Atto 594

Two markers, that is, osteopontin (OPN) and osteocalcin (OCL), were visualized with specific antibodies, that is, rabbit anti-osteopontin IgG and mouse anti-osteocalcin purchased from Life Technologies (Poland). Immunofluorescent staining was performed according to the general procedures provided by the manufacturer. Osteogenic cultures were incubated with primary antibodies for 1 hour at 37°C (mouse anti-rat osteocalcin, dilution 1 : 100; rabbit anti-rat osteopontin, dilution 1 : 100; Sigma). Secondary antibodies were labeled with atto-488 (goat anti-rabbit IgG, dilution 1 : 400; Sigma) and with atto-594 (goat anti-mouse IgG, dilution 1 : 400; Sigma). Samples were incubated with secondary antibodies at 37°C for 1 hour in dark. Appropriate negative controls were performed by incubating the specimens with only secondary antibodies, for excluding the nonspecific reaction. Samples were also counterstained with 4′,6-diamidino-2-phenylindole (DAPI) to visualize nuclei. Specimens were analyzed with inverted fluorescence microscope (Axio Observer A1, Carl Zeiss, Jena, Germany). Images were captured with Cannon PowerShot camera and merged using AxioVision 4.8 software (Carl Zeiss, Jena, Germany).

Normal human lung fibroblasts (NHLF) were transduced with lentiviral particles coding for ARF1 WT, ARF1 R99C or empty vector. 48 h later, the samples were washed with PBS and fixed in 4% paraformaldehyde solution (PFA) for 20 min at RT. Next, the cells were permeabilized and unspecific binding was blocked by incubation with blocking solution (3% (w/v) BSA and 0.3% (v/v) Triton X-100 in PBS) for 2 h at RT. The samples were incubated overnight at 4 °C with 1 μg/ml of the primary antibodies rabbit anti-GM130 (Cell Signaling, 12480) and mouse anti-STING (Novus Biologicals, AF6516) dissolved in diluted blocking solution (0.3% (w/v) BSA and 0.03% (v/v) Triton X-100 in PBS). After three washing steps with PBS, the samples were incubated with 1 μg/ml secondary goat anti-mouse antibody conjugated with Atto647N (Sigma-Aldrich, 50185), 1 µg/ml goat anti-rabbit antibody conjugated with Atto594 (Sigma-Aldrich, 77671) and anti-rat antibody conjugated with Alexa Fluor Plus 405 (Thermo Fisher Scientific, A48268, transfection control) dissolved in diluted blocking solution (0.3% (w/v) BSA and 0.03% (v/v) Triton X-100 in PBS) for 1 h at RT. Unbound antibodies were removed in three washing steps with PBS. For imaging, samples were kept in 97% 2,2′-thiodiethanol (TDE, Sigma Aldrich, 166782) solution in PBS, pH 7.5.