Sc 81611

Fibroblasts were seeded into 4 chamber-well slides (SPL Lifesciences, Korea). Cells were fixed for 15 minutes (RT) in a 4% paraformaldehyde +2% sucrose solution and then permeabilized for 3 minutes at RT using permeabilization buffer (0.5% Triton X-100, 50 mM NaCl, 300 mM sucrose, 20 mM HEPES pH 7.5, 3 mM MgCl2). Cells were then incubated with primary antibodies for 40 minutes at 37 °C (anti-progerin (1/50, sc-81611) and anti-lamin A/C (1/50, sc-20681), Santa Cruz Biotechnology). After washing, cells were incubated for 20 minutes at 37 °C with secondary antibodies (A11001, A11012, 1/400 Life Technologies). Nuclei were stained with DAPI for 10 minutes at room temperature (0.1 μg/mL, Thermofisher). Slides were mounted using FluorSave™ reagent (Merck Millipore) and observed on a fluorescence microscope (ApoTome.2 Zeiss).

Antibodies used for experiments included anti-GFP (1:1000; sc-9996; Santa Cruz Biotechnology, Dallas, TX, USA); anti-GST (1:5000; sc-138; Santa Cruz Biotechnology), anti-His (1:1000; 66005-1-lg; Proteintech, Rosemont, IL, USA), anti-Actin (1:10000; sc-47778; Santa Cruz Biotechnology), anti-Lamin A/C (1:10000; sc-376248; Santa Cruz Biotechnology), anti-Progerin (1:100; sc-81611; Santa Cruz Biotechnology), anti-Progerin (1:300; ab66587; Abcam, Cambridge, UK), anti-Ki67 (1:200; Ab15580; Abcam), anti-cyclin B1 (1:100; sc-594; Santa Cruz Biotechnology), and anti-p16-INK4A (1:500; 10883-1-AP; Proteintech).

Lamin A/C and progerin amounts in tissue samples including lung, liver, and kidney analysis of two Lmna^+/+, four untreated Lmna^G609G/+, and four D011-treated Lmna^G609G/+ mice were evaluated by western blotting. Proteins were extracted with RIPA lysis buffer supplemented with protease inhibitors. Tissues were sonicated and centrifuged at 15,000 rpm for 15 min at 4 °C. Protein concentration was determined by Coomassie-blue staining. Cellular lysates in SDS-containing sample buffer were inactivated by heating at 98 °C for 7 min. Proteins were loaded onto 8% SDS-PAGE gels and transferred to PVDF membranes. These membranes were blocked with 3% skimmed milk in TBS-T buffer for 1 h and incubated with primary antibodies including mouse monoclonal anti-progerin (1:100; sc-81611; Santa Cruz Biotechnology), mouse monoclonal anti-lamin A/C (1:500; MANLAC1; Developmental Studies Hybridoma Bank), and rabbit polyclonal anti-H3K9me3 (1;2000; Ab8898; Abcam) at 4°C overnight. Blots were then incubated with 1:10,000 goat anti-mouse (Pierce, Thermo Fisher Scientific) or 1:10,000 goat anti-rabbit (Pierce, Thermo Fisher Scientific) IgG (HRP conjugated) in 1% skimmed milk and washed with TBS-T buffer. Bands were developed using and Intron ECL detection system and quantified using Image J (National Institute of Health, NIH).