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: Journal of immunology (Baltimore, Md. : 1950)

Article Title: IFN mimetic as a therapeutic for lethal vaccinia virus infection: possible effects on innate and adaptive immune responses.

doi: 10.4049/jimmunol.178.7.4576

Figure Lengend Snippet: FIGURE 7. IFN- mimetic treatment results in activation of STAT1 and nuclear translocation of STAT1 and IFNGR1. a, Nuclear translocation of STAT1 and IFNGR1. WISH cells treated with 10 M lipo-IFN-95–132 (left columns), or lipo-IFN- (95–125) (right columns) were stained simultaneously with Abs to STAT1 and IFNGR1. Secondary Abs to STAT1 conjugated to Alexa 594 (top row), or to IFNGR1 conjugated to Cy-2 (bottom row) were used and analyzed by fluorescence microscopy. b, Phosphorylation of STAT1 by IFN mimetic. Cell extracts from WISH cells, untreated (lane 1), control peptide treated (lane 2), or IFN mimetic treated (lane 3) were electro- phoresed, transferred to Immobilon-P, and probed with an Ab to phospho- STAT1 (top row). The filter was stripped and reprobed with an Ab for total STAT1 (bottom row) to ensure equal loading of protein.

Article Snippet: Slides were then incubated for 1 h in blocking buffer containing rabbit polyclonal antisera against IFNGR1 (Santa Cruz Biotechnology) and goat polyclonal antisera to human STAT1 (R&D Systems).

Techniques: Activation Assay, Translocation Assay, Staining, Microscopy, Phospho-proteomics, Control

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: 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