Journal: The Journal of Clinical Investigation

Article Title: CRISPR/Cas9 screens identify LIG1 as a sensitizer of PARP inhibitors in castration-resistant prostate cancer

doi: 10.1172/JCI179393

Figure Lengend Snippet: ( A ) Immunoblot of LIG1 protein levels and cell viability measured with CCK8 assay in 22Rv1 cells transduced with the indicated inducible shRNAs. Cells were treated for 12 days with ethanol (EtOH), as control, or Dox to induce shRNA expression, and with DMSO, as control, or OLA. Data are presented as mean ± SD ( n = 3 biological replicates). P values were determined using a 2-way ANOVA and Bonferroni’s multiple-comparisons test. ( B ) Percentage of apoptotic cells measured by CellEvent caspase-3/7 assay in 22Rv1 cells transduced with the indicated sgRNA and treated with DMSO, as control, or OLA for 5 days. Data are presented as mean ± SD ( n = 3 biological replicates). P values were determined using 2-way ANOVA and Bonferroni’s multiple-comparisons test on control (sgNTC and sg EGFP ) and sg LIG1 samples. Images are representative of 22Rv1 cells treated with 4 μM OLA. Scale bar: 100 μm. ( C ) Immunoblot of PARP, cleaved PARP (Cl-PARP), cleaved caspase-3 (Cl-CASP3), and ACTB (used as loading control) in 22Rv1 transduced with the indicated sgRNAs and treated for 3 days with DMSO, as control, or OLA. * P ≤ 0.05; ** P ≤ 0.01; *** P ≤ 0.001; **** P ≤ 0.0001. Long exp: longer exposure.

Article Snippet: Genome-integrated sgRNA sequences were amplified by PCR using TaKaRa Ex Taq DNA Polymerase (Takara Bio Group, catalog RR001A; primers listed in ) and double-size selection with AMPure XP (Beckman Coulter) was performed to purify the PCR product of interest.

Techniques: Western Blot, CCK-8 Assay, Transduction, Control, shRNA, Expressing

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

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: Mouse pooled genome-wide sgRNA library (Brie) sequences , Addgene , Addgene: 73632.

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: Mouse pooled genome-wide sgRNA library (Brie) sequences , Addgene , Addgene: 73632.

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: Mouse pooled genome-wide sgRNA library (Brie) sequences , Addgene , Addgene: 73632.

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