oligonucleotides shrna sequences  (Addgene inc)

Journal: bioRxiv

Article Title: EWSR1::ETS-low cells promote metabolic reprogramming of the tryptophan-kynurenine-AHR axis, immunosuppression, and poor outcome in Ewing sarcoma

doi: 10.1101/2025.05.16.654502

Figure Lengend Snippet: a) Contingency table showing distribution of IPASS score and AHR activity score-based clusters in EwS tumors (n=166). Two-tailed Fisher’s exact test. b) Comparative analysis of inferred pro-inflammatory immune cell infiltration and c) immunosuppressive MDSCs and regulatory T cells infiltration between AHR-low (K1) and -high (K2) activity scoring EwS tumors. Horizontal bars represent the mean and whiskers the SEM. P -values were determined by Wilcoxon test. n=166. d) Relative cell viability MHH-ES-1/TR/shEF1 and TC-106/TR/shERG and e) fold change of interferon (IFN)-γ levels of MHH-ES-1/TR/shEF1 after 24 h co-culture with NK-92 cells. Horizontal bars represent the mean and whiskers the SEM. n=5 independent biological experiments. P- values were determined by two-tailed Mann-Whitney test. f) Relative AHR mRNA and g ) protein expression after establishing single cell clones with independent DOX-inducible shRNA targeting AHR (255, 286) in MHH-ES-1/TR/ and TC-106/TR/ cells (AU = arbitrary units). n=4−6 biologically independent experiments. Relative densitometry is shown, calculated from western blots of lysates from n=4 biologically independent experiments. Two-tailed Mann-Whitney test. h) Relative cell viability MHH-ES-1/TR/shAHR255 bulk and single clone #4 after 24 h co-culture with NK-92 cells. Horizontal bars represent the mean and whiskers the SEM, n=6 biologically independent experiments. P -values were determined by two-tailed Mann-Whitney test.

Article Snippet: AHR silencing (transcript ID: Human NM_001621.5) was achieved by cloning the shRNA oligonucleotide sequences into the Tet-pLKO-puro backbone (Addgene #21915, USA).

Techniques: Activity Assay, Two Tailed Test, Co-Culture Assay, MANN-WHITNEY, Expressing, Clone Assay, shRNA, Western Blot

Journal: The Journal of Immunology Author Choice

Article Title: Efficacy against Lung Cancer Is Augmented by Combining Aberrantly N -Glycosylated T Cells with a Chimeric Antigen Receptor Targeting Fragile X Mental Retardation 1 Neighbor

doi: 10.4049/jimmunol.2300618

Figure Lengend Snippet: A (to our knowledge) novel H441-targeting CAR specifically recognizes FMR1NB. (A) Schematic of the screening procedure. T cells transfected with a pool of CARs from a CAR library and with an IL-2 promoter–luciferase vector were incubated with H441 cells for 24 h. After incubation, samples with higher luciferase activity than control samples were selected and used for subsequent rounds of selection. (B) Luciferase activity of isolated CAR T cells incubated with H441 cells, as determined using a luminometer (n = 4). *p < 0.05 versus control CAR T cells. (C) FMR1NB structure and epitope. A phage display assay was used to determine the amino acid sequence of the first loop of FMR1NB bound to scFv-Fc protein. (D) Left, Precipitation immunoblot of scFv-Fc proteins. scFv-Fc proteins were precipitated with protein A–agarose and immunoblotted with anti-human IgG (Fc) Ab-HRP. Right, scFv-Fc binding. Binding of scFv-Fc-biotin (0–10 μg/ml) or control Fc-biotin (0–10 μg/ml) to H441 cells (1 × 105) was detected by streptavidin-FITC and analyzed by an overlay assay (n = 4). The fold increases in mean fluorescence intensity (MFI) were calculated and plotted against Fc protein concentration. *p < 0.05 versus control cells. (E) Luciferase activity of isolated CAR T cells incubated with the first loop of synthetic FMR1NB peptides, as determined using a luminometer (n = 4). *p < 0.05 versus control CAR T cells. (F) Left, Immunoblot of proteins prepared from H441 cells using anti-FMR1NB Ab derived from the CAR scFv (anti-FMR1NB [scFv]), followed by anti-human IgG (Fc) Ab-HRP or commercially available anti-FMR1NB Ab (anti-FMR1NB [Abnova]), and followed by anti-rabbit IgG ABHRP. Right, Representative flow cytometry showing the distribution of H441 cells expressing FMR1NB detected with control IgG, anti-FMR1NB (scFv), and anti-FMR1NB (Abnova) Abs. (G) Immunohistochemistry of lung adenocarcinoma tissue. Left, Cancer cells show greater staining with anti-FMR1NB (scFv) than normal lung tissue. Scale bar, 200 μm. Right, Cancer cells were heterogeneously stained with anti-FMR1NB (scFv). Scale bar, 10 μm.

Article Snippet: RNA interference shRNA oligonucleotides specific for the target sequence of human FMR1NB were designed as below, then annealed and ligated into expression vector pcDNA6.2-GW-miR (Invitrogen).

Techniques: Transfection, Luciferase, Plasmid Preparation, Incubation, Activity Assay, Selection, Isolation, Sequencing, Western Blot, Binding Assay, Overlay Assay, Fluorescence, Protein Concentration, Derivative Assay, Flow Cytometry, Expressing, Immunohistochemistry, Staining

Journal: The Journal of Immunology Author Choice

Article Title: Efficacy against Lung Cancer Is Augmented by Combining Aberrantly N -Glycosylated T Cells with a Chimeric Antigen Receptor Targeting Fragile X Mental Retardation 1 Neighbor

doi: 10.4049/jimmunol.2300618

Figure Lengend Snippet: Anti-FMR1NB CAR T cell effector activity requires FMR1NB expression on target cells. (A) Schematic of a protocol for an in vitro cytotoxic activity assay of anti-FMR1NB CAR T cells against luciferase-expressing cancer cells. T cells were transduced with lentivirus containing anti-FMR1NB CAR or control CAR at 3 d and incubated with cancer cells for 7 d. Luciferase activities of resident cancer cells were measured after a 16-h incubation with transduced T cells. (B) Killing of H441 cells by anti-FMR1NB CAR T cells. Left, Representative flow cytometry results showing the distribution of H441 cells expressing FMR1NB. Right, Cytotoxic activity of anti-FMR1NB CAR T cells against H441 cells at E:T ratios of 5:1, 10:1, and 25:1 (n = 10). Percentage activity was estimated by dividing the luciferase activity of H441 cells incubated with anti-FMR1NB CAR T cells by that of H441 cells treated with control CAR T cells, with an E:T ratio of 5:1. *p < 0.05 versus control CAR T cells. (C) Production of cytokines after CAR T cell killing of H441 cells. Secretion of cytokines by anti-FMR1NB CAR T cells incubated with H441 cells for 16 h, as determined by ELISA (n = 4). *p < 0.05 versus control CAR T cells. (D) Killing of H441, miaPaCa, and HUEhT-1 cells by anti-FMR1NB CAR T cells. Left, Representative flow cytometry results showing the distribution of cancer cells expressing FMR1NB. Right, Cytotoxic activity of anti-FMR1NB CAR T cells against cancer cells (n = 6). Percentage activity was estimated by dividing the luciferase activity of cancer cells incubated with anti-FMR1NB CAR T cells by that of tumor cells treated with control CAR T cells, with an E:T ratio of 5:1. *p < 0.05, **p < 0.01 versus control CAR T cells. (E) Killing of FMR1NB-overexpressing cancer cells by anti-FMR1NB CAR T cells. Left, Representative flow cytometry results showing the distribution of cancer cells expressing FMR1NB. Right, Cytotoxic activity of anti-FMR1NB CAR T cells against cancer cells expressing FMR1NB (n = 6). Percentage activity was estimated by dividing the luciferase activity of tumor cells incubated with anti-FMR1NB CAR T cells by that of tumor cells treated with control CAR T cells, with an E:T ratio of 5:1. *p < 0.05, **p < 0.01 versus control CAR T cells. (F) Killing of FMR1NB-deficient H441 cells by anti-FMR1NB CAR T cells. Left top, Representative flow cytometry results, representing transfection efficiency, showing the distribution of H441-sh-FMR1NB cells expressing GFP. Left bottom, Representative flow cytometry results showing the distribution of H441-sh-FMR1NB cells expressing FMR1NB. Right top, Immunoblot of proteins prepared from H441 cells and H441-sh-FMR1NB cells using anti-FMR1NB Ab followed by anti-rabbit IgG Ab-HRP. Right bottom, Cytotoxic activity of anti-FMR1NB CAR T cells against H441-sh-FMR1NB cells (n = 6). Percentage activity was estimated by dividing the luciferase activity of tumor cells incubated with anti-FMR1NB CAR T cells by that of tumor cells treated with control CAR T cells, with an E:T ratio of 5:1.

Article Snippet: RNA interference shRNA oligonucleotides specific for the target sequence of human FMR1NB were designed as below, then annealed and ligated into expression vector pcDNA6.2-GW-miR (Invitrogen).

Techniques: Activity Assay, Expressing, In Vitro, Luciferase, Transduction, Incubation, Flow Cytometry, Enzyme-linked Immunosorbent Assay, Transfection, Western Blot

Journal: The Journal of Immunology Author Choice

Article Title: Efficacy against Lung Cancer Is Augmented by Combining Aberrantly N -Glycosylated T Cells with a Chimeric Antigen Receptor Targeting Fragile X Mental Retardation 1 Neighbor

doi: 10.4049/jimmunol.2300618

Figure Lengend Snippet: 2DG treatment augments the antitumor function of anti-FMR1NB CAR T cells both in vitro and in vivo. (A) Killing of H441 cells by anti-FMR1NB CAR T cells or 2DG-treated anti-FMR1NB CAR T cells. Cytotoxic activity of CAR-treated anti-FMR1NB T cells or 2DG-treated anti-FMR1NB T cells against tumor cells (n = 6) is shown. Percentage activity was estimated by dividing the luciferase activity of H441 cells incubated with anti-FMR1NB CAR T cells by that of H441 cells incubated with control CAR T cells, with an E:T ratio of 5:1. *p < 0.05 between indicated groups. (B) Schematic of an intervention protocol for anti-FMR1NB CAR T cells in a NOG mouse xenograft model involving implantation of H441 cells. NOG mice were injected i.v. with 1 × 106 H441 cells. The tumors were imaged on day 7, and on the indicated days, mice were randomized into four groups based on i.v. injection with control CAR T cells, anti-FMR1NB T cells, 2DG-treated T cells, and 2DG-treated anti-FMR1NB T cells (1 × 107). Bioluminescence images of mice were obtained on the indicated days. (C) Pseudocolor images of mice obtained by luciferase-based bioluminescence imaging. On the indicated days, d-luciferin was injected i.p. and bioluminescence images were obtained. (D) Signal intensity of luciferase-based bioluminescence imaging was quantified and plotted (n = 4). *p < 0.05, **p < 0.01 between indicated groups. (E) Survival of mice bearing H441 injected with the four types of CAR T cells described in (B). *p < 0.05 between indicated groups. (F) Representative flow cytometry results and summary bar graphs showing the distribution of CD4+ or CD8+ T cell subsets in tumors. Top, Flow cytometry results of TILs isolated from mouse xenografts showing plots of CD4+ and CD8+ cells in tumors, excluding double-negative cells. After excluding nonlymphocytes based on forward and side scatter (FSC, SSC), live CD3+ cells were selected. The CD8 by CD4 gate distinguishes CD8+ and CD4+ T cells and excludes double-negative and double-positive autofluorescent cells. Bottom, Summary bar graphs showing percentages of CD4+ and CD8+ T cells in tumors, and percentages of CD4+ and CD8+ T cells before injection in each group (n = 4). *p < 0.05 versus control T cells.

Article Snippet: RNA interference shRNA oligonucleotides specific for the target sequence of human FMR1NB were designed as below, then annealed and ligated into expression vector pcDNA6.2-GW-miR (Invitrogen).

Techniques: In Vitro, In Vivo, Activity Assay, Luciferase, Incubation, Injection, Imaging, Flow Cytometry, Isolation