Journal: Journal for immunotherapy of cancer

Article Title: Orlistat facilitates immunotherapy via AKT-FOXO3a-FOXM1-mediated PD-L1 suppression.

doi: 10.1136/jitc-2024-008923

Figure Lengend Snippet: Figure 3 Orlistat suppressed PD-L1 via inhibition of FOXM1 signaling. (A) Volcano plot of the expression of the reported transcription factors that regulated the expression of PD-L1. These TFs include STAT1, ATF3, IRF2, TFEB, IRF1, YY1, STAT3, JUN, HSF1, NFκB, SP1, FOXP3, BRD4, EZH2, MYC, HIF-1α, and FOXM1. (B, C) qRT-PCR measurement of mRNA expression of FOXM1 after orlistat (40 µM) treatment for 0 hour, 24 hours, or 48 hours in AGS and HCT116 (B), MC38 and CT26 cells (C). (D) WB analysis of FOXM1 protein levels after orlistat treatment for different times (0 hour, 24 hours, 48 hours) in AGS, HCT116, MC38, and CT26 cells. (E) Representative images of immunohistochemical staining of FOXM1 protein expression in resected tumors at day 25. Scale bar=50 µm. (F) The expression of PD-L1 mRNA in AGS and MC38 cells after transfection with FOXM1 overexpression or knockdown. (G) WB for PD-L1 protein levels after FOXM1 overexpression or knockdown in AGS and MC38 cells. (H) Schematic representation of the PD-L1 gene promoter regions and primer pairs used for ChIP assays. (I) ChIP-qPCR assay was used to detect the binding of FOXM1 to the PD-L1 promoter at the (+222 to +450 bp) region in AGS cells. (J) ChIP-qPCR analysis of the impact of orlistat (40 µM) on the binding of FOXM1 to the PD-L1 promoter. (K) The luciferase activity of PD-L1 promoter after FOXM1 overexpression with or without orlistat (40 µM). HEK293T cells in 24-well plates were transfected with FOXM1 plasmids or control. The luciferase activity was measured 36 hours later. *p<0.05, **p<0.01, ***p<0.001.

Article Snippet: Following activation, STAT1 (TargetMol, TMPJ- 00961) was immobilized on the sensor chip surface using a sodium acetate buffer (pH 5.0).

Techniques: Inhibition, Expressing, Quantitative RT-PCR, Immunohistochemical staining, Staining, Transfection, Over Expression, Knockdown, ChIP-qPCR, Binding Assay, Luciferase, Activity Assay, Control

Journal: Journal for immunotherapy of cancer

Article Title: Orlistat facilitates immunotherapy via AKT-FOXO3a-FOXM1-mediated PD-L1 suppression.

doi: 10.1136/jitc-2024-008923

Figure Lengend Snippet: Figure 8 Orlistat upregulates ISGs and MHC-I through activating the STAT1 pathway. (A) Gene set enrichment plots of the antigen processing and presentation hallmarks. (B) Gene set enrichment plots of the JAK-STAT signaling pathway hallmarks. (C–E) WB analysis of Y701-phosphorylated STAT1 (p-STAT1-701) and total STAT1 in AGS and MC38 cells at different times (C) and concentrations (D) after orlistat administration, and tumors collected from mice-bearing subcutaneous MC38 tumors with or without orlistat treatment (E). (F–K) Quantitative PCR analysis of mRNA expression of IFNα, IFNβ, and ISGs or antigen processing and presentation genes in purified MC38 syngeneic tumors cells treated with control or orlistat (F), MC38 (H, I), and AGS (J, K) cell lines. (L) IB analysis and quantitative data of STAT1 in DMSO control vs orlistat-treated cells. AGS cells were treated with cycloheximide (CHX) 100 mg/mL, and collected at the indicated times. (M) Orlistat administration (40 µM) increased the thermal stability of STAT1 protein as determined by thermal stability shift assay (n=3). (N, O) SPR sensorgrams (N) and steady-state curves (O) for the orlistat analyte (2-fold dilutions; 50–1.56 µM) binding to the STAT1 protein ligand. (P) Schematic diagram depicting orlistat-mediated inhibition of PD-L1 expression in gastric cancer and colon cancer cells and promoting the expression of ISGs and MHC-I **p<0.01, ***p<0.001.

Article Snippet: Following activation, STAT1 (TargetMol, TMPJ- 00961) was immobilized on the sensor chip surface using a sodium acetate buffer (pH 5.0).

Techniques: Real-time Polymerase Chain Reaction, Expressing, Control, Shift Assay, Binding Assay, Inhibition

Journal: Iranian Journal of Medical Sciences

Article Title: Epstein-Barr Virus Promotes Tumorigenicity and Worsens Hodgkin Lymphoma Prognosis by Activating JAK/STAT and NF-κB Signaling Pathways

doi: 10.30476/IJMS.2023.97287.2896

Figure Lengend Snippet: The sequence of primers used for the real-time polymerase chain reaction

Article Snippet: The expression levels of target genes were then estimated using the 2 −ΔΔCT method., Primers STAT1 (#HP210040), JAK2 (#HP208201), IRF-1 (HP205934), PD-L1 (#HP210654), IFN-γ (#HP200586), NF-κB (#HP207409), Bcl-xL (#HP234144), COX-2 (#HP200900), GAPDH (#HP205798), and β-catenin (#KN208947) were purchased from OriGene Technologies (Beijing, China).

Techniques: Sequencing

Journal: Iranian Journal of Medical Sciences

Article Title: Epstein-Barr Virus Promotes Tumorigenicity and Worsens Hodgkin Lymphoma Prognosis by Activating JAK/STAT and NF-κB Signaling Pathways

doi: 10.30476/IJMS.2023.97287.2896

Figure Lengend Snippet: The mRNA expression of JAK2/STAT1 and NF-κB signaling pathways in patients with EBV-positive and EBV-negative Hodgkin lymphoma

Article Snippet: The expression levels of target genes were then estimated using the 2 −ΔΔCT method., Primers STAT1 (#HP210040), JAK2 (#HP208201), IRF-1 (HP205934), PD-L1 (#HP210654), IFN-γ (#HP200586), NF-κB (#HP207409), Bcl-xL (#HP234144), COX-2 (#HP200900), GAPDH (#HP205798), and β-catenin (#KN208947) were purchased from OriGene Technologies (Beijing, China).

Techniques: Expressing

Journal: EBioMedicine

Article Title: Mediator Kinase Phosphorylation of STAT1 S727 Promotes Growth of Neoplasms With JAK-STAT Activation

doi: 10.1016/j.ebiom.2017.11.013

Figure Lengend Snippet: Genome-wide mapping of STAT1 pS727 reveals occupancy at super-enhancers. A. GSEA plot shows enrichment of CA-upregulated genes in the top non-promoter STAT1 pS727 regions. B. Venn diagram shows the top STAT1 pS727 peaks overlap significantly with genes upregulated by CA (≥ 1.2-fold), P = 1.05e − 10 and H3K27Ac SEs, P = 1.49e − 60 (hypergeometric tests). C. ChIP-seq tracks at the GATA2 locus comparing normalized STAT pS727, total STAT1, CDK8, and H3K27ac signals (in reads per million) in SET-2 cells. D. Model for the mechanism of growth inhibition by CA in JAK-STAT activated neoplasms. See also Fig. S5.

Article Snippet: Primary antibodies included: STAT1 (CST #9172), STAT1 (R&D Systems #PAF-ST1), STAT1 pS727 (CST #9177), STAT1 pY701 (CST #9167), CDK8 (CST #4101 or #4106), CDK19 (Sigma #HPA007053), CDK9 (CST #2316), FLAG (Sigma F1804), PARP (CST #9532), actin (Sigma #A5060), GAPDH (Santa Cruz sc-47724).

Techniques: Genome Wide, ChIP-sequencing, Inhibition

Journal: EBioMedicine

Article Title: Mediator Kinase Phosphorylation of STAT1 S727 Promotes Growth of Neoplasms With JAK-STAT Activation

doi: 10.1016/j.ebiom.2017.11.013

Figure Lengend Snippet: CA suppresses growth of JAK2-mutant AML by inhibiting phosphorylation of STAT1 S727. A. Immunoblots after 2 h treatment showing dose-dependent inhibition of STAT1 S727 phosphorylation by CA in SET-2 cells. Ruxolitinib (Rux), a JAK1/2 inhibitor, inhibits tyrosine phosphorylation of STAT1 at 1 μm. Combined treatment with 1 μm ruxolitinib and 50 nM CA. Representative blots shown of at least three biological replicates. B. Competitive growth experiments show that SET-2 and UKE-1 cells overexpressing the phosphomimetic mutant STAT1 S727E (ZsGreen) outcompete cells expressing wild-type STAT1 (mCherry) in the presence of CA (mean ± s.e.m. ratio, n = 3 biological replicates). Representative growth curves shown for experiments repeated at least three times. C. A homozygous CRISPR-Cas9-edited clone expressing STAT1 S727E is resistant to CA compared to a control CRISPR knockout line targeting AAVS1. Solid lines represent calculated non-linear fit (STAT1 S727E(GI 50 ) = 37 nM, sgAAVS1(GI 50 ) = 0.3 nM). Dots represent the mean ± s.e.m. ratio of n = 3 biological replicates. D. CA-induced upregulation of CD61 can be inhibited by STAT1 S727E overexpression. CD61 mRNA transcript levels were measured by ddPCR (mean ± Poisson error, n = 5 biological replicates, experiment performed twice, P = 0.000091, two-tailed t -test). E. STAT1 S727A overexpression causes upregulation of CD41 on the cell surface compared to wild-type STAT1 in SET-2 cells. Representative flow cytometry traces shown and quantification plotted on the right (mean ± s.e.m., n = 4 biological replicates). See also Fig. S2.

Article Snippet: Primary antibodies included: STAT1 (CST #9172), STAT1 (R&D Systems #PAF-ST1), STAT1 pS727 (CST #9177), STAT1 pY701 (CST #9167), CDK8 (CST #4101 or #4106), CDK19 (Sigma #HPA007053), CDK9 (CST #2316), FLAG (Sigma F1804), PARP (CST #9532), actin (Sigma #A5060), GAPDH (Santa Cruz sc-47724).

Techniques: Mutagenesis, Phospho-proteomics, Western Blot, Inhibition, Expressing, CRISPR, Control, Knock-Out, Over Expression, Two Tailed Test, Flow Cytometry

Journal: EBioMedicine

Article Title: Mediator Kinase Phosphorylation of STAT1 S727 Promotes Growth of Neoplasms With JAK-STAT Activation

doi: 10.1016/j.ebiom.2017.11.013

Figure Lengend Snippet: Depletion of STAT1α or the STAT1 TAD induces growth arrest in JAK2-mutant AML cells. A. Diagram depicts location of various sgRNAs used to target STAT1. Dotted lines represent locations of the Cas9 cut site for each guide. Inset, part of the TAD (transactivation domain) DNA and protein sequence, with the CDK8 recognition motif highlighted in blue and serine 727 in red. B. CRISPR-Cas9-mediated knockout (KO) of STAT1 using sg1 and sg9n shows efficient depletion of STAT1 by immunoblot. STAT1-KO (mCherry) cells show resistance to CA compared to control AAVS1-KO (AmCyan) cells (mean ± s.e.m. ratio of n = 3 biological replicates). Representative growth curves shown for experiments repeated at least three times. C. The pie charts represent the percentage of frameshit versus in-frame mutations generated in pools of cells edited with guide sg5, sg4, sg11, or sg9n. SET-2 or UKE-1 cells were edited two independent times with each sgRNA and sequenced; one experiment is shown. D. Model for the role of STAT1 in proliferation. A proliferative state is maintained when STAT1α/β are present or absent. Growth arrest is induced when the TAD of STAT1α is inhibited by CA or when STAT1α is knocked out. See also Figs. S3 and S4.

Article Snippet: Primary antibodies included: STAT1 (CST #9172), STAT1 (R&D Systems #PAF-ST1), STAT1 pS727 (CST #9177), STAT1 pY701 (CST #9167), CDK8 (CST #4101 or #4106), CDK19 (Sigma #HPA007053), CDK9 (CST #2316), FLAG (Sigma F1804), PARP (CST #9532), actin (Sigma #A5060), GAPDH (Santa Cruz sc-47724).

Techniques: Mutagenesis, Sequencing, CRISPR, Knock-Out, Western Blot, Control, Generated

Journal: EBioMedicine

Article Title: Mediator Kinase Phosphorylation of STAT1 S727 Promotes Growth of Neoplasms With JAK-STAT Activation

doi: 10.1016/j.ebiom.2017.11.013

Figure Lengend Snippet: CA upregulates the expression of differentiation programs in MPN cells, partly through STAT1. A. Scatterplot of differentially expressed genes in SET-2 cells with CA or ruxolitinib treatment (RNA-seq) shows large differences between the transcriptional effects of the two compounds. Ruxolitinib data from . Best-fit linear regression line in blue, R 2 = 0.000047. B. Graph shows enriched molecular function and biological process terms related to transcription factor activity and differentiation from gene ontology analysis (DAVID) of 261 CA-upregulated genes. C. Table shows positive enrichment of three MK differentiation signatures with genes upregulated by CA treatment, but not ruxolitinib treatment (GSEA). D. GSEA plots show that CA upregulates positively-regulated STAT1 target genes, not negatively-regulated STAT1 target genes (published signatures from ). E. STAT1 S727E overexpression (probed for GATA2 , UBASH3B ) or knockin (probed for rest) partially rescues upregulation of some SE-associated genes in SET-2 cells by ddPCR (mean ± s.e.m. of Poisson error of two independent experiments, n = 6 biological replicates, two-tailed t -tests). See also Fig. S5 and Table S1.

Article Snippet: Primary antibodies included: STAT1 (CST #9172), STAT1 (R&D Systems #PAF-ST1), STAT1 pS727 (CST #9177), STAT1 pY701 (CST #9167), CDK8 (CST #4101 or #4106), CDK19 (Sigma #HPA007053), CDK9 (CST #2316), FLAG (Sigma F1804), PARP (CST #9532), actin (Sigma #A5060), GAPDH (Santa Cruz sc-47724).

Techniques: Expressing, RNA Sequencing, Activity Assay, Over Expression, Knock-In, Two Tailed Test

Journal: Cell reports

Article Title: PARP7 inhibition stabilizes STAT1/STAT2 and relieves experimental autoimmune encephalomyelitis in mice

doi: 10.1016/j.celrep.2025.116130

Figure Lengend Snippet: (A) Immunoblot analysis of STAT1 and STAT2 protein levels in PARP7 WT and KO MEF cells. The experiment was repeated twice. (B) Co-immunoprecipitation (coIP) analysis showing that STAT1 and STAT2 interact with PARP7. FLAG-PARP7 was ectopically expressed in HEK293T cells. FLAG-PARP7 was immunoprecipitated and then blotted for endogenous STAT1 and STAT2. The experiment was repeated twice. (C and D) Immunoblot analysis of ADP-ribosylated STAT1 (C) and STAT2 (D) in PARP7 WT and KO MEF cells. Cells were lysed in the presence of biotin-NAD + or NAD + . Proteins that were ADP-ribosylated by biotin-NAD + were pulled down with streptavidin beads and then blotted for STAT1 or STAT2. Samples treated with NAD + were used as a negative control. Both experiments were repeated three times. (E) PARP7 WT, but not the H532A mutant, ADP-ribosylates STAT1. GFP-tagged empty vector (EV), WT PARP7, or the H532 mutant were co-transfected with FLAG-STAT1 into HEK293T cells. FLAG-STAT1 was pulled down to detect interaction with PARP7 (blotted with GFP antibody) and ADP-ribosylation (blotted with ADP-ribose antibody). The experiment was repeated twice. See also .

Article Snippet: Recombinant human STAT1 , MedChemExpress , Cat# HY- P71335.

Techniques: Western Blot, Immunoprecipitation, TNKS1 Histone Ribosylation Assay, Negative Control, Mutagenesis, Plasmid Preparation, Transfection

Journal: Cell reports

Article Title: PARP7 inhibition stabilizes STAT1/STAT2 and relieves experimental autoimmune encephalomyelitis in mice

doi: 10.1016/j.celrep.2025.116130

Figure Lengend Snippet: (A) HEK293T cells were transfected with FLAG-WT or H532A mutant PARP7 overnight. FLAG-PARP7 was detected by immunofluorescence with anti-FLAG (green) antibodies. Endogenous STAT1 was detected by immunofluorescence with Alexa Flour 647. Nuclei were stained with Hoechst stain (blue) (scale bar: 2 μm). Right: quantification of STAT1 and PARP7 co-localization using Pearson’s correlation coefficient. Data are presented as mean ± SD. *** p < 0.001, unpaired t test. (B) HEK293T cells were transfected with FLAG-WT or H532A mutant PARP7 overnight. FLAG-PARP7 was detected by immunofluorescence with anti-FLAG (green) antibodies. Endogenous STAT2 was detected by immunofluorescence with Alexa Flour 647. Nuclei were stained with Hoechst stain (blue) (scale bar: 2 μm.). Right: quantification of STAT2 and PARP7 co-localization using Pearson’s correlation coefficient. Data are presented as mean ± SD. *** p < 0.001, unpaired t test. All experiments were repeated twice. See also .

Article Snippet: Recombinant human STAT1 , MedChemExpress , Cat# HY- P71335.

Techniques: Transfection, Mutagenesis, Immunofluorescence, Staining

Journal: Cell reports

Article Title: PARP7 inhibition stabilizes STAT1/STAT2 and relieves experimental autoimmune encephalomyelitis in mice

doi: 10.1016/j.celrep.2025.116130

Figure Lengend Snippet: (A) Western blot analysis of STAT1 and STAT2 in PARP7 WT or KO MEF cells after treatment of MG132 at 20 μМ (left) and cycloheximide (CHX) at 50 μМ (right). The experiment was repeated three times. (B) Western blot analysis of STAT1 and STAT2 in PARP7 WT or KO MEF cells after bafilomycin A1 treatment at 5 μМ. The experiment was repeated three times. (C) coIP analysis of p62 and PARP7 WT or H532A catalytic mutant. FLAG-tagged PARP7 WT and H532A mutant were transfected into HEK293T cells, and coIP was performed to detect interactions between p62 and PARP7 WT or H532A mutant. The experiment was repeated three times. (D) Co-localization of hemagglutinin (HA)-tagged p62 with GFP-tagged PARP7 WT or H532A mutant in HEK293T cells. HA-p62 was detected by immunofluorescence with anti-HA (red) antibody. Nuclei were stained with Hoechst stain (scale bar: 5 μm). The experiment was repeated twice. (E) PARP7 promotes STAT1 and STAT2 ubiquitination. HEK293T cells were co-transfected with FLAG-STAT1 (left) or FLAG-STAT2 (right) and GFP-tagged PARP7 WT or H532A mutant. STAT1 or STAT2 was immunoprecipitated by anti-FLAG affinity resins, and the ubiquitination of STAT1 or STAT2 was analyzed by western blot. The experiment repeated twice. (F) PARP7 promotes p62 interaction with STAT1 and STAT2. HEK293T cells were co-transfected with FLAG-STAT1 (left) or FLAG-STAT2 (right) and GFP-tagged PARP7 WT or H532A mutant. STAT1 or STAT2 was immunoprecipitated by anti-FLAG affinity resin and blotted for p62. The experiment was repeated twice. (G) p62 knockdown diminishes the difference in STAT1 and STAT2 levels between PARP7 WT and KO MEF cells. STAT1 and STAT2 protein levels in PARP7 WT and KO MEF cells with or without p62 knockdown were analyzed by western blots. The experiment was repeated twice. (H) Proposed model depicting the negative regulation of STAT1 and STAT2 by PARP7. PARP7 binds and ADP-ribosylates STAT1 and STAT2. The ADP-ribosylation recruits E3 ubiquitin ligases, leading to the ubiquitination of STAT1 and STAT2. The ubiquitinated STAT1 and STAT2 then recruits p62, leading to autophagy-mediated degradation. Created with BioRender.com . See also – .

Article Snippet: Recombinant human STAT1 , MedChemExpress , Cat# HY- P71335.

Techniques: Western Blot, Mutagenesis, Transfection, Immunofluorescence, Staining, Ubiquitin Proteomics, Immunoprecipitation, Knockdown

Journal: Clinical Science (London, England : 1979)

Article Title: Interferon regulatory factor 1 (IRF1) inhibits lung endothelial regeneration following inflammation-induced acute lung injury

doi: 10.1042/CS20220876

Figure Lengend Snippet: ( A ) Representative immunoblots and densitometric quantification of Stat1 Ser727 phosphorylation levels in MLECs isolated from Ifr1 fl/fl control mice pre- and post-LPS. ( B ) Representative immunoblots and densitometric quantification of Stat1 Ser727 phosphorylation levels in MLECs in Ifr1 fl/fl control mice and endothelial cell-specific Irf1 knockout mice at baseline. ( C ) Representative immunoblots and densitometric quantification of Stat1 Ser727 phosphorylation and Irf1 protein levels in the indicated MLEC cell lines following LPS (10 μg/ml) treatment. ( D ) Representative immunoblots and densitometric quantification of STAT1 Ser727 phosphorylation and IRF1 protein levels in the indicated HLMVEC cell lines following LPS (10 μg/ml) treatment. ( E ) Schematic of the human IRF1 promoter region depicting the two highly-conserved STAT1 binding site at −44 ∼ −56 bp and −177 ∼ −188 bp. The WT and MUT GAS1 sequences used in panel ( G ) are provided. ( F ) HLMVECs subjected to vehicle or LPS conditions for 8 hours, followed by ChIP-qPCR assays for detection of STAT1 binding to the two binding sites within the IRF1 promoter region. (G) HLMVECs co-transfected with control or STAT1 plasmid along with one of three luciferase (Luc) reporter gene constructs. Schematic representations of Luc constructs are indicated. All experiments: n = 6 mice or 6 independent biological replicates per group. Data represented as means ± SDs. * P <0.05, ** P <0.01 [(A, C, D) one-way ANOVA with Bonferroni post-hoc tests, and Log-rank Mantel-Cox tests; (B) two-tailed Student’s t -tests; (E, H, I) two-way ANOVA with Bonferroni post-hoc tests].

Article Snippet: For in vitro gene overexpression, the pMXs-ms- Irf1 , pMXs-ms- IRF1 , pMXs-ms- Stat1 , and pMXs-ms- STAT1 plasmids were generated by respectively cloning the mouse Irf1 cDNA clone (NM_008390, cat. no. MC200482, Origene), human IRF1 cDNA clone (NM_002198, cat. no. SC118744, Origene), murine Stat1 cDNA clone (BC004808, cat no. MC200236, Origene), or human STAT1 cDNA clone (NM_007315, cat. no. SC115595, Origene) into the pMXs-GW backbone (plasmid 18656, Addgene).

Techniques: Western Blot, Isolation, Control, Knock-Out, Binding Assay, Transfection, Plasmid Preparation, Luciferase, Construct, Two Tailed Test