Ve 822 s7102

Tumor tissue samples with their matched normal tissue were obtained from the International DIPG/DMG Registry as described previously [34 (link)]. Briefly, patients with DIPG were informed and consented to the IRB-approved protocol (Study ID: 2016-3357) at Cincinnati Children’s Hospital Medical Center. Diagnosis of DIPG was based on clinical symptoms and imaging characteristics on pretreatment magnetic resonance imaging (MRI). Primary normal human foreskin fibroblasts (HFF) strains (ATCC CRL-2091), human cervical carcinoma cell lines (HeLa), and the human osteosarcoma cell lines (Saos-2) (ATCC HTB-85) were purchased from ATCC. HFF and HeLa cells were cultured in DMEM (Gibco) supplemented with 10% FBS, while Saos-2 cells were cultured in McCoy’s 5A medium supplemented with 15% FBS. Pediatric GBM cell lines, CCHMC-DIPG-1, R0315-GBM, SJ-HGGX42 and HSJD-GBM002, were cultured in neurosphere stem cell media as described elsewhere [35 (link),36 (link)]. KNS42 cells were cultured in DMEM (Gibco) supplemented with 10% FBS. KNS42 cells were obtained from the JCRB (Japan Cancer Research Resources) cell bank. HSJD-GBM002 cells were generous gifts from Dr. Angel Montero Carcaboso. SJ-HGGX42 cells were obtained from PBTP (https://pbtp.stjude.cloud). ATR inhibitor VE-822 (S7102) and CHK1 inhibitor (Chir-124) (S2683) were purchased from Selleckchem.

A volume of 2 mL of complete medium containing 1000 cells was seeded in each well of a 12-well plate and incubated for about 2 weeks. For drug sensitivity colony formation assay, cells were treated with the indicated drug concentrations every 48 h. Once colonies formed in DMSO control conditions, plates were fixed with 4% paraformaldehyde, stained with crystal violet and the number of colonies was counted. Colony formation efficiency indicated by area of colonies treated relative to area of control colonies was used to measure survival between wells. RSR inhibitors and chemotherapy drugs for drug sensitivity assay were purchased from Selleck: VE-821 (S8007) targeting ATR, VE-822 (S7102) highly selective and potent derivatives of VE-821, Rabusertib (S2626) targeting Chk1, Cisplatin (S1166), Carboplatin (S1215), Dacarbazine (S1221). The quantifications presented were obtained from at least three independent biological replicates.