brunello whole genome ko library  (Addgene inc)

Journal: Journal for immunotherapy of cancer

Article Title: Genome-wide CRISPR/Cas9 screen reveals factors that influence the susceptibility of tumor cells to NK cell-mediated killing.

doi: 10.1136/jitc-2024-010699

Figure Lengend Snippet: Figure 1 Genome-wide CRISPR/Cas9 screening identifies genes involved in NKp44-mediated specific killing of tumor cells by KHYG-1 cells. (A) Genome-wide CRISPR/Cas9 screening design. HCT-116 Cas9+ cells were transduced with the knockout sgRNA Brunello library. Collection of mutant cells was subjected to lysis by WT KHYG-1 or NKp44-deficient KHYG-1 in the presence of α-NKp44 mAb or not (Round 1). Mutant cells that survived lysis by KHYG-1 were subjected to a second round (Round 2) of co-culture with WT KHYG-1 (condition 1), in the presence of α-NKp44 mAb (condition 2) or with NKp44-deficient KHYG-1 (condition 3). The abundance of sgRNA in the collection, in the surviving cells in Round 1 and in the surviving cells in Round 2 were determined by sequencing (see Material and methods). (B) Venn diagram of hits obtained with KHYG-1 co-culture (condition 1, see online supplemental table 1), KHYG-1+αNKp44mAb (condition 2, see online supplemental table 2) and NKp44- deficient KHYG-1 cells (condition 3, see online supplemental table 3). (B) Scatter plot showing the ranking of hits enriched in the NKp44-dependent killing of tumor cells by KHYG-1 cells by MAGeCK score and false discovery rate (see online supplemental table 4). FDR, false discovery rate; mAb, monoclonal antibody; sgRNA, single guide RNA; WT, wild-type.

Article Snippet: Human Brunello CRISPR KO pooled library was provided by the CRISPR Screen LabTech platform (Addgene #73178).

Techniques: Genome Wide, CRISPR, Transduction, Knock-Out, Mutagenesis, Lysis, Co-Culture Assay, Sequencing

Journal: Journal for immunotherapy of cancer

Article Title: Genome-wide CRISPR/Cas9 screen reveals factors that influence the susceptibility of tumor cells to NK cell-mediated killing.

doi: 10.1136/jitc-2024-010699

Figure Lengend Snippet: Figure 5 Relative requirement of NK cell effector functions for NK cell-mediated tumor lysis over time. WT and Fas-deficient HCT-116 Cas9+ cells were co-cultured with WT KHYG-1 or perforin-deficient KHYG-1 cells or with IL-2 activated primary NK cells from four donors, as indicated. The extent of specific lysis was determined in a standard 4-hour assay (short-term) (A) or as the CRISPR screen condition (long-term, 72 hours) (B). P values: ****<0.0001; **=0.024 in (A) and **=0.0037 in (B). Unpaired t-test. Data result from the pool of two to three independent experiments. IL, interleukin; KO, knockout; NK, natural killer; WT, wild-type.

Article Snippet: Human Brunello CRISPR KO pooled library was provided by the CRISPR Screen LabTech platform (Addgene #73178).

Techniques: Lysis, Cell Culture, CRISPR, Knock-Out

Journal: Nature Communications

Article Title: Bone morphogenetic protein (BMP) signaling determines neuroblastoma cell fate and sensitivity to retinoic acid

doi: 10.1038/s41467-025-57185-y

Figure Lengend Snippet: A ATRA IC 50 values for all non-hematological cancer cell lines screened by GDSC1. Each dot represents a cell line ( n = 759 in total). Box bounds show 25th and 75th percentiles, horizontal lines within the interquartile range (IQR) boxes indicate the median values for each group, and whiskers represent 1.5*IQR from the IQR values. Cancer types with ≤ 3 cell lines are not included. B Quantified flow cytometry results showing apoptosis in CHP-134, NB13, and D283 Med cell lines treated with ATRA (2 µM). Apoptotic cells were stained positive with annexin V and dead cells were stained positive with PI. Samples at day 9 for CHP-134 P = 1.67 × 10 −7 , NB13 P = 3.32 × 10 −6 ; samples at day 6 for D283 Med P = 1.35 × 10 −6 . C Western blots for cleaved PARP (c-PARP) expression in CHP-134, NB13, and D283 Med cell lines treated with ATRA (2 µM). D Enrichr clustergram of CRISPR screen results (for BioPlanet 2019 pathways and GO Molecular Function 2021). Input: top 1% of genes with lowest negative and positive RRA score (191 + 191 genes). The genes and their directionality are listed in Supplementary Data . Red indicates screen hits. E Scatter plot highlighting top CRISPR screen hits involved in BMP signaling, ranked by positive RRA score (left, top knockouts cause resistance to ATRA) or negative RRA score (right, top knockouts sensitize to ATRA). RARA is also highlighted. F Viability of CHP-134 cells under 3-day (left) or 6-day (right) treatment with ATRA in the presence of DMSO or 0.5 µM K02288. Cell viability was measured with MTS. Data represent the mean ± SD of 4 independent replicates. For arrow indicated points, P = 3.89 × 10 −6 for 3 days, P = 1.91 × 10 −8 for 6 days. G . Western blots for c-PARP expression in CHP-134 treated with indicated compounds for 3 days (2 µM ATRA and 0.5 µM K02288). H Quantified flow cytometry results showing apoptosis in CHP-134 treated as in (G) for 3 days (left) and 6 days (right). For panel B and H, data represent the mean ± SD of 3 independent replicates. For panels C and G, GAPDH and β-Actin were used as loading control, the representative results of three independent experiments are shown. Two-sided t-test was used for panels B, F, and H. Source data are provided as a Source Data file.

Article Snippet: The Human Brunello CRISPR KO library was a gift from David Root and John Doench (Addgene #73179) .

Techniques: Flow Cytometry, Staining, Western Blot, Expressing, CRISPR, Control

Journal: Nature Communications

Article Title: Bone morphogenetic protein (BMP) signaling determines neuroblastoma cell fate and sensitivity to retinoic acid

doi: 10.1038/s41467-025-57185-y

Figure Lengend Snippet: A Pearson correlations of the expression of all genes with ATRA sensitivity in the cell lines we rescreened for 6 days. Gene expression data were from GDSC (left, P = 4.2 × 10 −5 for SMAD9 ) and Depmap (right, P = 4.8 × 10 −4 for SMAD9 ). P -values are from two-sided Pearson’s correlation tests. B Scatter plot of SMAD9 RNA expression from GDSC (left) and Depmap (right) plotted against ATRA 6-day IC 50 values. P -values were calculated with two-sided Pearson correlation tests. The shaded area represents the Pearson correlation standard error. C Scatter plot of median RRA score of the 655 druggable genes from targeted CRISPR screens in 10 neuroblastoma cell lines. D Western blots showing BMP signaling activity (level of phosphorylated SMAD1/5/9) in neuroblastoma cell lines after FK506 and rapamycin treatment. Cells were treated for 4 h. GAPDH and β-Actin were used as a loading control, and the representative results of three independent experiments are shown. E Heatmaps of percent inhibition of cell viability in CHP-134 cells (left) conferred by drug-only treatments (values within dotted lines) and combination treatments (all other values) with ATRA and FK506. Ten-point doses were used at indicated concentrations with 1:3-fold dilutions for each drug. Each matrix represents the average of three independent experiments. Data were normalized as a function of percent cell viability using inter-plate controls. ATRA and rapamycin combination in the TGW cell line was shown on the right side. F Heatmap matrices of synergy scores derived from respective cell death values in (E). Synergy scores “ZIP δ” were calculated based on the zero interaction potency (ZIP) model, which can be interpreted similarly to BLISS synergy. G Heatmap showing the maximum ZIP δ synergy scores (color scale) of ATRA and FK506 combinations or ATRA and rapamycin combinations in 16 cell lines. Neuroblastoma cell lines are highlighted in red. H Correlation of max ZIP synergy scores of ATRA + FK506 (x-axis) and ATRA + rapamycin (y-axis) screens in the cell lines shown in ( G ). The P -value was calculated with two-sided Pearson correlation tests. The shaded area represents the Pearson correlation standard error. I Bar plot confirming synergy between ATRA and FK506 in CHP-134. Cells were treated for 6 days. Cell viability was measured with MTS assay and normalized to DMSO-treated samples, which were defined as 0% inhibition of cell growth. Data represent the mean ± SD of 4 independent replicates. Two-sided t-test for ATRA vs ATRA + 1 µM FK506, P = 1.29 × 10 −7 . J Like (I) but in the TGW cell line. Two-sided t-test for ATRA vs ATRA + 5 µM FK506, P = 4.71 × 10 −6 . K Barplot showing synergy between ATRA and FK506 in CHP-134 upon ACVR1 knockdown. Cells were transduced with lentiviral control shRNA or 3 independent ACVR1 shRNAs. Cells were treated with 0.05 µM ATRA and 0.1 µM FK506 for 6 days. Inhibition of cell growth was calculated as in ( I ). Data represent the mean ± SD of 4 independent replicates. In ATRA + FK506 group, two-sided test for control vs 0498, P = 3.22 × 10 −15 ; control vs 2778, P = 4.7 × 10 −14 ; control vs 3321, P = 1.24 × 10 −16 . Source data are provided as a Source Data file.

Article Snippet: The Human Brunello CRISPR KO library was a gift from David Root and John Doench (Addgene #73179) .

Techniques: Expressing, Gene Expression, RNA Expression, CRISPR, Western Blot, Activity Assay, Control, Inhibition, Derivative Assay, MTS Assay, Knockdown, Transduction, shRNA

Journal: Nature Communications

Article Title: Bone morphogenetic protein (BMP) signaling determines neuroblastoma cell fate and sensitivity to retinoic acid

doi: 10.1038/s41467-025-57185-y

Figure Lengend Snippet: A Immunofluorescent staining for CHP-134 cells treated with indicated compounds (2 µM ATRA, 0.5 µM K02288) for 6 days. MAP2 and NEFH expression and neurite structures are shown in red. Nuclei were stained with DAPI (blue). Representative results of 3 independent replicates are shown. Scale bar = 50 µm. B MAP2 expression in CHP-134 and NB13 cells treated for 3 days (2 µM ATRA, 0.5 µM K02288). Four isoforms of MAP2 (labeled a/b and c/d) are shown. C c-PARP expression in NB13 treated for 3 days (2 µM ATRA and 0.1 µM K02288 for NB13). D Quantified flow cytometry results showing apoptosis in NB13 treated for 6 days (2 µM ATRA, 0.1 µM K02288). E MAP2 and c-PARP expression in CHP-134 treated for 6 days (2 µM ATRA, 3 µg/ml noggin (NOG)). All samples were derived from the same experiment and all antibodies were incubated with the same membrane. F .Quantified flow cytometry results showing apoptosis in CHP-134 treated the same as in ( E ). G MAP2 and c-PARP expression in CHP-134 with SMAD9 CRISPR knockout ( SMAD9 KO). Cells were treated with DMSO or 2 µM ATRA (6 days for MAP2 and 3 days for c-PARP). The samples for MAP2 proteins and c-PARP are from 6-day and 3-day treated cells, respectively. The 6-day treated samples were ran in one gel and the 3-day treated samples were ran in another gel. Control, negative control gRNA with no target in the human genome; All, a mixture of 3 independent gRNAs targeting SMAD9 ; 2972, a single gRNA targeting SMAD9 . H c-PARP expression in CHP-134 and NB13 treated for 3 days (2 µM ATRA and 0.1 µM FK506 for CHP-134, 0.05 µM ATRA and 5 µM FK506 for NB13). I Quantified flow cytometry results showing apoptosis in CHP-134 treated for 3 days (left) and 6 days (right). FK, FK506. J Quantified flow cytometry results showing apoptosis in NB13 treated for 6 days. FK, FK506. For panels B , C , E , G , and H , β-Actin and GAPDH were used as loading control. Representative results of 3 independent replicates are shown. For panels D , F , I , and J , data represent the mean ± SD of 3 biologically independent replicates. P -values were calculated with two-tailed t-tests. Source data are provided as a Source Data file.

Article Snippet: The Human Brunello CRISPR KO library was a gift from David Root and John Doench (Addgene #73179) .

Techniques: Staining, Expressing, Labeling, Flow Cytometry, Derivative Assay, Incubation, Membrane, CRISPR, Knock-Out, Control, Negative Control, Two Tailed Test

Journal: Nature Communications

Article Title: Bone morphogenetic protein (BMP) signaling determines neuroblastoma cell fate and sensitivity to retinoic acid

doi: 10.1038/s41467-025-57185-y

Figure Lengend Snippet: A Immunofluorescent staining for TGW cells treated for 5 days (0.2 µM ATRA, 0.5 µM K02288). MAP2 and NEFH expression and neurite structures are shown in red. Nuclei were stained with DAPI (blue). Representative results of 3 biologically independent replicates are shown. Scale bar = 50 µm. B MAP2 expression in TGW and SK-N-SH cells treated for 6 days (0.02 µM ATRA and 0.5 µM K02288 for TGW, 5 µM ATRA and 0.5 µM K02288 for SK-N-SH). Four isoforms of MAP2 are shown. C Viability of TGW cells treated with ATRA for 6 days in the presence of DMSO or K02288. Cell viability was measured with MTS. Data represent the mean ± SD of 4 independent replicates. At the arrow indicated point, P = 7.51 × 10 −5 for 0.05 µM K02288, P = 3.99 × 10 −6 for 0.2 µM K02288. D Senescent cells form blue crystals in TGW and SK-N-SH cells treated with 0.2 µM and 2 µM ATRA, respectively. Scale bar = 50 µm. E Senescence levels quantified by intensity of fluorescence. TGW and SK-N-SH cells were treated for 0 (DMSO), 3, 6, and 9 days with 0.2 µM and 2 µM ATRA, respectively. Cell lysates containing 10, 20, and 40 µg protein from each time point were collected for β-gal activity analysis. P values for the arrow indicated samples are 2.11 × 10 −5 , 1.18 × 10 −4 , 4.38 × 10 −4 , 6.03 × 10 −5 (from left to right). F Senescence in TGW and SK-N-SH treated for 6 days (0.2 µM ATRA and 0.5 µM K02288 for TGW, 5 µM ATRA and 0.5 µM K02288 for SK-N-SH). Scale bar = 100 µm. G Senescence levels quantified by intensity of fluorescence in TGW and SK-N-SH cell lines treated the same as in ( F ). P values for ATRA vs ATRA + K02288 are 1.32 × 10 −4 for TGW, 6.08 × 10 −5 for SK-N-SH. H MAP2 expression in TGW with SMAD9 CRISPR knockout ( SMAD9 KO). Cells were treated with DMSO or 0.2 µM ATRA for 6 days. Control, negative control gRNA with no target in the human genome; All, a mixture of 3 independent gRNAs targeting SMAD9; 2972, a single gRNA targeting SMAD9 . I Senescence in TGW with SMAD9 KO. Cells were treated with 0.2 µM ATRA for 6 days. Scale bar = 50 µm. J Senescence levels quantified by intensity of fluorescence in TGW with SMAD9 KO. Cells were treated with 0.2 µM ATRA for 6 days. 20 µg protein from each sample was used. P values are 1.46 × 10 −3 for KO-All, 4.75 × 10 −3 for KO-2972. K Senescence in TGW and SK-N-SH treated for 6 days (0.05 µM ATRA and 1 µM FK506 for TGW, 0.5 µM ATRA and 1 µM FK506 for SK-N-SH). Scale bar = 100 µm. L Senescence levels quantified by intensity of fluorescence in TGW and SK-N-SH cell lines treated the same as in (K). P values for ATRA vs ATRA + K02288 are 0.019 for TGW, 2.24 × 10 −3 for SK-N-SH. For panel B and H , β-Actin and GAPDH were used as loading control. Representative results of 3 independent replicates are shown. For panels D , F , I , and K , the representative results of 3 independent replicates are shown. For panels E , G , J , and L , data represent the mean ± SD of 3 independent replicates. P -values were calculated with two-tailed t-tests. Source data are provided as a Source Data file.

Article Snippet: The Human Brunello CRISPR KO library was a gift from David Root and John Doench (Addgene #73179) .

Techniques: Staining, Expressing, Fluorescence, Activity Assay, CRISPR, Knock-Out, Control, Negative Control, Two Tailed Test