Changes in cellular viability were expressed as the percent of the annexinV negative/Hoechst negative fraction. SDS-PAGE, NBGE and immunoblotting Cells were washed twice with PBS, collected into Laemmli SDS sample lysis buffer (2% SDS, 50?mM Tris-Cl, 10% glycerol in double distilled H2O) and sonicated (2??15?s at 1? amplitude with 15?s cooling interval) using Soniprep 150 (MSE, London, UK). non-treated animals. Mechanistically, we found that the susceptibility of senescent cells to MitoTam is usually linked to a very low expression level of adenine nucleotide translocase-2 (ANT2), inherent to the senescent phenotype. Restoration of ANT2 in senescent cells resulted in resistance to MitoTam, while its downregulation in non-senescent cells promoted their MitoTam-triggered Neuropathiazol elimination. Our study files a novel, translationally intriguing role for an Neuropathiazol anticancer agent targeting mitochondria, that may result in a new strategy for the treatment of age-related diseases and senescence-associated pathologies. for 3?min. The pellet was resuspeneded in 200?l of annexin V buffer containing 0.3?l of annexinV-Dyomics 647(Apronex, Vestec, Czech Republic), and incubated for 20?min at 4?C. Hoechst 33258 (5?g/ml, Invitrogen, Carlsbad, CA, USA) was added before analysis. The cells were analyzed for viability using the LSRFortessa flow cytometer (San Jose, CA, USA). Changes in cellular viability were expressed as the percent of the annexinV unfavorable/Hoechst unfavorable fraction. SDS-PAGE, NBGE and immunoblotting Cells were washed twice with PBS, collected into Laemmli SDS sample lysis buffer (2% SDS, 50?mM Tris-Cl, 10% glycerol in double distilled H2O) and sonicated (2??15?s at 1? amplitude with 15?s cooling interval) using Soniprep 150 (MSE, London, UK). Protein concentration was estimated using the BCA method (Pierce Biotechnology, IL, Rockford, USA). Cell lysates were supplemented with 100?mM DTT and 0.01% bromophenol blue before separation by SDS-PAGE. The same amount of protein (50C70?g) was loaded into each well. Proteins were transferred onto a nitrocellulose membrane using wet transfer and detected by specific antibodies combined with horseradish peroxidase-conjugated secondary antibodies (goat anti-rabbit, goat anti-mouse). Peroxidase activity was detected by SuperSignal West Femto Extended Duration Substrate (Thermo Fisher Scientific, Waltham, MA, USA). -actin was used as a loading standard. Native blue gel electrophoresis was performed as described [50]. Detection of senescence-associated beta-galactosidase activity SA–gal activity was detected as previously described [51] with slight modifications. Cells were washed once with PBS, fixed with 0.5% glutaraldehyde (in PBS; pH 7.2), and washed in PBS (pH 6.0) supplemented with 1?mM MgCl2. Cells were stained PLCG2 with the X-gal answer (1?mg/ml X-gal, 0.12?mM K3Fe[CN]6, 0.12?mM K4Fe[CN]6, 1?mM MgCl2 in PBS at pH 6.0) at 37?C for 3C5?h. For tissue staining, tissue was cut into small pieces (2C3?mm3) and fixed in 1% formaldehyde/0.2% glutaraldehyde at 4?C for 1?h. Tissue was stained with the X-gal answer as described above. For statistical evaluation, tissue was cut into 80?m sections. -galactosidase signal was detected using light microscope Neuropathiazol (Leica, Mannheim, Germany) and evaluated using the Photoshop and ImageJ programme as an average from five sections/sample. Indirect immunofluorescence Cells produced on glass coverslips were fixed with 4% formaldehyde and permeabilized with 0.1% Triton X-100 in two consecutive actions, each at room temperature for 15?min. After washing with PBS, cells were incubated in 10% FBS (diluted Neuropathiazol in PBS) for 30?min to block unspecific signals. After this step, cells were incubated with diluted primary antibodies at room heat for 1?h and then extensively washed with PBS/0.1% Tween 20. The incubation with secondary antibodies was performed at room heat for 1?h. To counterstain nuclei, coverslips Neuropathiazol were mounted in Mowiol made up of 4,6-diamidino-2-phenylindole (Sigma) and viewed by a fluorescence microscope (Leica DMRXA). siRNA-mediated gene knock-down Cells were transfected with siRNAs using Lipofectamine RNAiMAX (Invitrogen, Carlsbad, CA, USA) following the manufacturers instructions. siRNA against ANT2 (sense sequence: ANT2#1: 5-GCU UUA ACG UGU CUG UGC Att-3; ANT2#2: 5-GCU UUA ACG UGU CUG UGC Att-3) was purchased from Applied Biosystems (Foster City, CA, USA). Non-targeting siRNA (Silencer? Select Unfavorable Control No. 1, #4390843) were used as a negative control (siNC). Quantitative real time PCR (qRT-PCR) Total RNA was isolated using.