Sic001

Cells were transfected with 100nM (final concentration) siRNAs against human NOTCH1 (SASI_Hs01_00052328), human NOTCH3 (SASI_ Hs01_00101285) or a non-targeting siRNA as control (SIC001) (Sigma, St Louis, MO, USA) using Oligofectamine (Invitrogen, Carlsbad, CA), according to the manufacturer’s recommendations. Twenty-four hours later, the medium was replaced with fresh complete growth medium and transfected cells were harvested at different time points.

Cells were plated either at 2.75 million per 35-mm dish for RNA and protein analyses, or 2 million per well in a six-well plate for amino acid transport assays. Following 18 h of culture, PHT cells were transfected with 100 nmol/L of siRNA targeting p38 MAPK (Sigma-Aldrich, SASI_Hs01_00018467) or nontargeting Scrambled (Scr) siRNA (SIC001, Sigma-Aldrich) using Dharmafect2 transfection reagent (ThermoScientific, Waltham, MA, USA) according to the manufacturer’s protocol and as reported previously (Aye et al. 2014a (link)).

MCF7 and BT474 cells were cultured in RPMI1640 supplemented with 10% fetal bovine serum and 1% penicillin and streptomycin. SKBR3 cells were cultured in Dulbecco’s Modified Eagle Medium supplemented with 10% fetal bovine serum and 1% penicillin and streptomycin. siRNA transfections were performed using RNAiMAX (Invitrogen), and plasmid transfections were performed using Lipofectamine 3000 (Invitrogen) according to the manufacturer’s instructions. The sequence of dsDNA90 was described previously [71 (link)], transfected using Lipofectamine 2000 (Invitrogen) according to the manufacturer’s instructions. siRNA universal negative control 1 and 2 were purchased from Sigma (SIC001 and SIC002). siKDM5A targeting sequences were described previously [29 (link)]. Other siRNA targeting sequences were as follows: siKDM5B-1, CAGTGAATGAGCTCCGGCA; siKDM5B-2, GGAGCTGACATTGCCTCAA; siKDM5C-1, GGAGGAAGGTGGTTATGAA; and siKDM5C-2, GGAGGAAGGTGGTTATGAA.
Histone extraction was conducted as described previously [66 (link)].