Journal: Cell reports
Article Title: DNA damage response deficiency enhances neuroblastoma progression and sensitivity to combination PARP and ATR inhibition.
doi: 10.1016/j.celrep.2025.115537
Figure Lengend Snippet: Figure 6. The ATR inhibitor ceralasertib enhances olaparib-mediated inhibition of DDR-deficient NB cells (A) Cell-viability analysis in human NB cell lines following siRNA-mediated gene knockdown of DDR genes and treatment with olaparib (0.05–800 mM) + cera- lasertib (500 nM). Error bars represent mean ± standard deviation. p values represent comparisons of non-linear fits between siDDR and siControl (siCTRL) cells treated with olaparib + ceralasertib. Comparisons of best-fit values for all treatment groups are shown in Table S8. (B) Western blot analysis of phosphorylated CHK1 (p-CHK1, Ser345), phosphorylated H2A.X (Ser345, y-H2AX), and cleaved PARP in cells following control (siCTRL), PALB2 (siPALB2), and BRCA2 (siBRCA2) knockdown. Vinculin is used as loading control. Relative protein levels are quantified below each lane and normalized to vinculin followed by vehicle control drug-treated cells within each siRNA treatment. (C) casper;prkdc/ zebrafish engrafted with EGFP;MYCN;tp53/;brca2+/ NB following DMSO vehicle control and olaparib (50 mg/kg, 4 consecutive days/ week) + ceralasertib (40 mg/kg, 4 consecutive days/week) treatments. (legend continued on next page)
Article Snippet: REAGENT or RESOURCE SOURCE IDENTIFIER Antibodies PCNA (D3H8P) Cell Signaling RRID: 13110 Tyrosine Hydroxylase (TH) Pel-Freez Biologicals RRID: P40101-150 EGFP Abcam RRID: ab6673 yH2AX phosphoSer139 GeneTex RRID: GTX127342 Alexa Fluor 555 donkey anti-goat IgG Thermo Scientific RRID: A21432 Cleaved-caspase 3 (Asp175, 5A1E) Cell Signaling RRID: 9664 phospho-Rb Ser807/811 Cell Signaling RRID: 9308 Rb GeneTex RRID: GTX54191 Rb (4H1) Cell Signaling RRID: 9309 phospho-Chk1 Ser345 (133D3) Cell Signaling RRID: 2348 ATM (D2E2) Cell Signaling RRID: 2873 BARD1 (E 11) Santa Cruz RRID: sc-74559 BRCA1 (ABX9F) Cell Signaling RRID: 14823 BRCA2 (D9S6V) Cell Signaling RRID: 10741 PALB2 (E9R2W) Cell Signaling RRID: 30253 cleaved-PARP (D214) Cell Signaling RRID: 9541 Vinculin (V284) Millipore RRID: 05–386 Actin Cell Signaling RRID: 4968 Chemicals, peptides, and recombinant proteins Olaparib AbMole RRID: AZD2281 Olaparib MedChemExpress RRID: HY-10162 Temozolomide (TMZ) AbMole RRID: M2129 Palbociclib Selleckchem RRID: S1579 Ceralasertib Selleckchem RRID: S7693 Tetracycline Sigma RRID: T3258 recombinant Cas9 protein PNA Bio RRID: CP01 Critical commercial assays Signal Stain Boost IHC Detection Reagent Cell Signaling RRID: 8114 EnGen sgRNA Synthesis Kit NEB RRID: E3322 Ultra II Directional mRNA prep kit for Illumina NEB RRID: E7760 Ultra II DNA Library Prep Kit for Illumina NEB RRID: E7645 Deposited data RNA sequencing Gene Expression Omnibus GEO: GSE283976 WGS sequencing Sequence Read Archive (NCBI) SRA: PRJNA1214744 Experimental models: Cell lines IMR-32 American Type Culture Collection (ATCC) RRID: CCL-127 SK-N-AS American Type Culture Collection (ATCC) RRID: CRL-2137 Kelly gift from Dr. Patrick Reynolds (Texas Tech University Health Sciences Center) N/A SH-EP gift from Dr. Patrick Reynolds (Texas Tech University Health Sciences Center) N/A (Continued on next page) 18 Cell Reports 44, 115537, April 22, 2025
Techniques: Inhibition, Knockdown, Standard Deviation, Western Blot, Control
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