primary anti stat1  (Cell Signaling Technology Inc)

Journal: International Journal of Biological Sciences

Article Title: IGF2BP2 Drives Thyroid Cancer Dedifferentiation Through m6A-Dependent STAT1 mRNA Destabilization

doi: 10.7150/ijbs.121503

Figure Lengend Snippet: STAT1 was a m6A target of IGF2BP2 in thyroid cancer associated with the dedifferentiation. (A) Distribution of IGF2BP2-binding peak of RIP-seq. (B) The upset plot shows the intersection of RNA-seq, RIP-seq, and MERIP seq ( GSE199205 ). (C) IGF2BP2 binding peaks in STAT1 transcripts visualized by IGV. Relative RNA level of STAT1 in PTC following IGF2BP2 overexpression (D) and ATC upon IGF2BP2 knockdown (E). (F) Western blot detected the protein level of STAT1 in PTC cells transfected with overexpressing lentiviruses carrying IGF2BP2. (G) The protein levels of STAT1 were measured by western blot analysis in ATC cells transfected with lentiviruses carrying sh- IGF2BP2 . (H) Kaplan-Meier analysis of overall survival of THCA patients using the KM Plotter online tool. P values were determined using a two-tailed unpaired Student's test (* P < 0.05, ** P < 0.01, *** P < 0.001).

Article Snippet: Anti-STAT1 primary antibodies (Cell Signaling Technology, USA,14994T) were diluted in antibody buffer (1% BSA, protease inhibitors) and incubated overnight at 4 °C, followed by species-matched secondary antibodies (ChiTag Goat anit-Rabbit IgG antibody, N269) conjugated to Protein A/G (1:500, Thermo Fisher, USA).

Techniques: Binding Assay, RNA Sequencing, Over Expression, Knockdown, Western Blot, Transfection, Two Tailed Test

Journal: International Journal of Biological Sciences

Article Title: IGF2BP2 Drives Thyroid Cancer Dedifferentiation Through m6A-Dependent STAT1 mRNA Destabilization

doi: 10.7150/ijbs.121503

Figure Lengend Snippet: STAT1 activated the transcription of thyroid differentiation genes in thyroid cancer and mediated the differentiation and stemness. (A-B) Relative mRNA (A) and protein (B) level of thyroid differentiation-related genes measured by qRT-PCR in STAT1 -knockdown TPC1 cells. (C-D) The mRNA (C) and protein (D) variation of dedifferentiation molecules was measured by western blot in CAL62 cells. (E) Layout and MFI of CD133 expression in TPC1 cells determined by flow cytometry. (F-G) The transcript (F) and protein (G) levels of stemness markers in si- STAT1 TPC1 cells. (H) Layout and MFI of CD133 expression in CAL62 cells determined by flow cytometry. (I-J) The transcript (I) and protein (J) levels of stemness markers in STAT1 -OE CAL62 cells. (K) CUT&Tag was performed with STAT1 antibody in TPC1 and CAL62 cells. Heatmap showing the genomic distribution of STAT1 flanking TSSs in TPC1 and CAL62 cells. (L) IGV tracks for thyroid differentiation genes from CUT&Tag. (M-N) Dual-luciferase reporter assay of p GL3-basic, TSHR, SLC26A4, SLC5A5, TPO, PAX8, FOXE1, and NKX2.1 promoter activity driven by STAT1 or pcDNA3.1 transfection in wild-type TPC1 and CAL62 tumor cells. Data are relative to Renilla luciferase activity. n = 3. P values were determined using a two-tailed unpaired Student's test (* P < 0.05, ** P < 0.01, *** P < 0.001).

Article Snippet: Anti-STAT1 primary antibodies (Cell Signaling Technology, USA,14994T) were diluted in antibody buffer (1% BSA, protease inhibitors) and incubated overnight at 4 °C, followed by species-matched secondary antibodies (ChiTag Goat anit-Rabbit IgG antibody, N269) conjugated to Protein A/G (1:500, Thermo Fisher, USA).

Techniques: Quantitative RT-PCR, Knockdown, Western Blot, Expressing, Flow Cytometry, Luciferase, Reporter Assay, Activity Assay, Transfection, Two Tailed Test

Journal: International Journal of Biological Sciences

Article Title: IGF2BP2 Drives Thyroid Cancer Dedifferentiation Through m6A-Dependent STAT1 mRNA Destabilization

doi: 10.7150/ijbs.121503

Figure Lengend Snippet: IGF2BP2 regulated stabilization of STAT1 transcript via an m6A-dependent manner. (A-B) TPC1-OE and CAL62-KD cells were treated with 5 μg/mL actinomycin D (ActD) for 0, 2, 4, 6, 8, and 10 h, followed by RT-qPCR. (C-F) TPC1 and CAL62 cell lysates were immunoprecipitated with IGF2BP2 or m6A antibody and control immunoglobulin G (IgG) to detect STAT1 mRNA expression and validated by agarose electrophoresis. (G) Sketch map shows the m6A -enriched sites of STAT1 transcript. (H-I) Dual-luciferase assay of STAT1 reporter activity driven by STAT1-A, B, and C transfection in vector and IGF2BP2 overexpression TPC1 cells as well as IGF2BP2 knockdown CAL62 cells. Data are relative to Renilla luciferase activity. n = 3. (J) Sketch map shows the dual-luciferase reporter plasmid construction for STAT1 m6A site validation. (K-L) Dual-luciferase assay of STAT1 reporter activity driven by STAT1-A mutant, and B mutant transfection in vector and IGF2BP2 overexpression TPC1 cells as well as IGF2BP2 knockdown CAL62 cells. Data are relative to Renilla luciferase activity. n = 3. (M-N) Co-IP and Western blot analysis of TPC1 (M) and CAL62 (N) cells demonstrate that IGF2BP2 is physically associated with CNOT1. (O-P) The relative STAT1 mRNA abundance and protein expression in si- CNOT1 TPC1 (O) and CAL62 cells (P). (Q-R) TPC1 (Q) and CAL62 (R) si- CNOT1 cells were treated with 5 μg/mL actinomycin D (ActD) for 0, 2, 4, 6, 8, and 10 h, followed by RT-qPCR. P values were determined using a two-tailed unpaired Student's test (* P < 0.05, ** P < 0.01, *** P < 0.001). * Method note: TPC1 and CAL62 RIPs were performed in separate batches with different starting cell numbers (10 × 10⁶ vs 6 × 10⁶). Equal amounts of eluted mRNA (500 ng) were reverse-transcribed; qPCR revealed 3.1 cycles higher Cp in CAL62, indicating ~8.6-fold lower template abundance. 10 µL PCR product was loaded for TPC1 and 5 µL for CAL62 (same cycle number). Bar graphs display within-cell-line IP/IgG enrichment ratios, absolute abundance cannot be compared across lines.

Article Snippet: Anti-STAT1 primary antibodies (Cell Signaling Technology, USA,14994T) were diluted in antibody buffer (1% BSA, protease inhibitors) and incubated overnight at 4 °C, followed by species-matched secondary antibodies (ChiTag Goat anit-Rabbit IgG antibody, N269) conjugated to Protein A/G (1:500, Thermo Fisher, USA).

Techniques: Quantitative RT-PCR, Immunoprecipitation, Control, Expressing, Electrophoresis, Luciferase, Activity Assay, Transfection, Plasmid Preparation, Over Expression, Knockdown, Biomarker Discovery, Mutagenesis, Co-Immunoprecipitation Assay, Western Blot, Two Tailed Test, Reverse Transcription

Journal: International Journal of Biological Sciences

Article Title: IGF2BP2 Drives Thyroid Cancer Dedifferentiation Through m6A-Dependent STAT1 mRNA Destabilization

doi: 10.7150/ijbs.121503

Figure Lengend Snippet: STAT1 reversed the vicious dedifferentiation, and stemness promoted by IGF2BP2 in thyroid cancer. (A) The TPC1-OE cells were transfected with pLvx-STAT1 plasmids confirmed by Western blotting. (B-C) Thyroid differentiation factor expressions were measured by qRT-PCR (B) and Western blot (C) in TPC1 cell lines. (D) CAL62-KD cells were interfered with si- STAT1 , the transfection efficiency was confirmed by Western blotting. (E-F) Thyroid differentiation factor expressions were measured by qRT-PCR (E) and Western blot (F) in TPC1 cell lines. (G) The CSCs CD133 features were measured by were assessed by flow cytometry in TPC1 cells. (H-I) The RNA (H) and protein (I) levels of stemness markers in TPC1 cells. (J) The CSCs CD133 features were measured by were assessed by flow cytometry in CAL62 cells. (K-L) The mRNA (K) and protein (L) levels of CSCs markers were measured by western blot analysis. (M-O) Growth curve and tumor weight of subcutaneous xenografts models with CAL62 cells. (P) Representative immunohistochemical (IHC) staining of IGF2BP2, STAT1, SLC5A5, and CD133 in xenograft tumors from each experimental group. Scale bar, 200 μm (applicable to all images). (Q) IHC H-scores for SLC5A5 and CD133 expression are quantified and presented as the mean ± SD in the adjacent bar graphs. P values were determined using a two-tailed unpaired Student's test (* P < 0.05, ** P < 0.01, *** P < 0.001).

Article Snippet: Anti-STAT1 primary antibodies (Cell Signaling Technology, USA,14994T) were diluted in antibody buffer (1% BSA, protease inhibitors) and incubated overnight at 4 °C, followed by species-matched secondary antibodies (ChiTag Goat anit-Rabbit IgG antibody, N269) conjugated to Protein A/G (1:500, Thermo Fisher, USA).

Techniques: Transfection, Western Blot, Quantitative RT-PCR, Flow Cytometry, Immunohistochemical staining, Immunohistochemistry, Expressing, Two Tailed Test

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