Sm568p

Human dermal fibroblasts were subjected to γ-irradiation as described above and seeded in 6-well plates on coverslips. Alternatively, cells were transfected with miR-15b inhibitors in the absence or presence of siRNA duplexes against SIRT4 as described below. Cells were fixed four days later in 4% paraformaldehyde for 20 min and permeabilized with 0.2% Triton X-100 for 20 min followed by a blocking step with 4% BSA/0.05% saponin for 30 min at room temperature. Subsequently, cells were costained with primary antibodies against the mitochondrial marker MTCO2 (abcam, ab3298; 1:500), SIRT4 (Santa Cruz Biotechnology, Inc., sc-135053; 1:200), and α-Tubulin (Acris antibodies, SM568P, 1:500) overnight at 4°C. Secondary antibodies (Alexa Fluor 488-conjugated goat anti-mouse IgG, Alexa Fluor 543-conjugated goat anti-rabbit IgG, and Alexa Fluor 633-conjugated goat anti-rat IgG) were from Life Technologies and used at a dilution of 1:500 for 1 hr at room temperature. Analyses were performed with a LSM510-Meta confocal microscope (Zeiss) equipped with 40/1.3 or 63/1.4 immersion objectives and excitation wavelengths of 488 nm, 543 nm, and 633 nm. Cells were examined and pictures were taken at the Z-stack level (0.5 μm) and when indicated further analysed using the ImageJ software v1.49k as described in the suppl. Materials & Methods part.