Propidium iodide

The cell cycle of the melanoma cell lines MEL-JUSO and SK-MEL-28 treated with siLMNB1 and siLBR (72 h) was analysed by flow cytometry. The cells were transfected for 72 h with siLMNB1 or siLBR and siCtrl, respectively. For each treatment, 200,000 cells were sown, harvested and fixed in 70% ice-cold methanol for at least 1 h. Then, the cells were washed twice with 0.2% BSA/PBS. After centrifugation (4000 rpm, 4 min), the cells were resuspended in 482.5 µL of 0.2% BSA/PBS, followed by addition of 10 µg/µL Rnase to each tube and incubation for 20 min at 37 °C. Just before the measurement, 12.5 µL of propidium iodide (1 mg/mL PromoCell, Heidelberg, Germany) were added to each tube, gently mixed, and incubated for 30 min. propidium iodide is a fluorescent dye which intercalates into double-stranded nucleic acid, and therefore the DNA content could be measured. Analysis was carried out using a BD LSRFortessa^™ X20 flow cytometer, and flow cytometry data were analysed using the BD FACSDIVA software (version 8.0, BD Bioscience, San Jose, CA, USA).

CPI-613 and hydroxychloroquine were purchased from AdooQ BioScience (Irvine, CA, USA) and FUJIFILM Wako Pure Chemical Corporation (Osaka, Japan), respectively.
The number of cells undergoing apoptosis was quantified using FITC-conjugated annexin V and propidium iodide (PI; PromoCell GmbH, Heidelberg, Germany). Briefly, 5 × 10⁴ cells were harvested under different culture conditions, washed, resuspended in binding buffer, mixed with annexin V-FITC and PI, and analyzed as previously described 17 (link). In several experiments, we examined the apoptotic status of non-treated cells.

NVP-AEW541 and NVP-BEZ235 were gifts from Novartis (Basel, Switzerland). Lapatinib, KU0063794 and LY294002 were from Cayman Chemical (Michigan, USA). Cisplatin was purchased from Sigma-Aldrich (Steinheim, Germany). 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) was purchased from Serva (Heidelberg, Germany). Propidium iodide was from PromoCell (Heidelberg, Germany). Roswell Park Memorial Institute (RPMI) media 1640, Dulbecco’s Modified Eagle Medium (DMEM), fetal bovine serum (FBS), penicillin/streptomycin [10,000 U/ml; 10 mg/ml] and trypsin-EDTA (0.05% trypsin, 0.02% EDTA in PBS) were purchased from PAN Biotech (Aidenbach, Germany). Primary antibodies were purchased from R&D Systems (Wiesbaden, Germany) (pIGF1R, IGF1R, p-EGFR, EGFR, p-ErbB2, ErbB2, p-ErbB3, ErbB3) or Santa Cruz Biotechnology (Heidelberg, Germany) (p-Akt, Akt, β-Actin, PARP). HRP-conjugated secondary antibodies were from R&D Systems. All other reagents and chemicals were from VWR BDH PROLABO (Darmstadt, Germany).