p53 crispr cas9 single knockout cells sko  (Addgene inc)

Journal: Molecular and Cellular Biology

Article Title: A Genome Wide CRISPR Screen Reveals That HOXA9 Promotes Enzalutamide Resistance in Prostate Cancer

doi: 10.1080/10985549.2024.2401465

Figure Lengend Snippet: Genome-wide CRISPR knock out screen identifies stemness and epigenetic related genes as important for cell viability under enzalutamide treatment. (A) Schematic of genome-wide CRISPR knockout screen workflow. LNCaP cells were transduced with a pooled lentivirus sgRNA library targeting over 18,000 protein coding genes. Infected cells were divided into three treatment and three control replicates and treated with enzalutamide or DMSO for approximately 2 months. (B) Genomic DNA was extracted from cells from initial (T 0 ) and final timepoints (T f ), sgRNA coding sequences were PCR amplified and sequenced. MAGEK was used to compare sgRNA sequence abundance across treatments and timepoints. (C) Volcano plot of sgRNA abundance scored for each gene in enzalutamide treated cells relative to DMSO at T f . Genes diminished in this analysis are labeled as “sensitive” and illustrated with gray spots, while genes enriched in this comparison are labeled as “resistant” in purple. Select genes or controls are annotated. (D) Volcano plot of sgRNA abundance scored for each gene at the T f and T 0 timepoints in DMSO vehicle treated cells. (E) Venn diagram comparing overlap and number of genes positively enriched in the resistant and proliferative categories. All genes considered have a log 2 fold change > 1, P value < 0.05. Representative genes from each set are labeled. (F) Venn diagram of the number of negatively enriched genes from the screen (log 2 fold change < −1, P value < 0.05). Representative genes from each set are shown. (G) Venn diagram with number of GO processes found within each negatively enriched gene set annotated. Number and percentage of GO terms related to stemness, differentiation or embryos and histones or epigenetics are highlighted within the enzalutamide sensitive and essential gene regions.

Article Snippet: To generate RB and p53 CRISPR-Cas9 single knockout cells (SKO), RB1 and TP53 sgRNA sequences highlighted in Supplementary Table 6 were cloned in a LentiCRISPRv2 (Addgene # 52961) backbone.

Techniques: Genome Wide, CRISPR, Knock-Out, Transduction, Infection, Control, Amplification, Sequencing, Labeling, Comparison

Journal: Molecular and Cellular Biology

Article Title: A Genome Wide CRISPR Screen Reveals That HOXA9 Promotes Enzalutamide Resistance in Prostate Cancer

doi: 10.1080/10985549.2024.2401465

Figure Lengend Snippet: RB-p53 DKO cells are resistant to enzalutamide (EZ) and upregulate stemness genes, such as HOXA9. (A) Upper: representative Western blot highlighting CRISPR-Cas9 induced knockout of RB in RB-p53 DKO pools. Cell lysates from LNCaP WT and RB-p53 DKO pools probed for RB. Lower: representative Western blot highlighting CRISPR-Cas9 induced knockout of p53 in RB-p53 DKO pools. Cell lysates from LNCaP WT and RB-p53 DKO pools treated with etoposide or vehicle and probed for p53. (B) Alamar blue cell viability assay for LNCaP WT and DKO pools treated with various concentrations of enzalutamide for 6 days. Representative nonlinear regression line for each cell type is shown and generated by taking the mean viability values of each concentration from biological replicates. IC 50 values were obtained by taking the mean best-fit IC 50 value of biological replicates for each cell type and compared using one-way ANOVA. N = 5 or 6 biological replicates. (C) Representative images from colony forming assay of LNCaP WT and DKO pools. White arrows point to representative colonies stained with crystal violet. (D) Analysis of colony forming assay from C. Number of colonies were counted using ImageJ software (N = 3). (E) Volcano plot of genes differentially expressed in RNA-seq analysis of DKO cells relative to LNCaP WT (N = 4 for each). Relative expression of negative controls RB1 and TP53 are labelled and identified in black. Relative expression of HOXA9 is labelled and identified in red. (F) RNA-seq expression heat map of representative neuroendocrine (NE), stemness (Stem), epithelial-to-mesenchymal transition (EMT) and androgen receptor signature (AR Sig.) genes significantly and differentially expressed in DKO cells relative to LNCaP WT (N = 4 replicates). (G) Comparative mRNA expression of labelled NE genes and HOXA9 in LNCaP WT, RB and p53 SKO and DKO clone C1 measured by RT-PCR. Bars show ΔΔCq values (mean ± SEM, N = 3 replicates). One-way ANOVA. (H) Venn diagram comparing overlap of genes significantly upregulated in DKO cells (log 2 fold change > 1, P < 0.05) and negatively enriched in the CRISPR screen at the final timepoint (log 2 fold change < −1, P < 0.05). Where applicable: **** P < 0.0001, *** P < 0.001, ** P < 0.01, * P < 0.05, ND, not detected; ns, not significant.

Article Snippet: To generate RB and p53 CRISPR-Cas9 single knockout cells (SKO), RB1 and TP53 sgRNA sequences highlighted in Supplementary Table 6 were cloned in a LentiCRISPRv2 (Addgene # 52961) backbone.

Techniques: Western Blot, CRISPR, Knock-Out, Viability Assay, Generated, Concentration Assay, Staining, Software, RNA Sequencing, Expressing, Reverse Transcription Polymerase Chain Reaction

Journal: Molecular and Cellular Biology

Article Title: A Genome Wide CRISPR Screen Reveals That HOXA9 Promotes Enzalutamide Resistance in Prostate Cancer

doi: 10.1080/10985549.2024.2401465

Figure Lengend Snippet: RB-p53 DKO cells are more sensitive to the HOXA9 inhibitor DB818 and show synergy in combination with enzalutamide. (A) mRNA expression of HOXA9 target gene FLT3 in LNCaP WT and RB-p53 DKO-C1 cells measured by RT-PCR. Bars show ΔΔCq values (mean ± SEM, n = 3 replicates). Unpaired Welch’s t test. (B and C) mRNA expression of FLT3 in LNCaP (B) and DKO-C1 (C) cells treated with various concentrations of the HOXA9 inhibitor DB818. (D) Western blot of cell lysates from LNCaP WT and DKO-C1 treated as in A–C and probed for FLT3. (E) Alamar blue cell viability assay for LNCaP and DKO-C1 cells treated with various concentrations of DB818 for 6 days. Representative nonlinear regression lines and IC 50 values obtained using method described above. Unpaired Welch’s t test. N = 6 biological replicates. (F and G) Synergy maps for LNCaP (F) and DKO-C1 (G) cells treated with combinations of DB818 and EZ. Synergistic (red) and antagonistic (green) dose regions are highlighted. The most synergistic areas (MSA) for each cell type is shown as a dashed box on map. Calculated ZIP synergy scores for each cell type are also shown. (H and I ) Quantification of viability values from F and G when treated with single drug (EZ, enzalutamide or DB, DB818) or combination of both drugs at indicated doses in LNCaP (H) or DKO C1 (I) cells. N = 6 biological replicates. One-way ANOVA. Where applicable: **** P < 0.0001, *** P < 0.001, ** P < 0.01, * P < 0.05, ns, not significant.

Article Snippet: To generate RB and p53 CRISPR-Cas9 single knockout cells (SKO), RB1 and TP53 sgRNA sequences highlighted in Supplementary Table 6 were cloned in a LentiCRISPRv2 (Addgene # 52961) backbone.

Techniques: Expressing, Reverse Transcription Polymerase Chain Reaction, Western Blot, Viability Assay