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Chen et al.
Supplementary MaterialsSupplementary Materials
Supplementary Table S1: Ibrutinib sensitivity screening with NCI-60 panel
Cell line Time zero Control −8.0 −7.0 −6.0 −5.0 −4.0 GI50 TGI LC50
Supplementary Figure S1. Response of MDA-MB-361 cells to multiple inhibitors. MD-MB-361 cells were plated in 96-well plates overnight before treatment with inhibitors for 72 hours. CellTiter-Glo® was used for quantifying cell growth. The assay was repeated 3 times, and the error bars represent SD.
Supplementary Figure S2. Response of BT-474 (A) and SK-BR-3 (B) cells to treatment with different ErbB kinase inhibitors. The cells were treated with inhibitors continuously for 72 hours, and alamarBlue® assay was used to quantify cell growth.
Supplementary Figure S3. Response of cell signaling pathways to treatment with ibrutinib in multiple cell lines. Cells were treated for 1 hour with ibrutinib. 50,000 cells were loaded in each lane.
Supplementary Figure S4. Ibrutinib inhibited the growth of BT-474 xenograft tumors and related signaling pathways in the tumor samples. (A) Inhibition of BT-474 xenograft growth in NOD-SCID mice by ibrutinib administered at once -daily doses of 3 mg/kg (▲) or 16 mg/kg (▼) compared to vehicle control (▀). Significance for differences from control is indicated by *(P<0.05) or **(P<0.01). (B) Comparison of pharmacokinetic parameters among different strains of mice dosed at 16 mg/kg and 48 mg/kg, PO. (C) Selected images for pERK staining from the BT-474 tumors dosed at 16 and 48 mg/kg, PO, and the data analysis. *P<0.05 and **P<0.01. Error bars represent standard deviation (SD). (D) Relative phosphorylation of AKT (pAKT/AKT) for harvested BT-474 tumors as for (C) at different timepoint. The density of each band was normalized to the first band of vehicle. The results for 48 mg/kg were graphed. Error bars represent SD.
Supplementary Figure S5. Ibrutinib irreversibly inhibited SK-BR-3 cells, and bound covalently to EGFR and HER4 in rapid dilution assays. (A) SK-BR-3 cells were treated with ibrutinib at 0.1 and 0.5 µM for varied times before washing twice with fresh media or media with drug, as for no-washout group. Cells were cultured for another 2 hours after washout before harvesting. (B) The growth of SK-BR-3 cells was still inhibited 6 days after original treatment with ibrutinib for 1 hour using wash-out step. Cells were plated in 10 cm plate in triplicate, and washout was repeated twice. The cells were counted with Coulter counter. *P<0.05, compared with control. Potency of irreversible binding to EGFR (C) and HER4 (D) by ibrutinib, afatinib, and neratinib with rapid dilution assays. The value of kobs quantifies the potency of covalent bond formation. BTK was assayed as a comparison (C). The labeled time indicates pre-incubation time for test compound and enzyme.