Capivasertib

To assess the impact of miR-4649-5p and PIP5K1C on cellular growth, 3 × 10³ SUM159, 5 × 10³ MDA-MB-231, and 6 × 10³ BT-20 cells were seeded per well of a 96-well plate (one plate for each time point) and transiently transfected with the miRNA mimic, PIP5K1C siRNA and their respective controls in six replicates using HiPerFect Transfection Reagent (Qiagen) according to the reverse transfection protocol of the manufacturer. To investigate potential additive effects of miR-4649-5p mimic and PIP5K1C or AKT inhibition, cells were additionally treated with 10 μM of the PIP5K1C inhibitor UNC3230 (MedChemExpress, Monmouth Junction, NJ, USA), 0.5 μM of the AKT inhibitor capivasertib (MedChemExpress) or equal volumes of DMSO as vehicle control. Cells were incubated for 24, 48, 72, and 96 h. At each time point, WST-1 proliferation reagent (Roche) was added to the wells in a 1:10 ratio according to the manufacturer’s instructions. After incubation at 37 °C for 60 or 120 min (depending on the cell line), colorimetric changes were measured using a SPECTROstar Omega (BMG Labtech, Ortenberg, Germany) at a wavelength of 450 nm with a reference wavelength of 620 nm.

The NSCLC A549 and NCI-H1299 human cells were purchased from the American General Cultural Protection Center and cultured in RPMI-1640 complete media supplemented with 10% fetal bovine serum (Gibco, MA, USA). Fibrin gels were obtained from Sea Run Holdings, Inc. (NJ, USA). Neutralizing antibodies against integrin β1 and integrin β3 were obtained from Selleck Chemicals (NJ, USA). The AKT inhibitor capivasertib was obtained from MedChemExpress (NJ, USA). Cisplatin (Cis) and paclitaxel (PTX) were purchased from Sangon Biotech (Shanghai, China).