Lamin a c

Cells were lysed with RIPA buffer (25 mM Tris, pH7.4; 150 mM NaCl; 1% NP-40; 0.5% sodium deoxycholate and 0.1% SDS) supplemented with protease inhibitors (Roche, 4693132001) and phosphatase inhibitors (Roche, 4906845001) and sonicated with Bioruptor Plus at High setting. Cell lysates were resuspended in 4× sample buffer (200 mM Tris, pH 6.8; 8% SDS; 40% glycerol; 0.4% bromophenol blue and 20% 2-mercaptoethanol) and heated at 95 ℃ for 5 min. Antibodies used were OPA1 (67589), DRP1 (8570), Mitofusin-1 (14793), Mitofusin-2 (11925), H2AX (2595), γH2AX (2577), H3 (9715), H3K4me1 (5326), H3K4me3 (9751), IRF3 (11904), p-IRF3 (S396) (29047), TBK1 (3504), p-TBK1 (S172) (5483), p65 (8242) and p-p65 (S536) (3033) from Cell Signaling; H3K4me2 (39679), H3K9me1 (39887), H3K9me2 (39683), H3K9me3 (39765), H3K27me1 (61015), H3K27me2 (39919), H3K27me3 (39155), H3K36me1 (61351), H3K36me2 (39255), H3K36me3 (61101) from Active Motif; α tubulin (sc-23948) from Santa Cruz; Lamin A/C (GTX101127) from GeneTex. α tubulin was in 1:20000 dilution. For Lamin A/C and all histone-related antibodies, antibodies were used in 1:5000 dilution. Others were in 1:1000 dilution.

A standard immunocytochemical protocol was used as previously described [40 (link), 45 (link), 47 (link)–49 (link)]. Histone H2B-GFP-transfected hBM-MSCs were plated onto collagen IV (Sigma‒Aldrich)-coated plastic or glass coverslips and maintained in neural induction media. Cells were rinsed with PBS and fixed in freshly prepared 4% paraformaldehyde (PFA; Sigma‒Aldrich). Fixed cells were blocked for 2 h in PBS containing 10% normal horse serum (Gibco) and 0.25% Triton X-100 (Sigma) and incubated overnight at 4 °C with antibodies against β-III-tubulin (TUJ1; 1:500, Covance), fibrillarin (1/300, Abcam) and lamin A/C (1/300, GeneTex) in PBS containing 1% normal horse serum and 0.25% Triton X-100. The next day, the cells were rinsed and incubated with secondary antibodies conjugated with Alexa Fluor® 488 (anti-rabbit; 1:500, Molecular Probes) and Alexa Fluor® 594 (anti-mouse; 1:500, Molecular Probes). Cell nuclei were counterstained with DAPI (0.2 mg/ml in PBS, Molecular Probes). Alexa Fluor 488® phalloidin (Molecular Probes) was used to selectively stain F-actin. Data are representative of ten independent experiments per condition.

Cells were lysed in RIPA buffer (Thermo Scientific) or cytoplasmic and nuclear fractions were obtained with an NE-PER kit (Thermo Scientific). Proteins were quantified with bicinchoninic acid assay (Thermo Scientific). Samples were mixed in a 4X Laemmli buffer (BioRad, Hercules, CA, USA) with 2-mercaptoethanol and heated for 5 min at 95 °C. Then, 25 µg of protein was used for electrophoresis in a 10 or 12% polyacrylamide gel. Proteins were transferred into nitrocellulose membranes (BioRad), which were blocked for 1 h at room temperature using an Li-Cor blocking buffer (Licor, Lincoln, NE, USA). Primary antibodies for HDAC3 (1:750, GeneTex), H3K9ac (1:1000, SCBT, Dallas, TX, USA), H3K9me2 (1:1000, GeneTex), Lamin A/C 1:2000 (GeneTex), Lamin A 1:1000, GAPDH (1:3000, SCBT, Dallas, TX, USA), or H3 (1:1000, GeneTex) were incubated overnight, and then incubated with secondary antibodies (Li-Cor, Lincoln, NE, USA). Membranes were developed by fluorescence in the Odyssey scanner (Li-Cor, Lincoln, NE, USA). Quantification was performed using ImageJ software Version 1.53t.

Cells were treated with or without 25, 50, or 100 μg/mL of O-PMs and harvested with RIPA buffer (H.M. Biological, Taoyuan, Taiwan) supplemented with protease and phosphatase inhibitors (Thermo Fisher Scientific, MA, USA). In addition, cytoplasmic and nuclear proteins were extracted using a Nuclear Extraction Kit (Cayman Chemical, MI, USA). Thirty micrograms of protein were subjected to sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). The membranes were incubated overnight at 4 °C with primary antibodies against fibronectin, ETS-1 (1: 2000 dilution, Abcam, Cambridge, UK), E-cadherin, phosphorylated-NF-κB p65 (1:2000 dilution, Cell Signaling Technology, MA, USA), Lamin A + C, α-tubulin, β-actin (1:2000 dilution, GeneTex, CA, USA), or vimentin (1:2000 dilution, Santa Cruz Biotechnology, TX, USA). The anti-GAPDH antibody (1:10000 dilution, Tools, New Taipei City, Taiwan) was used as the loading control. Images were visualized by UVP BioSpectrum 815 imaging system (UVP, CA, USA), and the intensity of each band was quantified using ImageJ software.