Trypsin

The study was approved by the ethical committee of Motol University Hospital, Prague, Czech Republic. All surgical tissue samples were obtained from HNSCC patients after they signed the informed consent documents. Patients were completely clinically examined, and tumour samples were taken from verified HNSCC under general anaesthesia; the inclusion criteria were as follows: histologically confirmed squamous cell carcinoma and no previous oncologic treatment; each tissue specimen underwent pathology quality control. Hematoxylin and eosin-stained sections from each sample were subjected to independent pathology review to confirm squamous cell carcinoma histological consistence. Tumour samples with approximately >60% tumour nuclei and <20% necrosis were considered sufficient for the following analyses. The tissue material harvested during surgery was divided into two pieces, the first was stored in an RNAlater (Ambion, Austin, TX, USA) for later RNA analyses. The second portion of the sample was placed into the cultivation medium (MEM) with the addition of 1% trypsin and 1% antibiotic-antimycotic solution (Santa Cruz Biotechnology, Dallas, TX, USA). The material was maintained at cold temperatures for 16–24 h to enable trypsin diffusion. Primary cell lines were subsequently prepared.

MNNG/HOS cells were inoculated in 6-well plates with 1×10⁶ /L cells per well and incubated for 24h, then different kinds of management of the respective plasmids was executed. 24h later, the cells were harvested and washed twice by PBS, digested by trypsin (Santa Cruz, USA) and dissociated into single cell suspension. After that the cells were fixed by 70% acetic acid ethanol for 1h at 4°C, re-suspended in 100 ul propidium iodide (PI; Sigma, USA) solution and incubated for 30 min in the dark. FACS Calibur (BD, USA) was used to analyze the cell cycle.