G418 disulfate salt

The MCF7 breast cancer cell line, consisting of the fluorescent protein Venus fused to p53, is the same as previously described [9 (link), 38 (link)], a gift from Galit Lahav, Harvard Medical School. Cells were maintained in 95% humidity at 37 degrees Celsius and cultured in RPMI (Invitrogen) media with 10% FBS (Invitrogen), 1% PenStrep (Invitrogen 0.045 units/mL Penicillin, 0.045 units/mL Streptomycin). After the first splitting following resurrection from liquid nitrogen, stably integrated MCF7 cells were maintained at 20 mL volume in petri dishes with 400ug/mL G 418 disulfate salt (400ng/mL, Sigma). In a 12 well plate (Griener), 80,000 MCF7 p53-Venus cells were plated on the afternoon before transfection. The following morning, the cells were treated with Neocarzinostatin (Sigma) and transfected with 3ul JetPRIME (Polyplus) mix with 25nM of the microRNA inhibitors 192, 29a and 34a (Qiagen) according to the manufacture’s protocol. To stimulate the activity of p53 we added the radiomimetic drug Neocarzinostatin (NCS), which induces the particular lesion of DNA double-strand breaks and elicits p53 to oscillate [35 (link), 38 (link)]. Following the addition of 0.8 μl NCS per well from 0.5 mg/mL stock (Sigma) and either with or without the transfection of microRNA inhibitors we commenced a time-lapse microscopy at 37 degrees Celsius with humidified 5% CO2.

pcDNA3-HA was kindly provided by N. Sonenberg [53 (link)]. Human wild-type PDCD4 was cloned in frame with a triple HA sequence into pKH3 vector to generate pKH3-HA-PDCD4. pKH3-HA-PDCD4 (S67A), pKH3-HA-PDCD4 (S76A), pKH3-HA-PDCD4 (S457A), pKH3-HA-PDCD4 (S67/457A), pKH3-HA-PDCD4 (S76/457A), and pKH3-HA-PDCD4 (S67/76/457A) plasmids were constructed by overlap extension PCR mutagenesis using plasmid pKH3-HA-PDCD4 as template.
MDA-MB-231 cells were transfected using Lipofectamine 3000 (Invitrogen, Grand Island, NY, USA) according to the manufacturer's protocol. To select PDCD4 over-expressing MDA-MB-231 cells, cells were co-transfected with PDCD4 plasmids and pcDNA3-HA vector (harboring neomycin resistance gene), and selected in media containing geneticin (500 μg/ml; G418 disulfate salt; Sigma-Aldrich).

Establishing the cell lines by lentivirus transduction was carried out by using the published procedure^{28 ,55 (link),56 (link)}. HEK293T cells at 85–90% confluency were co-transfected with 21 μg pTRIPZ (M) containing the fusion gene, 21 μg psPAX2, and 10.5 μg pMD2.G by using calcium phosphate precipitation. At the time of 12 h after transfection, the medium was replaced with 10 ml DMEM supplemented with 10% FBS, 2 mM l-glutamine, and 100 units per ml penicillin G sodium. At the time of 50 h after medium change, the medium was harvested to transduce cells in the presence of 8.0 µg per ml polybrene (H9268; Sigma-Aldrich, St Louis, MO). For co-transducing multiple genes, lentiviruses were produced separately and mixed at the time of transduction. At the time of 72 h after transduction, infected cells were selected by using 1.0–2.0 μg per ml of puromycin (P8833; Sigma-Aldrich, St Louis, MO) and/or 600–800 μg per ml G418 disulfate salt (A1720, Sigma-Aldrich, St Louis, MO). Unless otherwise indicated, for live-cell single-molecule imaging experiments, the fusions were expressed at the basal level without administrating doxycycline.