Alexa flour 594

Hela Cells were seeded at 5×10⁴ per well on glass coverslips in 24-well plates 1 day before transfection. Cells were transfected to express corresponding proteins for 18 hr, and then the medium was replenished with fresh medium containing 500 nM Mitotracker Red (Thermo Fisher Scientific, cat# M22425). After incubated at 37°C for 30 min, cells were fixed by 4% formaldehyde solution for another 30 min at 4°C. Fixed cells were permeabilized by 0.2% Triton X-100 solution for 5 min, and blocked with 4% goat serum for 30 min at 37°C. Flag-tagged proteins were stained with a specific antibody (Sigma-Aldrich, cat# F1804) at a dilution of 1:50. Incubation with primary antibodies was performed overnight at 4°C, and then cells were stained with secondary antibodies conjugated to Alexa Flour 594 or Alexa Flour 488 (Thermo Fisher Scientific) at a dilution of 1:500 for 1 hr at room temperature. After staining for nucleus with Hoechst, images were acquired using a Zeiss LSM 880 confocal microscope. The colocalization of Ceg3 with mitochondria was quantitated by measuring the Pearson correlation coefficient values with the ImageJ software (http://rsb.info.nih.gov/ij/).

Rabbit antibodies against phospho-Akt (Ser473), phospho-Akt (Thr308), Akt, phospho-p85 (Tyr458), p85, phospho-GSK-3β (Ser9), GSK-3β, phospho-mTOR (Ser2448), mTOR, glyceraldehyde 3-phosphate dehydrogenase (GADPH), and inhibitors LY294002, LY303511 and wortmannin were purchased from Cell Signaling Technology (Beverly, MA, United States). Mouse anti-FLAG antibody and anti-GFP were provided from Thermo Fisher Scientific (1:1000, Thermo Fisher Scientific, Shanghai, China). Secondary infrared dye 800CW goat anti-rabbit and anti-mouse IgGs were purchased from LI-COR Biosciences (Lincoln, NE, United States). And Alexa Flour 594 was purchased from Thermo Fisher Scientific (Thermo Fisher Scientific, Shanghai, China).

To evaluate the induction of AECII senescence by macrophages, AECII cells were co-cultured with polarized BMDM using Transwell inserts (Nunc-140652, 0.4 μm, Thermo Fisher). Briefly, 5x10⁵ macrophages polarized with vehicle or IL13 were seeded on the bottom of well (Nunc 12-well culture plate), 4x10⁴ AECII into the insert, and co-cultured with 2 ml DMEM media containing 10% FBS and 1% antibiotics for 72 hours. AECII on membrane was washed with PBS twice, applied to β-galactosidase activity assay (Abcam). AECII was visualized with anti-prosurfactant protein C antibody (Abcam, Cambridge, MA, USA) and a secondary antibody conjugated to Alexa Flour 594 (Thermo Fisher). AECII on membrane was placed on glass slide (VWR, Radnor, PA, USA), and mounted with ProLong antifade reagent containing DAPI (Thermo Fisher). The number of senescent AECII was counted on five 20× fields per mouse (n ≥ 5).

For immunofluorescence microscopy, cells were plated on cover slides and fixed in 4% paraformaldehyde for 15 min. The fixed cells were permeabilized on 0.5% Triton X-100 for 30 min, and blocked with 10% goat serum for another 30 min, then incubated with Vimentin (Santa Cruzs), Ki67 (Proteintech) overnight at 4 °C. After extensively washing with PBS, secondary antibody, the donkey anti-goat IgG conjugated with Alexa Flour 594 (ThermoFisher, A-11058), was applied to the slides for 1 h. After three times washing, cell nuclei were stained with DAPI (Sigma) for 5 min. The slides were photographed by confocal microscopy.
For IHC, paraffin-fixed tissues were deparaffinized, rehydrated and treated with 0.01 M sodium citrate for antigen retrieval. Endogenous peroxidase activity was blocked by 0.3% (v/v) hydrogen peroxide and then incubated with 0.3% Triton X-100 for 15 min. After blocking with 10 % goat serum for 1 h. The tissues were incubated with primary antibodies at 4 °C overnight. After extensive washing with PBS, tissues were incubated with secondary antibodies (Abcam, ab6884) for 1 h. Diaminobenzidine hydrogen peroxide was used to show staining signaling, then the sections were counterstained with hematoxylin.