p53 crispr–cas9 knockout plasmid sc-419469  (Santa Cruz Biotechnology)

Journal: Frontiers in Oncology

Article Title: PALB2 deficiency may sensitize H3K27M-mutant pediatric HGG cells to BMN673/talazoparib

doi: 10.3389/fonc.2025.1589396

Figure Lengend Snippet: PALB2 deficiency increases the sensitivity of H3K27M-mutant Res259 cells to BMN673. (A) List of genes mutated in the Res259 and SF188 cell lines, respectively. (B) Left: Western blot analysis showing the expressions of PALB2 in the four indicated cell lines: WT Res259 cells (WT), WT Res259 cells with PALB2 deficiency (Wko), H3K27M-mutant Res259 cells (H3K27M) and H3K27M-mutant Res259 cells with PALB2 deficiency (Hko). Right: Dose–response curves for H3K27M-mutant Res259 cells with PALB2 deficiency and parental H3K27M-mutant Res259 cells treated with BMN673. (C) Left: Western blot analysis showing the expressions of ARID1A in the four indicated cell lines. Right: Dose–response curves for H3K27M-mutant Res259 cells with ARID1A deficiency and parental H3K27M-mutant Res259 cells treated with BMN673. (D) Left: Western blot analysis showing the expressions of P53 in the four indicated cell lines. Right: Dose–response curves for H3K27M-mutant Res259 cells with P53 deficiency and parental H3K27M-mutant Res259 cells treated with BMN673. (E) Left: Dose–response curves for H3K27M-mutant Res259 cells with PALB2 deficiency and WT Res259 cells with PALB2 deficiency treated with BMN673; Right: Dose–response curves for H3K27M-mutant Res259 cells with ARID1A deficiency and WT Res259 cells with ARID1A deficiency treated with BMN673. The data are shown as the mean ± SD from three independent experiments.

Article Snippet: The P53 CRISPR–Cas9 knockout plasmid (sc-419469, Santa Cruz Biotechnology, USA) or ARID1A CRISPR–Cas9 knockout plasmid (sc-400116, Santa Cruz Biotechnology, USA), both comprising a pool of three guide RNAs (gRNAs), and the corresponding homology-directed DNA repair (HDR) plasmid containing an HDR template with a dual selection marker (red fluorescent protein (RFP) and puromycin resistance gene) were used.

Techniques: Mutagenesis, Western Blot

Journal: iScience

Article Title: p21 , ccng1 , foxo3b , and fbxw7 contribute to p53 -dependent cell cycle arrest

doi: 10.1016/j.isci.2025.112558

Figure Lengend Snippet: puma −/− ; noxa −/− ; p21 −/− zebrafish are not predisposed to spontaneous tumors (A) Kaplan-Meier tumor-free survival of p53 −/− (blue curve, N = 96, T50 = 261 days) zebrafish compared with puma −/− ; noxa −/− ; p21 −/− (called pnp −/− , N = 43, green) and wildtype allele ( N = 96, orange). (B) Kaplan-Meier tumor-free survival of BRAF V600E ; p53 −/− (blue curve, N = 44, T50 = 271 days) zebrafish compared with BRAF V600E ; puma −/− ; noxa −/− ; p21 −/− (called BRAF V600E ; pnp −/− , N = 42, green), BRAF V600E ; p21 −/− ( N = 52, green), and wildtype allele ( N = 44, orange). Long-rank statistic test was done. ∗∗∗∗, p -value between p53 −/− and pnp −/− < 0.0001 and p -value between p53 −/− and p53 +/+ < 0.0001. ∗∗∗∗, p -value between BRAF V600E ; p53 −/− and BRAF V600E ; pnp −/− < 0.0001, p -value between p -value between BRAF V600E ; p53 −/− and BRAF V600E ; p21 −/− < 0.0001, and BRAF V600E ; p53 −/− and BRAF V600E ; p53 +/+ < 0.0001.

Article Snippet: The p53 knockout (KO) allele was obtained from Jackson Labs (Strain #002101) and maintained on a C57BL6/J genetic background (Strain #000664), which were also purchased from Jackson Labs. Mice had ad libitum access to food and water under 12:12 light/dark cycle at 21-22°C.

Techniques:

Journal: iScience

Article Title: p21 , ccng1 , foxo3b , and fbxw7 contribute to p53 -dependent cell cycle arrest

doi: 10.1016/j.isci.2025.112558

Figure Lengend Snippet: Loss of puma , noxa , and p21 provide resistance to p53-mediated induction of apoptosis and partially resistance to p53-mediated cell-cycle arrest (A) Experimental workflow showing how samples were harvested. 29-, 27- and 24-h post fertilization (hpf) wildtype, puma −/− ; noxa −/− , pnp −/− and p53 −/− zebrafish embryos were treated with 30 Gy IR-irradiation and fixed at 1-, 3-, 6-, 9- and 12-h post IR-treatment (hpi, 1hpi, 3hpi and 6hpi panels). (B) Representative images of anti-active Caspase-3 staining on 30-hpf zebrafish embryos for each group. Arrows in WT points out active apoptotic area in head region at 3 and 6 hpi. Scale bar: 500μM. (C) Representative images of phospho-histone H3 (pH3)-stained 30-hpf (1 and 3 hpi) or 36-hpf (12 hpi) zebrafish embryos for each group. Experimental design showing in A and A. Scale bar: 500μM. (D) Quantification of pH3 positive cells in treated and untreated WT, pnp −/− and p53 −/− embryos for each group. Each dot represents an individual. The average number of pH3+ cells (Mean) were indicated in each group. Bars represent mean ± SEM. ∗, p < 0.05. ∗∗∗, p < 0.001.∗∗∗∗, p < 0.0001.

Article Snippet: The p53 knockout (KO) allele was obtained from Jackson Labs (Strain #002101) and maintained on a C57BL6/J genetic background (Strain #000664), which were also purchased from Jackson Labs. Mice had ad libitum access to food and water under 12:12 light/dark cycle at 21-22°C.

Techniques: Irradiation, Staining

Journal: iScience

Article Title: p21 , ccng1 , foxo3b , and fbxw7 contribute to p53 -dependent cell cycle arrest

doi: 10.1016/j.isci.2025.112558

Figure Lengend Snippet: Loss of p21 partially rescues p53 -dependent mdm2 -null induced cell-cycle arrest (A) The conceptional diagram of mdm2 -null induced embryonic lethality. Loss of mdm2 elevates p53 protein levels to induce downstream targets and effector functions to render the lethality. (B) Representative gross images of 24-hpf mdm2 +/+ , mdm2 −/− ; puma −/− ; noxa −/− ; p21 −/− ( mpnp −/− ) and mdm2 −/− ; p53 −/− embryos. Scale bar: 500μM. (C) pH3-stained mdm2 +/+ , mdm2 −/− ; mpnp −/− embryos at 12-, 16-, 20- and 24-hpf. Scale bar: 200μM. (D) Quantification of pH3 positive cells at 12 hpf (Top panel) and at 24 hpf (bottom panel). Each dot represents an individual. Bars represent mean ± SEM. ∗∗∗, p < 0.001.∗∗∗∗, p < 0.0001. Not statistical significance between mdm2 −/− and mpnp −/− at 24 hpf.

Article Snippet: The p53 knockout (KO) allele was obtained from Jackson Labs (Strain #002101) and maintained on a C57BL6/J genetic background (Strain #000664), which were also purchased from Jackson Labs. Mice had ad libitum access to food and water under 12:12 light/dark cycle at 21-22°C.

Techniques: Staining

Journal: iScience

Article Title: p21 , ccng1 , foxo3b , and fbxw7 contribute to p53 -dependent cell cycle arrest

doi: 10.1016/j.isci.2025.112558

Figure Lengend Snippet: Defining IR induced zebrafish early responsive p53 -upregulated genes (A and B) Volcano plots showing 30-hpf zebrafish pnp −/− embryos with the treated versus untreated at 1 (A) and 3 hpi (B). The cutoff was set as fold change ≥2 or ≤ −2 and p value <0.05. Upregulated DEGs were color-labeled with magenta and the downregulated were labeled with blue. The gene symbol of some TOP DEGs was indicated on the plot. The -log 10 ( p -value) of phlda3 , foxo3b and mdm2 treated versus untreated at 3 hpi is above 300. Their log 2 (Fold change) values were pointed out (top right square). (C) Schematic of the method used to create Venn diagrams for p53 -upregulated DEGs in pnp −/− at 1 and 3 hpi (left panel). Venn graphs for the DEGs (right panel). The cut-off is fold change ≥1.5 and q < 0.05. (D) Venn graph showing 264 p53 -induced genes in pnp −/− between 1 and 3 hpi. (E) Representative plots showing well-established p53 targets, including gadd45aa , mdm2 and ccng1 , in pnp −/− but not p53 −/− datasets in response to IR treatment.

Article Snippet: The p53 knockout (KO) allele was obtained from Jackson Labs (Strain #002101) and maintained on a C57BL6/J genetic background (Strain #000664), which were also purchased from Jackson Labs. Mice had ad libitum access to food and water under 12:12 light/dark cycle at 21-22°C.

Techniques: Labeling

Journal: iScience

Article Title: p21 , ccng1 , foxo3b , and fbxw7 contribute to p53 -dependent cell cycle arrest

doi: 10.1016/j.isci.2025.112558

Figure Lengend Snippet: Defining conserved p53-upregulated genes in zebrafish and mouse (A) Venn graphs representing p53 -upregulated DEGs in mouse p53 +/+ at 1 and 3 hpi. The cut-off is fold change ≥1.5 and q < 0.05. (B) Venn graph showing 1,617 p53 -induced genes in mouse p53 +/+ between 1 and 3 hpi. (C) The diagram showing the analysis on mouse orthologs of p53-upregulated DEGs in pnp −/− zebrafish embryos at 1 and 3 hpi. For 264 p53-upregulated DEGs defined in zebrafish at 1 or 3 hpi, 247 of them are with mouse orthologs. Among them, 226 genes have one ortholog, and 21 of them are with multiple orthologs. 12 did not define orthologs in mouse. Five of them are non-coding genes. And 247 zebrafish p53-upregulated DEGs are corresponding to 323 mouse orthologs and 1,804 mouse paralogs. Among them, 232 genes are upregulated in mouse WT but not in p53 −/− treated versus untreated. Finally, defining 137 zebrafish p53 -induced DEGs are also conserved upregulated by p53 in mouse.

Article Snippet: The p53 knockout (KO) allele was obtained from Jackson Labs (Strain #002101) and maintained on a C57BL6/J genetic background (Strain #000664), which were also purchased from Jackson Labs. Mice had ad libitum access to food and water under 12:12 light/dark cycle at 21-22°C.

Techniques:

Journal: iScience

Article Title: p21 , ccng1 , foxo3b , and fbxw7 contribute to p53 -dependent cell cycle arrest

doi: 10.1016/j.isci.2025.112558

Figure Lengend Snippet: Comparing 137 conserved p53 dependent IR induced genes with DEGs in mpnp −/− datasets (A) Venn graph displaying the overlapping genes between 137 conserved UP DEGs in both zebrafish and mouse with IR-irradiation and 2,582 upregulated (UP) DEGs in mpnp −/− versus sibling controls at 18 hpf. Experimental timeline showing the timepoint that distinguish mpnp −/− embryos from sibling controls and how to harvest RNA samples at the time (Top panel). (B) Heatmap showing transcriptional changes for 2,582 DEGs over time in early mpnp −/− datasets (8, 10, 12, 14, and 16 hpf). Each timepoint was measured in duplicate, and the averages of the duplicates were used for each group. Z-scores, calculated from TPM values calculated across all samples, were used to standardize gene expression levels before clustering. The experimental workflow for sample collection is shown in the top panel. Note that mutant samples at each timepoint were diluted 4-fold by their sibling controls (¼ mpnp −/− and ¾ sibling controls). Genes with similar expression patterns over time were grouped into eight clusters, with their trends and cluster sizes shown in the right panel. (C) Line graphs showing the kinetics of 8 out of 24 GOIs in early mpnp datasets, with the dashed line indicating a fold change of 2. Expected counts calculated with RSEM were used for creating line graphs. Line graphs for the remaining 16 GOIs are shown in B.

Article Snippet: The p53 knockout (KO) allele was obtained from Jackson Labs (Strain #002101) and maintained on a C57BL6/J genetic background (Strain #000664), which were also purchased from Jackson Labs. Mice had ad libitum access to food and water under 12:12 light/dark cycle at 21-22°C.

Techniques: Irradiation, Gene Expression, Mutagenesis, Expressing

Journal: iScience

Article Title: p21 , ccng1 , foxo3b , and fbxw7 contribute to p53 -dependent cell cycle arrest

doi: 10.1016/j.isci.2025.112558

Figure Lengend Snippet: fbxw7 , foxo3b and ccng1 G0 crispants mitigate p53 -mediated cell-cycle arrest (A) Representative images showing pH3-stained un-injected (control, un-inj) and injected mpnp −/− embryos at 21 hpf. Scale bar: 250μM. (B) Quantification of pH3 positive cells in injected mpnp −/− embryos for 24 GOIs. un-inj (negative control) and the p53 guides-injected ( p53 , positive control). (C) Representative images representing pH3-stained, IR-irradiation treated or untreated, four-guide injected pnp −/− embryos at 30 hpf. Scale bar: 500μM. (D) Quantification of pH3 positive cells in injected pnp −/− embryos for fbxw7 , foxo3b and ccng1 . p21 -inj (negative control) and p53 -inj (positive control). Each dot represents an individual. Bars represent mean ± SEM. ∗, p < 0.05. ∗∗, p < 0.01.∗∗∗, p < 0.001.∗∗∗∗, p < 0.0001.

Article Snippet: The p53 knockout (KO) allele was obtained from Jackson Labs (Strain #002101) and maintained on a C57BL6/J genetic background (Strain #000664), which were also purchased from Jackson Labs. Mice had ad libitum access to food and water under 12:12 light/dark cycle at 21-22°C.

Techniques: Staining, Injection, Control, Negative Control, Positive Control, Irradiation

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