Journal: Cell reports

Article Title: β-Amyloid Clustering around ASC Fibrils Boosts Its Toxicity in Microglia

doi: 10.1016/j.celrep.2020.02.025

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: Thereafter, cells were blocked using 5% normal goat serum (Vector Laboratories) in PTX for 20 min and primary antibodies were added for another 30 min. To check for ASC speck formation, the rabbit anti-ASC (1:250; clone AL177, AdipoGen) or mouse-specific rabbit anti-ASC/TMS1 (1:250, D2W8U, Cell Signaling Technology®) and rat anti-CD11b (1:250; Serotec by Bio-Rad) were used.

Techniques: Control, Recombinant, Magnetic Beads, Enzyme-linked Immunosorbent Assay, Bicinchoninic Acid Protein Assay, Quantitation Assay, Gel Extraction, Purification, Isolation, Software, Transmission Assay, Microscopy, Spectrophotometry, Fluorescence, Imaging

Journal: Molecular cell

Article Title: An Epstein-Barr virus protein interaction map reveals NLRP3 inflammasome evasion via MAVS UFMylation

doi: 10.1016/j.molcel.2023.05.018

Figure Lengend Snippet:

Article Snippet: Anti-ASC/TMS1 rabbit monoclonal antibody (E1E3I) , Cell Signaling Technology , cat# 13833; RRID: AB_2798325.

Techniques: Recombinant, Magnetic Beads, Transfection, Sequencing, Modification, Mass Spectrometry, Produced, Protease Inhibitor, Selection, Purification, Gel Extraction, SYBR Green Assay, Reporter Assay, Protein Quantitation, CRISPR, Amplification, Molecular Cloning, Plasmid Preparation, Software

Journal: Cancer Communications

Article Title: Lipid metabolism reprograming by SREBP1‐PCSK9 targeting sensitizes pancreatic cancer to immunochemotherapy

doi: 10.1002/cac2.70038

Figure Lengend Snippet: Combined effect of PD‐1 mAb and Fatostatin combination treatment is dependent on CD8 + T cells and SREBP1‐deficiency of cancer cells. (A) Overall survival of the PDAC patients following Sintilimab therapy stratified by median serum TC levels ( n = 43). (B‐F) C57BL/6 mice were implanted with 7 × 10 5 KPC cells and co‐treated with Fatastatin and PD‐1 mAb. (B) A schematic view of the treatment plan. (C) Kaplan‐Meier survival curves for each group (10 mice/group). (D) Tumor image and statistical analysis of orthotopic PDAC model (5 mice/group). (E‐F) Representative images and statistical results of tumor‐infiltrating lymphocytes (CD8 + T cells, GZMB + CD8 + T cells, and IFN‐γ + CD8 + T cells) are shown as indicated by flow cytometry (E) and IHC (F). (G‐I) C57BL/6 mice were implanted with 7 ×10 5 KPC cells and received Fatostatin, PD‐1 mAb, CD8α mAb or IgG isotype control (IgG2a) treatment. (G) A schematic view of the treatment plan. (H) Tumor image and statistical analysis of orthotopic PDAC model (6 mice/group). (I) Oil Red O staining was treated with Fatostatin or vehicle control in C57BL/6 mice xenograft tumor samples. (J) KEGG enrichment analysis of proteomics in KPC tumor treated with Fatostatin or vehicle control. (K‐M) C57BL/6 mice were implanted with 7 ×10 5 shSREBP1 KPC or shNC cells and received PD‐1 mAb or IgG2a treatment. (K) Kaplan‐Meier survival curves for each group (10 mice/group). (L) Tumor image and statistical analysis of orthotopic PDAC model (5 mice/group). (M) Statistical results of tumor‐infiltrating lymphocytes (CD8 + T cells, GZMB + CD8 + T cells) are shown as indicated by flow cytometry. (N) Tumor image and statistical analysis of C57BL/6 mice implanted with 7×10 5 SREBP1‐OE or Vector KPC cells and treated Fatostatin plus PD‐1 mAb. Results are presented as mean ± SEM. ns, not significant; * P < 0.05, ** P < 0.01, *** P < 0.001. PD‐1, programmed cell death protein 1; mAb, monoclonal antibody; SREBP1, sterol regulatory element binding proteins1; KPC, KrasG12D/Trp53R172H/Pdx1‐Cre; GZMB, granzyme B; KEGG, Kyoto Encyclopedia of Genes and Genomes; shNC, shRNA negative control; IFN‐γ, interferon‐γ; IHC, immunohistochemistry; OE, overexpression; SEM, standard error of the mean.

Article Snippet: A week post‐tumor implantation, the mice were divided into six groups: Vehicle + IgG2a (BE0089, BioXCell, West Lebanon, NH, USA) (150 μg per mouse; intraperitoneal injection; every 3 days), Vehicle + PD‐1 mAb (BE0273, BioXCell) (150 μg per mouse; intraperitoneal injection; every 3 days), PD‐1 mAb + TVB‐2640 (HY‐112829, MedChemExpress, New Brunswick, NJ, USA) (10 mg/kg; intragastric administration; every day), PD‐1 mAb + Lovastatin (HY‐N0504, MedChemExpress) (10 mg/kg; intragastric administration; every day), PD‐1 mAb + Simvastatin (HY‐17502, MedChemExpress) (50 mg/kg; intragastric administration; every day), PD‐1 mAb + Fatostatin (S8284, Selleck, Houston, TX, USA) (30 mg/kg; intragastric administration; every day).

Techniques: Flow Cytometry, Control, Staining, Plasmid Preparation, Binding Assay, shRNA, Negative Control, Immunohistochemistry, Over Expression

Journal: Cancer Communications

Article Title: Lipid metabolism reprograming by SREBP1‐PCSK9 targeting sensitizes pancreatic cancer to immunochemotherapy

doi: 10.1002/cac2.70038

Figure Lengend Snippet: SREBP1 binds directly to the PCSK9 promoter in PDAC. (A) Signal densities of CUT & Tag‐seq at PCSK9 gene loci in SW1990 cells, showing SREBF1 binding. (B) PCSK9 expression of KPC and PANC02 cells transfected with shSREBP1 or shNC, as determined by RNA‐seq analysis. (C‐D) RT‐PCR (C) and Western blotting (D) analysis of PCSK9 expression of KPC and PANC02 cell transfected with shSREBP1 or shNC. (E) ELISA assay analyzed the Secreted PCSK9 of PDAC cell lines transfected with shSREBP1 or shNC in the culture supernatant. (F‐G) qRT‐PCR (F) and Western blotting (G) analysis of PCSK9 expression of PDAC cell lines treated with or without Fatostatin at the indicated doses for 48 h. (H) SREBF1 binding to the PCSK9 promoter was determined by chromatin immunoprecipitation‐RT‐PCR in human PDAC cell lines (BxPC3 and SW1990). (I) Schematic representation of the PCSK9 promoter cloned into the pGL4.1 vector. Three predicted SREBF1 binding motifs are shown, and promoter constructs containing mutations in these 3 regions to cause SREBF1‐binding deficiency are generated. (J) Analysis of PCSK9 WT or mutant promoter activity in 293T cell lines transfected with SREBF1‐Flag. (K‐M) SREBP1 is positively correlated with PCSK9 in PDAC by multi‐color IHC (K), ordinary IHC (L) and TIMER database (M). (N) The correlation of SREBF1 and CD8 + T cell in PAAD from the TIMER database. (O) Kaplan‐Meier analysis of survival in PDAC according to the expression of PCSK9 in the group with decreased or enriched intratumoral CD8 + T cell infiltration. (P) Comparison of serum PCSK9 levels in patients with objective response versus non‐objective response to anti‐PD‐1 therapy ( n = 43). Results are presented as mean ± SEM, n = 3. And all results were repeated for three times. ns, not significant; P > 0.05, *P < 0.05, **P < 0.01, *** P < 0.001. CUT & Tag‐seq, cleavage under targets and tagmentation sequencing; SREBF1, sterol regulatory element binding factor 1; shNC, shRNA negative control; RNA‐seq, RNA sequencing; ELISA, enzyme‐linked immunosorbent assay; qRT‐PCR:quantitative reverse transcription polymerase chain reaction; PAAD, pancreatic adenocarcinoma; TIMER, tumor immune estimation resource; IHC, immunohistochemistry; PDAC, pancreatic ductal adenocarcinoma.

Article Snippet: A week post‐tumor implantation, the mice were divided into six groups: Vehicle + IgG2a (BE0089, BioXCell, West Lebanon, NH, USA) (150 μg per mouse; intraperitoneal injection; every 3 days), Vehicle + PD‐1 mAb (BE0273, BioXCell) (150 μg per mouse; intraperitoneal injection; every 3 days), PD‐1 mAb + TVB‐2640 (HY‐112829, MedChemExpress, New Brunswick, NJ, USA) (10 mg/kg; intragastric administration; every day), PD‐1 mAb + Lovastatin (HY‐N0504, MedChemExpress) (10 mg/kg; intragastric administration; every day), PD‐1 mAb + Simvastatin (HY‐17502, MedChemExpress) (50 mg/kg; intragastric administration; every day), PD‐1 mAb + Fatostatin (S8284, Selleck, Houston, TX, USA) (30 mg/kg; intragastric administration; every day).

Techniques: Binding Assay, Expressing, Transfection, RNA Sequencing, Reverse Transcription Polymerase Chain Reaction, Western Blot, Enzyme-linked Immunosorbent Assay, Quantitative RT-PCR, Chromatin Immunoprecipitation, Clone Assay, Plasmid Preparation, Construct, Generated, Mutagenesis, Activity Assay, Comparison, Sequencing, shRNA, Negative Control, Reverse Transcription, Polymerase Chain Reaction, Immunohistochemistry

Journal: Cancer Communications

Article Title: Lipid metabolism reprograming by SREBP1‐PCSK9 targeting sensitizes pancreatic cancer to immunochemotherapy

doi: 10.1002/cac2.70038

Figure Lengend Snippet: The combined effect of targeting PCSK9 and PD‐1 mAb combination treatment is dependent on CD8 + T cells. (A‐D) C57BL/6 mice were implanted with 7×10 5 shPCSK9 or shNC KPC cells received PD‐1 mAb or IgG2a control. (A) Tumor image and statistical analysis of orthotopic PDAC model (5 mice/group). (B) Kaplan‐Meier survival curves for each group (10 mice/group). (C‐D) Flow cytometry showing tumor‐infiltrating lymphocytes (CD8 + T cells, GZMB + CD8 + T cells) and tumor cell PD‐L1 expression. (E‐F) C57BL/6 mice were implanted with 7×10 5 shPCSK9 KPC or shNC cells received PD‐1 mAb or CD8α mAb treatment. (E) Tumor image and statistical analysis of orthotopic PDAC model (5 mice/group). (F) Flow cytometry and quantification of CD8 + splenocytes confirming immune cell depletion. (G‐H) C57BL/6 mice were implanted with 7×10 5 KPC cells and co‐treated with PCSK9 neutralizing antibodies (Evolocumab or Alirocumab) and PD‐1 mAb. (G) Tumor image and statistical analysis of orthotopic PDAC model (5 mice/group). (H) Kaplan‐Meier survival curves for each group (10 mice/group). (I‐L) The HuPBMC‐NCG PDX model was co‐treated with PCSK9 neutralizing antibodies and PD‐1 mAb. The schematic diagram shows the process of constructing the patient‐derived PDAC‐huPBMC mice model (Top) and treatment schedule (Bottom). (J‐K) Plots of tumor growth (J) and tumor weight (K) for each group (5 mice/group). (L) Representative images and statistical results of tumor‐infiltrating lymphocytes (CD8 + T cells, PD1 + CD8 + T cells, and Tim3 + CD8 + T cells) are shown as indicated by flow cytometry. (M) Kaplan‐Meier survival curves for tumor‐bearing GEMM mice co‐treated with PCSK9 neutralizing antibodies and PD‐1 mAb. Treatments began when the solid tumor was palpable. (N) Diagram illustrating SREBP1‐mediated PD‐L1 regulation. SREBP1 binds to the PD‐L1 promoter to suppress its transcription. Meanwhile, SREBP1 binds to the PCSK9 promoter to promote its transcription, then PCSK9‐mediated degradation of PD‐L1 in the lysosome. Fatostatin (red arrows) impairs the activation of SREBP‐1 by inhibiting the ER‐Golgi translocation of SREBPs, which increases the PD‐L1 expression. Results are presented as mean ± SEM. ns, not significant; P > 0.05, * P < 0.05, ** P < 0.01, *** P < 0.001. PD‐1, programmed death‐1 monoclonal antibody; mAb, monoclonal antibody; IgG2a, immunoglobulin G2a; PDAC, pancreatic ductal adenocarcinoma; shNC, shRNA negative control; PD‐L1, programmed death‐ligand 1; GZMB, granzyme B; HuPBMC, human peripheral blood mononuclear cells; PDX, patient‐derived xenograft; GEMM, genetically engineered mouse model; ER‐Golgi:Endoplasmic Reticulum – Golgi; SREBP1, sterol regulatory element binding protein 1; Tim3, T‐cell immunoglobulin and mucin‐domain containing‐3.

Article Snippet: A week post‐tumor implantation, the mice were divided into six groups: Vehicle + IgG2a (BE0089, BioXCell, West Lebanon, NH, USA) (150 μg per mouse; intraperitoneal injection; every 3 days), Vehicle + PD‐1 mAb (BE0273, BioXCell) (150 μg per mouse; intraperitoneal injection; every 3 days), PD‐1 mAb + TVB‐2640 (HY‐112829, MedChemExpress, New Brunswick, NJ, USA) (10 mg/kg; intragastric administration; every day), PD‐1 mAb + Lovastatin (HY‐N0504, MedChemExpress) (10 mg/kg; intragastric administration; every day), PD‐1 mAb + Simvastatin (HY‐17502, MedChemExpress) (50 mg/kg; intragastric administration; every day), PD‐1 mAb + Fatostatin (S8284, Selleck, Houston, TX, USA) (30 mg/kg; intragastric administration; every day).

Techniques: Control, Flow Cytometry, Expressing, Derivative Assay, Activation Assay, Translocation Assay, shRNA, Negative Control, Binding Assay

Journal: PLoS Pathogens

Article Title: Herpesvirus Genome Recognition Induced Acetylation of Nuclear IFI16 Is Essential for Its Cytoplasmic Translocation, Inflammasome and IFN-β Responses

doi: 10.1371/journal.ppat.1005019

Figure Lengend Snippet: List of antibodies used in this study.

Article Snippet: Mouse monoclonal antibody against ASC was from MBL International, Woburn, MA.

Techniques:

Journal: Molecular cell

Article Title: An Epstein-Barr virus protein interaction map reveals NLRP3 inflammasome evasion via MAVS UFMylation

doi: 10.1016/j.molcel.2023.05.018

Figure Lengend Snippet:

Article Snippet: Anti- ASC/TMS1 mouse monoclonal antibody , Proteintech , cat# 67494-1-Ig; RRID: AB_2882718.

Techniques: Recombinant, Magnetic Beads, Transfection, Sequencing, Modification, Mass Spectrometry, Produced, Protease Inhibitor, Selection, Purification, Gel Extraction, SYBR Green Assay, Reporter Assay, Protein Quantitation, CRISPR, Amplification, Molecular Cloning, Plasmid Preparation, Software