Millicell slide

2 × 10⁴ HB2 and HB2 FGFR2(-) cells per well of an 8-well Millicell slide (Millipore) were seeded in 400 µl of complete culture medium and incubated for 24 h at 37ºC. Next day, after fixation with 4% PFA for 10 min at RT, permeabilisation with 0.1% Triton X-100 at 4ºC and blocking with blocking buffer (3% BSA, 3% FBS in PBS) for 1 h at RT, cells were incubated with the desired concentrations of primary antibodies diluted in blocking buffer, overnight at 4ºC. Secondary antibodies conjugated with DyLight™ 488 or DyLight™ 594 (from Jackson ImmunoResearch) together with a counterstain for the nucleus with Hoechst were used for the visualisation of desired target proteins using scanning confocal microscope Leica HCS LSI (Leica).

In the immunofluorescence assays, the cells were cultured overnight in the Millicell slide (Millipore) and then exposed to different concentrations of thallium for 24 h. Cells were fixed in methanol for 20 min and acetone for 1 min. After blocking in 5% bovine serum albumin for 30 min, the cells were incubated with primary antibody diluted in a blocking solution for overnight. After washing twice with phosphate buffer saline (PBS) with Tween 20, the fluorophore-conjugated secondary antibody was added and incubated for 1 h. After washing, the samples were mounted with a mounting solution. RpL23a-GFP was amplified with forward (ATGAGATCTATGGCGCCGAAAGCGAAG) and reverse primers (GCAGAATTCTTAGATGATCCCAATTTTGTTG) and constructed in pEGFP-C1. The RpL23a-GFP was transfected with TransIT-X2 (Mirus). To detect the reactive oxygen species (ROS), the cells were stained with 2ʹ,7ʹ-dichlorofluorescin diacetate (DCFDA) (Sigma) or MitoSOX^TM (Thermo). Fluorescence was visualized on a microscope (AxioScope A1; Zeiss) fitted with a digital microscopy camera (AxioCam MRm Rev. 3), which was controlled with an AxioVision LE module Fluorescence Lite (Zeiss). The images were prepared using Photoshop (version CS3; Adobe).