Huh7 hepatocellular carcinoma cell line was sourced from JCRB cell bank (JCRB0403, Japan). Huh7, RD (CCL-136, ATCC, VA, USA) and NMuMG (CRL-1636, ATCC) cell lines were maintained in high glucose Dulbecco’s Modified Eagle Medium (DMEM, Thermo Fisher Scientific, Waltham, MA, USA) containing 10% fetal bovine serum (FBS, Hyclone, Logan, UT, USA), 2 mM l-glutamine and antibiotics. HT-1080 (CCL-121, ATCC) cell line was maintained in Eagle’s Minimum Essential Medium (MEM, Thermo Fisher Scientific) containing 10% FBS and supplemented with 2 mM l-glutamine, 1 mM sodium pyruvate, and antibiotics.
Generation of LMNA null cell line
A human lamin A Double Nickase Plasmid set (sc-400039-NIC, Santa Cruz Biotechnology, Dallas, TX, USA) was used to generate LMNA-knockout (LMNA_KO) cells. The lamin A Double Nickase Plasmid set consists of a pair of plasmids each encoding GFP/puromycin selection markers, and guide RNA (gRNA) sequences offset by approximately 20 bp to allow for specific Cas9-mediated double nicking of LMNA genomic DNA. Huh7 cells were transfected with the lamin A Double Nickase Plasmid set using Lipofectamine™ 2000 (Thermo Fisher Scientific) transfection reagent. Two days after the transfection, top 5% GFP-positive cells were sorted using a BD Influx (BD Biosciences, San Jose, CA, USA) cell sorter, and individual cells were plated into 96-well plates. Expression of lamin A/C in each single clone were determined by Western blot analysis and immunofluorescence staining using a lamin A/C antibody (ab108595, Abcam).
Antibodies and reagents
The manufacturers and dilutions of the antibodies used in Western blot analysis and immunofluorescence staining are listed in Additional file 1: Tables S3, S4, respectively. TGFβ1 was obtained from PeproTech (Rocky Hill, NJ, USA); the TGFβ type I receptor/ALK5 inhibitor SB-431542 was purchased from TOCRIS (Bristol, UK); cytochalasin D was obtained from Cayman Chemical (Ann Arbor, MI, USA); nocodazole was from Sigma-Aldrich (St. Louis, MO, USA); Actinomycin D was obtained from Thermo Fisher Scientific. To induce EMT, cells were treated with 10 ng/mL TGFβ1 in completed medium containing 5% FBS.
Plasmids and transfection
Complementary DNA (cDNA) of human SMAD2 (Genbank: BC014840) was obtained from transOMIC Technologies (Huntsville, AL, USA), amplified by PCR, and cloned into pcDNA3 vector (Thermo Fisher Scientific) with two HA tags inserted at the C-terminus of SMAD2 (i.e., SMAD2-HA). The nuclear-localizing green fluorescence protein (i.e., GFP-NLS) was constructed by inserting nuclear localization sequence (nucleotide sequence: 5ʹ-ccaaagaagaaacgcaaagtg-3ʹ; protein sequence: PKKKRKV) of SV40 Large T-antigen into 3ʹ end of pEGFP-C2 (Clontech). The expression vector of CFP-H2B (pH2b-CyFP) and YFP-lamin B1 (pYFP-laminB1) were sourced from Jan Ellenberg . The mCherry-H2B expression vector was modified from pH2b-CyFP by replacing CFP with mCherry cDNA. The mCherry-lamin A expression vector was obtained by cloning full-length lamin A into pZome-1-C vector with mCherry at 5ʹ end driven by a CMV (cytomegalovirus) promoter. The GFP-lamin A expression vector was constructed by cloning GFP at the N-terminus of lamin A in pcDNA3 vector. The Lipofectamine™ 2000 (Thermo Fisher Scientific) transfection reagent was used to deliver the expression plasmids into cells in accordance with the protocol provided by the manufacturer.
siRNAs and transfection
Sequences and/or manufacturers of the small interfering RNAs (siRNAs) used to deplete the expression of the targeted genes are listed in Additional file 1: Table S5. Cells were transfected with siRNAs via Lipofectamine™ RNAiMAX (Thermo Fisher Scientific) in accordance with the manufacturer’s protocol.
RNA extraction and real-time quantitative PCR (qRT-PCR)
Total mRNAs were isolated from cells using RNeasy mini kit (Qiagen, Hilden, Germany). Complementary DNAs were produced using the SuperScript® IV Reverse Transcriptase system (Thermo Fisher Scientific). qRT-PCR was carried out using Power SYBR Green master mix (Thermo Fisher Scientific). The qRT-PCR primers of H3.3 (i.e., H3-3A gene) was obtained from Qiagen (Cat. No. QT00247128). Gene expression levels were normalized to GAPDH using primers (forward: 5ʹ-GGAAGGTGAAGGTCGGAGTCA-3ʹ and reverse: 5ʹ-GTCATTGATGGCAACAATATCCACT-3ʹ).
Expression of proteins in cells were analyzed by Western blotting against specific antibodies summarized in Additional file 1: Table S3. Cells were lysed with RIPA buffer [50 mM HEPES, pH 7.3, 150 mM NaCl, 2 mM EDTA, 20 mM β-gylcerophosphate, 0.1 mM Na3VO4, 1 mM NaF, 0.5 mM DTT and protease inhibitor cocktail (Roche Applied Science, Indianapolis, IN, USA)] containing 0.5% NP-40 with mild sonication to extract nuclear envelope and chromatin proteins. Total cell lysates were further lysed in 1× SDS sample buffer containing β-mercaptoethanol, analyzed by SDS-PAGE, transferred to polyvinylidene fluoride (PVDF, Millipore) membranes, and blotted with primary antibodies. Corresponding horse radish peroxidase (HRP) or alkaline phosphatase (AP)-conjugated secondary antibodies (Sigma-Aldrich) were added, and the blots were developed by chemiluminescence in accordance with the manufacturer’s protocols.
Immunofluorescence staining and confocal microscopy
Cells were fixed in 4% paraformaldehyde for 30 min at room temperature and permeabilized with 0.5% Triton X-100 in phosphate buffered saline (PBS) for 30 min. For the immunofluorescence staining of histone H3.3, antigen retrieval was carried out by incubation in 100 °C citrate buffer (10 mM Citric Acid, pH 6.0) for 1 h, followed by incubation in 1% Triton X-100/PBS for 20 min. After two washes with PBS, cells were applied with 1% bovine serum albumin (BSA, Sigma-Aldrich)/PBS for 30 min at room temperature to block non-specific bindings. Then cells were incubated with primary antibodies (Additional file 1: Table S4) diluted in PBS for 1.5 h at room temperature. Fluorescent (Alexa-488, Alexa-568 or Alexa-633)-conjugated secondary antibodies (Thermo Fisher Scientific) at dilution 1/1000 were used for detection. For PLA experiments, cells seeded in 8-well chamber slides (Millicell EZ SLIDE, Millipore) were fixed with 4% paraformaldehyde in PBS for 15 min. Cells were permeabilized with 0.5% Triton X-100 in PBS for 30 min, and blocked with the Duolink® Blocking Solution for 1 h at 37 °C. Primary antibodies diluted in Duolink® Antibody Diluent where applied, and the slide were incubated for 1.5 h at room temperature. Detection of protein interactions was performed by following the manufacturer’s (Sigma-Aldrich) instructions. Cell nuclei were counterstained with Hoechst 33342 (Thermo Fisher Scientific) and mounted on slides using Prolong Gold antifade reagent (Thermo Fisher Scientific). Images were recorded using a Leica TCS SP5 confocal microscope (Leica, Wetzlar, Germany) equipped with HyD (hybrid detector). For live cell imaging, cells were incubated in a humidified chamber maintained at 37 °C and supplied with 5% CO2 (CU-109, Live Cell Instrument, Korea). Images were processed using Imaris 7.3 software (Bitplane, Zurich, Switzerland) and MetaMorph® (Molecular Devices, San Jose, CA, USA).
Immunogold staining and transmission electron microscopy (TEM)
Cells seeded on ACLAR® film were fixed in a mixture containing 0.1% glutaraldehyde and 1% paraformaldehyde (Electron Microscopy Sciences, Hatfield, PA, USA) for 2 h on ice. Crosslinking was quenched using 0.125 M glycine, followed by neutralization with 0.1 M ammonium chloride. Cells were treated with a series of cold methanol dilutions, and then embedded in LR-Gold reagent on a Leica EM AFS2 (Leica Microsystems, Wetzlar, Germany). The embedded samples were stored in a humidity control box at room temperature. For immunogold labeling, ultrathin sections of LR-Gold embedded samples were mounted on 200 mesh nickel grids covered with carbon-backed formvar film. The grids were first incubated with 3% normal sheep serum in PBS at room temperature for 15 min, and incubated with a mouse anti-H1 antibody (sc-8030, Santa Cruz, Dallas, TX, USA) for 60 min. After 6 sequential washes with 1% normal sheep serum in PBS, the grids were incubated for 60 min with 18 nm gold–IgG complexes. The grids were washed sequentially with 1% normal sheep serum in PBS and 3% normal rat serum in PBS, followed by incubation with a rabbit anti-H3K27me3 antibody (#9733, Cell Signaling, Danvers, MA, USA) for 60 min. After another 6 sequential washes with 1% normal rat serum in PBS, the grids were incubated for 60 min with 12 nm gold–IgG complexes. Following another sequential wash with 1% normal rat serum in PBS, the grids were incubated with 3% normal donkey serum in PBS at room temperature for 15 min, and incubated with a goat anti-Lamin B antibody (sc-6217, Santa Cruz) for 60 min. After washes with 1% normal donkey serum in PBS, the grids were incubated for 60 min with 6 nm gold–IgG complexes. The grids were then washed sequentially with 1% normal donkey serum in PBS, followed by two washes with triple distilled water. Finally, the grids were treated with 2% uranyl acetate and 30 mM lead citrate. The final immunogold labeled grids were examined and photographed using a FEI Tecnai T12 electron microscope.
Spots excised from the Coomassie blue-stained SDS-PAGE were digested using MS grade Trypsin Gold (Promega, Madison, WI) overnight at 37 °C. The tryptic digests were extracted using 10 μL Milli Q water initially, followed by two extractions using a total of 20 μL 50% acetonitrile/0.1% trifluoroacetic acid. The combined extracts were dried in a vacuum concentrator, and then dissolved in 1 μL of 5% acetonitrile/0.5% trifluoroacetic acid. A Thermo Scientific™ Orbitrap Fusion™ Lumos™ Tribrid™ Mass Spectrometer (Thermo Fisher Scientific) was used to detect electrospray ionization (ESI)–MS/MS and higher energy collisional dissociation (HCD)–MS/MS peptide signals. The MS/MS signal was analyzed using the MASCOT search engine (www.matrixscience.com).
Calculation of axial ratio (AR)
Nuclear morphology was quantified by calculating the axial ratio (AR) of each nucleus, determined from the nuclear staining of Hoechst 33342 cells. A custom Matlab code was developed to trace the nuclear perimeter, and applied elliptical Fourier analysis (EFA) to find the first 20 elliptic harmonics . AR was defined as the sum of the axes from the first 20 ellipses normalized by the first ellipse, subtracted by one. AR represents deviations from a perfect elliptical shape, where bigger ellipses from the later harmonics would result in a larger AR value.
Data and statistical analyses were performed using Microsoft Excel and Graphpad Prism software. Data were analyzed using two-tailed Student’s t test or Fisher’s exact test. P values below 0.05 were considered significant.