) Upper: Representative western blot analysis of STAT1 protein levels in OECM-1-WT/OECM-1-Ctx R (left) and CAL-27-WT/CAL-27-Ctx R (right) cells following treatment with cycloheximide (20 μg/mL) for the indicated times. β-actin was the loading control. Lower: Quantification of STAT1 protein levels. Data are presented as the mean ± SD. n = 3 per group. Statistical significance was determined using unpaired Student’s t test. ∗ p < 0.05; ∗∗∗ p < 0.001; ns, not significant. (B) Upper: Representative western blot analysis of STAT1 protein levels in OECM-1-Ctx R (left) and CAL-27-Ctx R (right) cells transfected with STAT1 (OECM-1-Ctx R -STAT1 and CAL-27-Ctx R -STAT1) and treated with proteasome inhibitor (MG132, 20 μM) for 18 h. Snail was the positive control for proteasomal degradation. Lower: Representative western blot analysis of STAT1 protein levels in OECM-1-Ctx R (left) and CAL-27-Ctx R (right) cells transfected with STAT1 (OECM-1-Ctx R -STAT1 and CAL-27-Ctx R -STAT1) and treated with lysosomal inhibitor (bafilomycin A1, 100 nM) or autophagic degradation inhibitor (hydroxychloroquine [HCQ], 20 μM). LC3B is a marker for monitoring autophagy. GAPDH was the loading control. The experiments were performed in triplicate. (C) Representative immunoprecipitation and western blot analyses of polyubiquitinated STAT1 in OECM-1-WT/OECM-1-Ctx R (left) and CAL-27-WT/CAL-27-Ctx R (right) cells transfected with STAT1. The cells were treated with MG132 (20 μM) for 6 h to inhibit proteasome degradation. The experiments were performed in triplicate. (D) Representative western blot analysis of total STAT1, Tyr701-phosphorylated STAT1, and Ser727-phosphorylated STAT1 in OECM-1-WT/OECM-1-Ctx R (left) and CAL-27-WT/CAL-27-Ctx R (right) cells transfected with STAT1. The cells were treated with MG132 (10 μM) for 16 h to inhibit proteasome degradation. GAPDH was the loading control. The experiments were performed in triplicate. (E) Representative immunoprecipitation and western blot analyses of polyubiquitinated STAT1 in OECM-1-Ctx R cells transfected with wild-type (WT) or Tyr701-unphosphorylatable mutant (Y701F) STAT1. Cells were treated with MG132 (10 μM) for 6 h to inhibit proteasomal degradation. The experiments were performed in triplicate. See also .

Article Snippet: Primary antibodies against STAT1 (Cell Signaling Technology, Danvers, MA, USA) were used, and Hoechst 33342 (Thermo Fisher Scientific, Waltham, MA, USA) was used for nuclear staining.

Techniques: Phospho-proteomics, Western Blot, Control, Transfection, Positive Control, Marker, Immunoprecipitation, Mutagenesis

Journal: Cell Reports Medicine

Article Title: Therapeutic stress triggers tumor STAT1 acetylation to disarm immunotherapy

doi: 10.1016/j.xcrm.2025.102448

Figure Lengend Snippet: Reduced transcriptional activity of STAT1 in cetuximab-resistant HNSCC via Lys637 acetylation (A) Representative western blot analysis of the indicated proteins in OECM-1-WT/OECM-1-CtxR (left) and CAL-27-WT/CAL-27-Ctx R (right) cells transfected with STAT1 and treated with or without IFN-γ (100 ng/mL) for 24 h. α-tubulin was the loading control. The experiments were performed in triplicate. (B) Mass spectrometric analysis of CAL-27-Ctx R cells, identifying acetylation at Lys637 of STAT1. (C) Sequence alignment showing the conservation of STAT1 Lys637 across various species. (D) Representative western blot analysis of CAL-27-Ctx R and OECM-1-Ctx R cells transfected with wild-type or unacetylatable mutant STAT1(K637R), treated with or without IFN-γ (100 ng/mL) for 24 h. GAPDH was the loading control. The experiments were performed in triplicate. (E) Representative co-immunoprecipitation and western blot analyses detecting lysine-acetylated STAT1 in CAL-27-Ctx R and OECM-1-Ctx R cells transfected with wild-type STAT1 or STAT1(K637R). The cells were treated with MG132 (10 μM) for 16 h. The experiments were performed in triplicate. (F) Representative electrophoretic mobility shift assay assesses the DNA binding of wild-type STAT1 or STAT1(K637R) in CAL-27-Ctx R cells. The cells were transfected with the corresponding vectors, treated with MG132 (10 μM, 16 h) and IFN-γ (100 ng/mL, 30 min). (G) Representative western blot analysis of the indicated proteins in U3A cells transfected with STAT1(K637R) or STAT1(K637Q) mutants and treated with IFN-γ (100 ng/mL) for 24 h. GAPDH was a loading control. The experiments were performed in triplicate. (H) Representative blot detecting dimerized STAT1 and Tyr701-phosphorylated STAT1 in U3A cells transfected with STAT1(K637R) or STAT1(K637Q) mutants treated with IFN-γ (100 ng/mL) with or without disuccinimidyl suberate (DSS) (2.5 μM) for 10 min. The experiments were performed in triplicate. See also .

Article Snippet: Primary antibodies against STAT1 (Cell Signaling Technology, Danvers, MA, USA) were used, and Hoechst 33342 (Thermo Fisher Scientific, Waltham, MA, USA) was used for nuclear staining.

Techniques: Activity Assay, Western Blot, Transfection, Control, Sequencing, Mutagenesis, Immunoprecipitation, Electrophoretic Mobility Shift Assay, Binding Assay

Journal: Cell Reports Medicine

Article Title: Therapeutic stress triggers tumor STAT1 acetylation to disarm immunotherapy

doi: 10.1016/j.xcrm.2025.102448

Figure Lengend Snippet: IFN-β and TNF-α as potential upstream regulators of STAT1 inactivation in cetuximab-resistant HNSCC (A) Schematic representation of the identification of upstream regulators using Ingenuity Pathway Analysis in OECM-1-Ctx R and CAL-27-Ctx R cells (left). Expression levels of the indicated genes based on RNA sequencing in OECM-1-Ctx R and CAL-27-Ctx R cells compared to parental cells (right). (B) ELISA of IFN-β (left) and TNF-α (right) concentrations in conditioned media from CAL-27 and CAL-27-Ctx R cells ( n = 3, with two technical replicates each). Data are presented as mean ± SD. Statistical analyses were performed using unpaired Student’s t test. ∗∗∗ p < 0.001. (C) Representative western blot of the indicated proteins in CAL-27-Ctx R cells transfected with STAT1 (CAL-27-Ctx R -STAT1) and treated with MG132 (10 μM) combined with JAK1 (left), JAK2 (middle), and TYK2 inhibitors (right) at the indicated concentrations for 16 h. GAPDH was a loading control. The experiments were performed in triplicate. (D) Representative western blot of the indicated proteins in CAL-27-Ctx R (left) and OECM-1-Ctx R (right) cells transfected with STAT1 (CAL-27-Ctx R -STAT1 and OECM-1-Ctx R -STAT1) and treated with MG132 (10 μM) and IFN-β-neutralizing antibody at indicated concentrations for 16 h. GAPDH was the loading control. The experiments were performed in triplicate. (E) Representative western blot of STAT1 Tyr701 phosphorylation in OECM-1-Ctx R (left) and CAL-27-Ctx R (right) cells transfected with STAT1 (CAL-27-Ctx R -STAT1 and OECM-1-Ctx R -STAT1) and treated with MG132 (10 μM) combined with an IFN-α-neutralizing antibody at indicated concentrations for 16 h. α-tubulin was the loading control. Experiments were duplicated. (F) Representative co-immunoprecipitation and western blot analyses to investigate the interaction between STAT1 and histone acetyltransferases in the CAL-27-Ctx R and OECM-1-Ctx R cells transfected with STAT1 (CAL-27-Ctx R -STAT1 and OECM-1-Ctx R -STAT1). The cells were then treated with MG132 (10 μM) for 16 h. The experiments were performed in triplicate. (G) Representative in vitro acetylation assay. Biotin-labeled synthetic peptides, corresponding to the sequence encompassing STAT1 lysine 637 (K637) or a mutant variant where K637 was substituted with arginine (K637R), were utilized. These peptides were incubated in the presence or absence of the histone acetyltransferase (PCAF) and with acetyl-coenzyme A (acetyl-CoA). Following the incubation, the reaction products were analyzed by dot blot for assessing acetylation levels. The experiments were performed in triplicate. See also .

Article Snippet: Primary antibodies against STAT1 (Cell Signaling Technology, Danvers, MA, USA) were used, and Hoechst 33342 (Thermo Fisher Scientific, Waltham, MA, USA) was used for nuclear staining.

Techniques: Expressing, RNA Sequencing, Enzyme-linked Immunosorbent Assay, Western Blot, Transfection, Control, Phospho-proteomics, Immunoprecipitation, In Vitro, Acetylation Assay, Labeling, Sequencing, Mutagenesis, Variant Assay, Incubation, Dot Blot

Journal: Laboratory Animal Research

Article Title: Fludarabine attenuates inflammation and dysregulated autophagy in alveolar macrophages via inhibition of STAT1/IRF1 pathway

doi: 10.1186/s42826-025-00245-7

Figure Lengend Snippet: The STAT1/IRF1 pathway is associated with LPS-induced ALI in mice. A RNA-seq data presenting comparisons of the expression of several genes, including IRF1 family genes, between the control and LPS groups of mouse lung tissue lysates ( n = 2 per group). B Expression of IRF family genes was measured as FPKM values. C Western blotting of IRF1 and IRF7 in mouse lung tissue (upper) and their densitometric ratios against β-actin (bottom) ( n = 3 per group). * p < 0.05 compared with the control group. ns: not significant. D Confocal images of immunostaining for IRF1 (green) in BAL cells. Scale bar, 10 μm ( n = 2 per group). E Western blotting of pSTAT1 (Tyr701), pSTAT1 (Ser727), total STAT1, and β-actin in mouse lung tissue (upper) and their densitometric ratios against STAT1 (bottom) ( n = 3 per group). The data are presented as mean ± SD

Article Snippet: Primary antibodies against pSTAT1 (Ser727), pSTAT1 (Tyr701), STAT1, IRF1, IRF7, LC3B, and β-actin were purchased from Cell Signaling Technology, (Danvers, MA, USA).

Techniques: RNA Sequencing, Expressing, Control, Western Blot, Immunostaining

Journal: Laboratory Animal Research

Article Title: Fludarabine attenuates inflammation and dysregulated autophagy in alveolar macrophages via inhibition of STAT1/IRF1 pathway

doi: 10.1186/s42826-025-00245-7

Figure Lengend Snippet: The STAT1/IRF1 inhibitor Fluda attenuates LPS-induced ALI in mice. A pSTAT1 (Tyr701), pSTAT1 (Ser727), STAT1, IRF1, and β-actin were detected in mouse lung tissue. B Densitometric ratios of pSTAT1 (Tyr701) and pSTAT1 (Ser727) against STAT1 and that of IRF1 against β-actin. C pSTAT1 (Tyr701), pSTAT1 (Ser727), STAT1, IRF1, and β-actin expression was detected in BAL cell lysates using western blotting. D Densitometric ratios of pSTAT1 (Tyr701) and pSTAT1 (Ser727) against STAT1 and that of IRF1 against β-actin ( n = 3). E Representative images of hematoxylin and eosin-stained lung sections from four experimental groups (× 200). Scale bar, 50 μm. F BAL cells were subjected to Giemsa staining and then observed under a microscope (× 200). Scale bar, 50 μm. G The lung injury score illustrated Fluda reduced LPS-induced ALI in mice ( n = 4 per group). H The numbers of macrophages and neutrophils in BALF. I Total protein in BALF was measured using the BCA assay. J MPO activity was measured in whole-lung lysates. K Inflammatory cytokines in BALF, including TNF-α, IL-6, IFN-γ, and IL-1β, were detected using ELISA. The data are presented as mean ± SD, n = 3 independent experiments were performed. * p < 0.05, ** p < 0.01, and *** p < 0.001 compared with the control group. † p < 0.05, †† p < 0.01, and ††† p < 0.001 compared with the LPS group

Article Snippet: Primary antibodies against pSTAT1 (Ser727), pSTAT1 (Tyr701), STAT1, IRF1, IRF7, LC3B, and β-actin were purchased from Cell Signaling Technology, (Danvers, MA, USA).

Techniques: Expressing, Western Blot, Staining, Microscopy, BIA-KA, Activity Assay, Enzyme-linked Immunosorbent Assay, Control

Journal: Laboratory Animal Research

Article Title: Fludarabine attenuates inflammation and dysregulated autophagy in alveolar macrophages via inhibition of STAT1/IRF1 pathway

doi: 10.1186/s42826-025-00245-7

Figure Lengend Snippet: STAT1/IRF1, iNOS, and NF-κB/ERK1/2 activation was attenuated by Fluda in RAW264.7 cells. A pSTAT1 (Tyr701), pSTAT1 (Ser727), IRF1, and STAT1 were detected in RAW264.7 cells. STAT1 and β-actin were used as loading controls. Densitometric ratios of pSTAT1 (Tyr701) and pSTAT1 (Ser727) against STAT1 and that of IRF1 against β-actin. B Relative Nos2 expression in RAW264.7 cells was measured using real-time PCR. C Western blotting for iNOS expression. Densitometric ratio of iNOS against β-actin. D NO release was determined by measuring the amount of nitrite in conditioned medium using Griess reagent. E IL-6 and ( F ) TNF-α levels in conditioned medium were detected using ELISA. G Western blotting for p-NF-κB (Ser536), p-ERK1/2, p-JNK1/2, and p-p38. Total NF-κB, total JNK1/2, total ERK1/2, total p38, and β-actin were used as loading controls. All blots were subjected to densitometric analysis and relative quantification. Data are presented as mean ± SD ( n = 3 per group). * p < 0.05, ** p < 0.01, and *** p < 0.001 compared with the control group. † p < 0.05, †† p < 0.01, and ††† p < 0.001 compared with the LPS group. ns: not significant

Article Snippet: Primary antibodies against pSTAT1 (Ser727), pSTAT1 (Tyr701), STAT1, IRF1, IRF7, LC3B, and β-actin were purchased from Cell Signaling Technology, (Danvers, MA, USA).

Techniques: Activation Assay, Expressing, Real-time Polymerase Chain Reaction, Western Blot, Enzyme-linked Immunosorbent Assay, Quantitative Proteomics, Control