Unc3230

Dulbecco’s modified Eagle’s medium (DMEM), minimum essential medium, EGF, LPA, verteporfin, crystal violet, poly-L-lysine, and anti-FLAG M2 Affinity agarose gel (A2220) were purchased from Sigma-Aldrich (St. Louis, MO, USA). Lipofectamine 2000, Lipofectamine RNAiMAX, Opti-MEM I, DAPI, goat serum, and Alexa Fluor-conjugated secondary antibodies were from Thermo Fisher Scientific (Waltham, MA, USA). UNC3230, AR7, and 6AN were purchased from MedChemExpress (Monmouth Junction, NJ, USA). Antibodies to α-tubulin (T5168), β-actin (A5316), vinculin (V4505), and FLAG-tag (F1804) were obtained from Sigma-Aldrich. Antibodies to HA-tag (#3724), Myc-tag (#2278), V5-tag (#13202), LATS1 (#9153), phospho-LATS1 Ser909 (#9157), YAP (#4912), phospho-YAP Ser127 (#4911), Merlin (#6995), and PIP5Kγ (#3296) were purchased from Cell Signaling Technology (Danvers, MA, USA). Antibodies against HA-tag (sc-7392), lamin B1 (sc-374015), Hsc70 (sc-7298), GFP (sc-9996), and GAPDH (sc-47724) were obtained from Santa Cruz Biotechnology (Dallas, TX, USA).

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.

1 H-indole-3-carbaldehyde (4-anilino-6-[4-morpholinyl]-1,3,5-triazin-2-yl)hydrazone (WX8) was from SPECS (MLS000543798). UNC3230 was from MedChemExpress (HY-110150). ONTARGET SMART-pools and non-targeting control siRNAs were designed by Dharmacon. Cell-Titer Glo kit was from Promega Biotechnology (G7570).