human soluble fas  (R&D Systems)

Journal: Advanced Science

Article Title: ACSS2/AATF Drives Soluble FasL‐Mediated CD8 + T Cell Apoptosis in Pancreatic Neuroendocrine Tumors

doi: 10.1002/advs.202506883

Figure Lengend Snippet: Increased ACSS2 catalyzes PNETs cells histone acetylation modification through pan‐acetylation regulation, and promotes FasL transcription and exocytosis of sFasL in PNET cells. A) ACSS2 enhances histone pan‐acetylation in PNET cell lines. After overexpression or knockdown of ACSS2, immunoblotting was performed with an antibody against ACSS2, using β‐actin as the loading control, and was performed with the antibodies against Ace‐H3, Ace‐H4, H3K9ac, H3K27ac, and H4K16ac, using H3 or H4 as the loading control. B) ACSS2 was overexpressed or knocked down in PNET cell lines, and acetyl‐CoA content was determined by fluorescence ELISA. The assay was repeated independently 3 times ( * p < 0.05, ** p < 0.01). C) Cellular acetyl‐CoA content assay in BON‐1 and QGP‐1 cells after treatment with acetate supplementation or ACSS2i, respectively. D) Cross‐analysis of differentially expressed genes in the four comparison pairs (ACSS2‐OE vs EV‐up, ACSS2‐KO1 vs Ctrl‐down, ACSS2‐KO2 vs Ctrl‐down, ACSS2i vs DMSO‐down) yielded a total of 69 common genes. E) Ranking of the total number of pathways enriched for common genes. The vertical coordinate is the total number of pathways enriched to, and the horizontal coordinate is the gene name. F) FasLG gene expression levels were analyzed at the RNA level as verified by RT‐qPCR assays in different treatment groups of ACSS2 in BON‐1 and QGP‐1 cell lines, respectively ( * p < 0.05, ** p < 0.01, **** p < 0.0001). G) ELISA assay of soluble FasL (sFasL) levels in BON‐1 and QGP‐1 cell culture supernatants from overexpression or silencing of ACSS2. Five independent repetitions for each group ( * p < 0.05, ** p < 0.01, *** p < 0.001). H) ELISA assay of sFasL levels in BON‐1 and QGP‐1 cell culture supernatants from different treatment groups. Five independent repetitions for each group ( * p < 0.05, ** p < 0.01).

Article Snippet: Dimethyl sulfoxide (DMSO, D2650, Sigma–Aldrich), acetate (S8750, Sigma–Aldrich), ACSS2 inhibitor (ACSS2i, vy‐3‐135, #1824637‐41‐3, MedChemExpress), FasL blocking antibody (FasL antibody) (ab186671), animal‐free recombinant human soluble Fas ligand (rh‐sFasL, HY‐P700052AF, MedChemExpress), and Kp7‐6 (HY‐ P10102 , MedChemExpress) follow the manufacturer's instructions.

Techniques: Modification, Over Expression, Knockdown, Western Blot, Control, Fluorescence, Enzyme-linked Immunosorbent Assay, Comparison, Gene Expression, Quantitative RT-PCR, Cell Culture

Journal: Advanced Science

Article Title: ACSS2/AATF Drives Soluble FasL‐Mediated CD8 + T Cell Apoptosis in Pancreatic Neuroendocrine Tumors

doi: 10.1002/advs.202506883

Figure Lengend Snippet: ACSS2‐mediated increase in sFasL in PNET cells induces apoptosis in Jurkat cells through non‐classical manner. A) Dimensionality reduction plot depicting the scRNA‐seq data, identifying a total of 10 major cell types across the tumor tissues. B) Dot plot showing the expression of classical cell type markers across identified cell subpopulations. Dot size represents the percentage of cells expressing each marker and color intensity reflecting average expression. C) Violin plot comparing the expression levels of FAS across major cell subpopulations, with T cells exhibiting significantly higher expression than other subpopulations. D) The uniform manifold approximation and projection (UMAP) representation of four cell subpopulations generated from sub‐clustering T cells. F) Dot plot showing the expression levels of FAS across four T cell subpopulations. G) CellChat infers intercellular communication networks between different cell types. H) The flowchart for the co‐culture of PNETs cells and Jurkat cells. Tumor cells and Jurkat cells were inoculated in the lower and upper chambers of a six‐well plate and co‐cultured for 48 h. Subsequently, Jurkat cells in the upper chamber were aspirated and flow assayed to observe the proportion of cells undergoing apoptosis. Each group of experiments was independently repeated three times. I) Tumor cells with specific ratios (tumor cell: Jurkat cell ratio of 5W:5 W, 10W:5 W, 25W:5 W, and 50W:5 W respectively) wer e co‐cultured with Jurkat cells according to the co‐culture model, and differences in apoptotic proportion of Jurkat cells caused by PNET cells from ACSS2‐OE versus ACSS2‐EV were examined ( ** p < 0.01). J) Multicolor immunofluorescence staining of paraffin‐embedded Rip1‐Tag2 mouse tissue sections for FasL (green), Fas (red), CD8 (white), ACSS2 (orange), and nuclei (DAPI, blue). Yellow arrows indicate magnified cells. To quantify CD8 ± Fas ± T cells or ACSS2 ± FasL ± tumor cells, 3 randomly photographed spot areas were taken on the 3 slides using a 40 × oil immersion objective, respectively. K) The expression scores of mIF of ACSS2, FASL, Fas, and CD8 in tumor and adjacent normal tissues of Rip1‐Tag2 mice models ( ** p < 0.01, n = 3).

Article Snippet: Dimethyl sulfoxide (DMSO, D2650, Sigma–Aldrich), acetate (S8750, Sigma–Aldrich), ACSS2 inhibitor (ACSS2i, vy‐3‐135, #1824637‐41‐3, MedChemExpress), FasL blocking antibody (FasL antibody) (ab186671), animal‐free recombinant human soluble Fas ligand (rh‐sFasL, HY‐P700052AF, MedChemExpress), and Kp7‐6 (HY‐ P10102 , MedChemExpress) follow the manufacturer's instructions.

Techniques: Expressing, Marker, Generated, Co-Culture Assay, Cell Culture, Multicolor Immunofluorescence Staining

Journal: Advanced Science

Article Title: ACSS2/AATF Drives Soluble FasL‐Mediated CD8 + T Cell Apoptosis in Pancreatic Neuroendocrine Tumors

doi: 10.1002/advs.202506883

Figure Lengend Snippet: Transcription‐promoting factor AATF synergizes with ACSS2 to increase FasL expression A) Silver staining of SDS‐PAGE gels indicated ACSS2‐interacting proteins. Flag‐ACSS2 expression stable BON‐1 cells were lysed and immunoprecipitated with anti‐Flag Ab or rabbit IgG control Ab, and then subjected to SDS‐PAGE gel and silver staining. B) Representative tandem MS spectrum of the peptide LLSFMAPIDHTTMNDDAR from AATF as determined by IP‐Mass Spec. C) AATF was further identified to bind with ACSS2 by immunoprecipitation. Flag‐ACSS2 expression stable BON‐1 cells were lysed and immunoprecipitation with anti‐Flag Ab or abbit IgG control Ab, and then subjected to immunoblotting using antibodies against AATF. D) Endogenous ACSS2 colocalized with AATF in PNET cells. Localization of AATF (green) and ACSS2 (red) in BON‐1 and QGP‐1 cells was detected by double immunofluorescence labeling and confocal microscopy. The merged image with the yellow signal represented their colocalization. E) A ChIP‐re‐ChIP assay was conducted using anti‐AATF antibody first (AATF) in BON‐1 and QGP‐1 cells. The eluents were then subjected to a second ChIP assay using anti‐Flag‐ACSS2 antibody (AATF + Flag‐ACSS2) or control IgG antibody (AATF + IgG) (n = 3). F) Dual luciferase validation of AATF positive regulation of FasL transcription. Schematic representations of the reporter and effector constructs used in the dual‐LUC assay. Firefly luciferase (LUC) driven by the FasLG promoter was used as the reporter. Renilla luciferase (REN) was used as an internal control. G) The binding sites of ACSS2 and AATF protein were simulated using AutoDock Vina v.1.2.2 molecular docking analysis, whose low binding energy is −11.3 kcal mol −1 . H) Surface plasmon resonance (SPR) analysis using a Biacore 8K system (Cytiva) quantified the binding kinetics and affinity between the analyte (AATF) and immobilized ligand (ACSS2). Six serially diluted concentrations of AATF (range: 12.5 – 400 n m ) were injected over ACSS2‐coupled CM5 sensor chips. Real‐time binding curves demonstrated concentration‐dependent responses, yielding an equilibrium dissociation constant (K D ) of 33.3 ± 1.7 n m , indicative of high‐affinity molecular recognition. I) Overexpression (or knockdown) of AATF in PNET cells could lead to increased (or decreased) sFasL levels. ELISA assay of sFasL concentration levels in BON‐1 cell culture supernatants from different treatment groups. Five independent repetitions for each group ( * p < 0.05, ** p < 0.01).

Article Snippet: Dimethyl sulfoxide (DMSO, D2650, Sigma–Aldrich), acetate (S8750, Sigma–Aldrich), ACSS2 inhibitor (ACSS2i, vy‐3‐135, #1824637‐41‐3, MedChemExpress), FasL blocking antibody (FasL antibody) (ab186671), animal‐free recombinant human soluble Fas ligand (rh‐sFasL, HY‐P700052AF, MedChemExpress), and Kp7‐6 (HY‐ P10102 , MedChemExpress) follow the manufacturer's instructions.

Techniques: Expressing, Silver Staining, SDS Page, Immunoprecipitation, Control, Mass Spectrometry, Western Blot, Immunofluorescence, Labeling, Confocal Microscopy, Luciferase, Biomarker Discovery, Construct, Binding Assay, SPR Assay, Injection, Concentration Assay, Over Expression, Knockdown, Enzyme-linked Immunosorbent Assay, Cell Culture

Journal: Advanced Science

Article Title: ACSS2/AATF Drives Soluble FasL‐Mediated CD8 + T Cell Apoptosis in Pancreatic Neuroendocrine Tumors

doi: 10.1002/advs.202506883

Figure Lengend Snippet: AATF acts as an intranuclear guide that binds ACSS2 and localizes to the FasLG promoter region to enhance transcription in concert with histone pan‐acetylation modification. A) AATF binding to the FasLG promoter region and promoting transcription requires synergistic ACSS2‐mediated histone acetylation modifications. ChIP qRT‐PCR detection of AATF occupancy on the promoters of FasLG in BON‐1 cells and QGP‐1 cells. ChIP‐level antibodies against acetylation‐modified H3 and H4 were used to specifically pull down H3ac/H4ac histones in each treatment group. Silencing of ACSS2 expression after overexpression of AATF reversed the increase in FasLG promoter region occupancy caused by AATF overexpression (siNC + AATF‐OE vs EV + siNC, siNC + AATF‐OE vs AATF‐OE + siACSS2). The decrease in FasLG promoter region occupancy after silencing ACSS2 expression was not reversed by overexpression of AATF (EV + siACSS2 vs EV + siNC, EV + siACSS2 vs AATF‐OE + siACSS2) ( ** p < 0.01, ns indicates not significant). B) Enhancement of FasLG transcription by ACSS2 requires endogenous guidance of AATF and pro‐transcriptional regulation of the FasLG promoter region by AATF. ChIP qRT‐PCR detection of AATF occupancy on the promoters of FasLG in BON‐1 cells and QGP‐1 cells. ChIP‐level antibodies against acetylation‐modified H3 and H4 were used to specifically pull down H3ac/H4ac histones in each treatment group. Silencing of AATF expression after overexpression of ACSS2 reversed the increase in FasLG promoter region occupancy caused by ACSS2 overexpression (siNC + ACSS2‐OE vs EV + siNC, siNC + ACSS2‐OE vs ACSS2‐OE + siAATF). The decrease in FasLG promoter region occupancy after silencing AATF expression was not reversed by overexpression of ACSS2 (EV + siAATF vs EV + siNC, EV + siAATF vs ACSS2‐OE + siAATF) ( ** p < 0.01, ns indicates not significant). C,D) ELISA assay of sFasL concentration levels in BON‐1 and QGP‐1 cell culture supernatants from different treatment groups. Consistency in intergroup comparisons of different cell lines. Five independent repetitions for each group ( ** p < 0.01, ns indicates not significant). E) The percentage of apoptotic Jurkat cells after co‐cultured with BON‐1 and QGP‐1 cells among distinct groups ( ** p < 0.01, ns indicates not significant). F) FasL antibody (0.1 µg mL −1 ) was detected after 48 h of incubation in the lower chamber of co‐cultured six‐well plates ( ** p < 0.01). G,H) Validation of expression levels of key pro‐apoptotic proteins in the Fas/FasL pathway. After overexpression of ACSS2 or AATF, immunoblotting was performed using FADD, cleaved caspase‐8, and cleaved caspase‐3 antibodies with β‐actin as upload control in BON‐1 (G) and QGP‐1 (H) cell lines, respectively.

Article Snippet: Dimethyl sulfoxide (DMSO, D2650, Sigma–Aldrich), acetate (S8750, Sigma–Aldrich), ACSS2 inhibitor (ACSS2i, vy‐3‐135, #1824637‐41‐3, MedChemExpress), FasL blocking antibody (FasL antibody) (ab186671), animal‐free recombinant human soluble Fas ligand (rh‐sFasL, HY‐P700052AF, MedChemExpress), and Kp7‐6 (HY‐ P10102 , MedChemExpress) follow the manufacturer's instructions.

Techniques: Modification, Binding Assay, Quantitative RT-PCR, Expressing, Over Expression, Enzyme-linked Immunosorbent Assay, Concentration Assay, Cell Culture, Incubation, Biomarker Discovery, Western Blot, Control

Journal: Advanced Science

Article Title: ACSS2/AATF Drives Soluble FasL‐Mediated CD8 + T Cell Apoptosis in Pancreatic Neuroendocrine Tumors

doi: 10.1002/advs.202506883

Figure Lengend Snippet: Detection of apoptotic proteases in primary T cells and histological validation of the correlation between ACSS2 and FasL expression in PNETs. A) Flow sorting procedure for primary CD8 + T cells. B,C) The percentage of apoptotic primary CD8 + T cells after co‐cultured with BON‐1 and QGP‐1 cells among distinct groups. Animal‐free recombinant human soluble Fas ligand (rh‐sFasL, 4 units/mL) was detected after 18 h co‐incubation of independent treatment ( * p < 0.05, ** p < 0.01, ns indicates not significant). D,E) Changes in the activity of caspase‐8 (D) and caspase‐3 (E) in BON‐1 and QGP‐1 cell lines were determined by fluorescence assay after the corresponding treatments. Acetate (1 m m ) was detected after 48 h of independent treatment in the upper and lower chambers of co‐cultured six‐well plates. FasL antibody (0.1 µg mL −1 ) was detected after 48 h of incubation in the lower chamber of co‐cultured six‐well plates ( * p < 0.05, ** p < 0.01, ns indicates not significant). F) The percentage of apoptotic primary CD8 + T cells after co‐cultured with PNET patient‐derived organoids (PDOs) among distinct groups. The ACSS2i (0.5 µM), anti‐FasL antibody (0.1 µg mL −1 ), or animal‐free recombinant human soluble Fas ligand (rh‐sFasL, 4 units/mL) was added to one well in a six‐well plate and then independently treated and co‐incubated for 48 h ( ** p < 0.01, ns indicates not significant). G) Bar plot showing the IFN‐γ positive spots/10 5 cells in the different suppression and co‐culturing conditions as indicated in the plot. TGF‐β treatment was added as a negative control for CD8 + T cell suppression. Error bars indicate mean ± SEM ( * p < 0.05, ** p < 0.01, ns indicates not significant).

Article Snippet: Dimethyl sulfoxide (DMSO, D2650, Sigma–Aldrich), acetate (S8750, Sigma–Aldrich), ACSS2 inhibitor (ACSS2i, vy‐3‐135, #1824637‐41‐3, MedChemExpress), FasL blocking antibody (FasL antibody) (ab186671), animal‐free recombinant human soluble Fas ligand (rh‐sFasL, HY‐P700052AF, MedChemExpress), and Kp7‐6 (HY‐ P10102 , MedChemExpress) follow the manufacturer's instructions.

Techniques: Biomarker Discovery, Expressing, Cell Culture, Recombinant, Incubation, Activity Assay, Fluorescence, Derivative Assay, Negative Control

Journal: bioRxiv

Article Title: ARID1A Mutations Protect Follicular Lymphoma from FAS-dependent Immune Surveillance by Reducing RUNX3/ETS1-Driven FAS-Expression

doi: 10.1101/2023.12.12.571212

Figure Lengend Snippet: A Schematic of the FASLG-induced apoptosis assay (soluble human recombinant FAS ligand treatment). B Percent AnnexinV-positive OCI-Ly8 (left panel) and OCI-Ly1 (right panel) clones with ARID1A WT, het, KO and overexpression of RUNX3 on het (het + RUNX3) after 24 h treatment with increasing dose of purified soluble human recombinant FAS ligand (N = 3). P-values are from a linear regression on square root transformed values tested against WT and Bonferroni-adjusted . C Schematic of the T-cell mediated killing assay. D Conjugate formation of OCI-Ly8 cells (CFSE + ) and CD8 + T-cells (VPD + ); double-positive conjugates (CSFE + /VPD + ) quantified as percentage of total CD8 + T-cells (VPD + ) (N = 5) at indicated conditions. E T cell mediated cytotoxicity; percentage of OCI-Ly8 cells (CFSE + ) undergoing apoptosis upon co-culture with CD8 + T cells quantified as percentage of all measured cells at indicated conditions (N = 5, biological replicates performed with different healthy T cell donors). P-value is from the paired Welch test. Het+RUNX3 was tested against het. Pooled data from biological replicates (N) are represented as mean ±SD .

Article Snippet: ARID1A wild-type and mutant cell lines were treated with 0, 3, 30, or 300 ng/mL purified soluble human recombinant FAS ligand (SUPERFASLIGAND®, Enzo Life Sciences, Inc., Farmingdale, New York, USA) for 24 h. The cells were assayed by flow cytometry (BD FACSCanto™ II).

Techniques: Apoptosis Assay, Recombinant, Clone Assay, Over Expression, Purification, Transformation Assay, Co-Culture Assay