human pooled genome wide sgrna library brunello sequences addgene addgene  (Addgene inc)

Journal: Cell

Article Title: A membrane-associated MHC-I inhibitory axis for cancer immune evasion

doi: 10.1016/j.cell.2023.07.016

Figure Lengend Snippet: (A-D) Representative histograms (left) and bar plots (right) showing the surface levels of HLA-A2:AFP (A) or HLA-A2 (B) in human THP-1-Cas9-AFP-BFP cells, and the surface levels of H-2Kb:OVA (C) or H-2Kb (D) in mouse RN2-Cas9-OVA-BFP cells transduced with the indicated sgRNAs. (n=3) (E and F) Schematic of the T cell activation assay (E) and bar plot showing the IL-2 secreted by the B3Z T cell hybridoma incubated with sgRNA-transduced RN2-Cas9-OVA-BFP cells (F). (n=3) (G and H) Schematic of the mouse T cell killing assay (G) and bar plot showing the percentages of sgRNA-transduced RN2-Cas9-OVA-BFP cells killed by the OT-I T cells (H). (n=3) (I and J) Schematic of the human T cell killing assay (I) and bar plot showing the percentages of sgRNA-transduced NY-ESO-1-expressing THP-1-Cas9 cells killed by the NY-ESO-1 TCR-T cells (J). (n=3) (K) Schematic of the in vivo validations of SUSD6 functions in a mouse syngeneic AML model. (L-P) Quantification of the tumor volumes (L and O) and Kaplan-Meier survival curves (M and P) of immunocompetent (L and M) or CD8+ T cell-depleted (O and P) mice transplanted with sgRNA-transduced C1498-Cas9-GFP cells as described in (K). (for L and M: n=4 for sgNT and n=6 for sgSusd6; for O and P: n=5 for sgNT and n=6 for sgSusd6) (R) Violin plot of SUSD6 mRNA levels in normal HSPCs, MDS cells, and AML cells with different karyotypes from patient samples. HSPCs, hematopoietic stem and progenitor cells; HSC, hematopoietic stem cell; MPP, multipotent progenitor; CMP, common myeloid progenitor; GMP, granulocyte-monocyte progenitor; MEP, megakaryocyte-erythrocyte progenitor; MDS, myelodysplastic syndromes. Data were obtained from BloodSpot.51 (S) Survival of AML patients with high or low expression of SUSD6 in the TCGA-LAML cohort. (T) Pearson correlation of SUSD6 expression levels in AML cells and T cell activation signature in CD8+ T cells from the bone marrow immuno-microenvironments from AML patients. Data were generated by single-cell RNA-seq.53 Data are presented as the mean ± SEM. ns, not significant; *, p< 0.05; **, p< 0.01; and ***, p< 0.001 by two-tailed unpaired Student’s t-test (A-D, F, H, and J), two-way ANOVA for the last time point (L and O), or Log-rank Mantel-Cox test (M and P). Rosa26-targeting sgRNA (sgRosa, for human) and non-targeting sgRNA (sgNT, for mouse) were used as controls.

Article Snippet: Human pooled genome-wide sgRNA library (Brunello) sequences , Addgene , Addgene: 73179.

Techniques: Transduction, Activation Assay, Incubation, Expressing, In Vivo, Generated, RNA Sequencing, Two Tailed Test

Journal: Cell

Article Title: A membrane-associated MHC-I inhibitory axis for cancer immune evasion

doi: 10.1016/j.cell.2023.07.016

Figure Lengend Snippet: (A) Representative western blots (left) and normalized band intensities (right) of HLA-A and B2m in sgRNA-transduced THP-1 cells. (n=5). (B) Schematic of the surface HLA-A2 internalization assay in sgRNA-transduced THP-1 cells. (C) Quantifications of the surface-remaining HLA-A2. (n=3) (D-F) Time course studies of the surface HLA-A2 expression on shRNA-transduced THP-1 cells treated with Cycloheximide (CHX, D), Bafilomycin A1 (BafA1, E), or Epoxomicin (Epox, F) (n=4). (G-J) Representative confocal images (top) and quantifications (bottom) of the surface-derived MHC-I colocalized with the plasma membrane markers (G and I) or the lysosomal marker (H and J) in THP-1 cells (G and H) or MutuDC cells (I and J) transduced with indicated shRNAs. At least 100 cells were quantified in each group. All scale bars: 5 μm. (K and L) Flow cytometric analyses of intracellular MHC-I storage (K) and recycle (L) in shRNA-transduced THP-1 cells as described in Figures S5H and S5I, respectively. (for K: n=6; for L: n=4) Data are presented as the mean ± SEM (A-F, K, and L) or box and whiskers with all data points (G-J). ns, not significant; ***, p<0.001 by two-tailed unpaired Student’s t-test (A), two-way ANOVA for the last time point (C-F, K, and L), or Mann-Whitney test (G-J). sgRosa and shRen were used as controls.

Article Snippet: Human pooled genome-wide sgRNA library (Brunello) sequences , Addgene , Addgene: 73179.

Techniques: Western Blot, Expressing, shRNA, Derivative Assay, Clinical Proteomics, Membrane, Marker, Transduction, Two Tailed Test, MANN-WHITNEY

Journal: Cell

Article Title: A membrane-associated MHC-I inhibitory axis for cancer immune evasion

doi: 10.1016/j.cell.2023.07.016

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: Human pooled genome-wide sgRNA library (Brunello) sequences , Addgene , Addgene: 73179.

Techniques: Control, Purification, Blocking Assay, Virus, Recombinant, Protease Inhibitor, Transfection, Immunoprecipitation, Activation Assay, Magnetic Beads, Reverse Transcription, SYBR Green Assay, Enzyme-linked Immunosorbent Assay, Double Knockout, shRNA, Real-time Polymerase Chain Reaction, Genome Wide, Plasmid Preparation, Software, Flow Cytometry