Allstar nc sirna

FAM188B expression was silenced by transfecting with FAM188B-specific siFAM188B (Qiagen, Germany) targeting the sequence 5′-CTGACCATTGACACCACCAA-3′, Allstar NC siRNA (Qiagen) at 5 nM using Lipofectamine RNAiMAX (Thermo Fisher Scientific, San Jose, CA, USA).
Cell cycle was analyzed by flow cytometry by staining with propidium iodide (Sigma-Aldrich). Nuclear fragmentation in siRNA-treated cells stained with Hoechst33342 (Sigma-Aldrich) was observed by confocal microscopy at ×400 magnification. Annexin V assays for the detection of apoptotic populations was carried out using a BD FITC annexin V apoptosis detection kit I (BD Pharmingen, San Jose, CA, USA) according to the manufacturer’s protocol.

ELF3 expression was silenced by transfecting with ELF3-specific siELF3 (Cat. No. SI04265660, Qiagen, Hilden, Germany) targeting the sequence 5′-CTGGACTGGATCAGCTACCAA-3′, FOXM1-specific siFOXM1 (Cat. No. SI04140808, Qiagen, Hilden, Germany) targeting the sequence 5’-AACATCAGAGGAGGAACCTAA-3′, (Supplementary Table S4) and Allstar NC siRNA (Cat. no. SI03650318, Qiagen, Germany) at 5 nM using Lipofectamine RNAiMAX (Cat. no. 13778150, Thermo Fisher Scientific, San Jose, CA, USA). siRNA transfections were done according to manufacturer’s instructions. For immunoblots, A549 and H1975 (2.0 × 10⁵) were seeded in 60 mm dishes, and siRNA-treated cells were harvested at 3 time points (24, 48, and 72 h) after siRNA transfection. For WST-1 assay, A549 and H1975 (3.0 × 10³) were seeded in a 96-well plate. Cells were incubated for 72 h before WST-1 assay (Cat. no. MK400, Takara, Japan). For measuring changes of cell viability, A549 and H1975 cells (3.0 × 10³) were seeded in a 96-well plate. Auranofin and gefitinib were treated for 48 h and OD was measured in 450 nm.