Patient material, ethics and consent for publication
The blood samples and epidermal tissue samples used in this study from patients and healthy donors were preprocessed in a standardized manner and were collected with written informed consent. Relevant experiments were approved by medical ethic committee (Shenzhen Medical Ethics Committee, Shenzhen Luohu People’s Hospital Medical Ethics Committee). Consent included the use of all de-identified patient data for publication. Participants were not compensated. Detail information of patients’ family and healthy donors were listed in Table S1.
Generation of human PBMC derived-induced pluripotent stem cells
Blood samples were collected from patients and healthy donors. PBMCs from blood samples were isolated by density-based centrifugal separation. PBMCs were grown in Stem Pro-34 (Gibco, 10639-011) SFM complete medium containing SCF (SinoBiological, 10451-H08B), IL-3 (SinoBiological, 11858-HNAE), and GM-CSF (Peprotech, AF-315-03) for 12 days, and 2×106 of PBMCs were electroporated with iPSC Reprogramming Vectors. After transfection, PBMCs were plated onto Matrigel-coated (Corning, 354277) 6 well culture-plates, and incubated in complete Stem Pro-34 medium containing cytokines for one day, N2B27 medium supplemented with 100 ng/ml bFGF (SinoBiological, 10014-HNAE) for one week, and finally Essential medium (Gibco, A15169-01) for two weeks. Colonies of iPSCs were manually picked 15–21 days after transfection, and transferred onto fresh Matrigel-coated 6 well culture-plates for expansion. iPSCs were passaged by 0.5mM EDTA for 8 min at 37 °C. iPSCs were split at 1:10 twice a week, and the cells were cultured in hPSC medium (Nuwacell, RP01001) at 37 °C in 5% CO2.
Generation of mesenchymal stem cells from human induced pluripotent stem cells
When iPSCs were 20% confluent, hPSC medium was replaced with a series of MSC differentiation Basal Medium (Nuwacell, RP01013). After 14 days in culture, cells were considered as passage 0. iMSCs were passaged by MSC Solase for 5 min at 37 °C, and were seeded 5000 cells/ml in hMSC medium (Nuwacell, RP02010) at 37 °C in 5% CO2. Further experiments were performed after flow cytometry analysis of MSC phenotypic characteristics at passage 4 (5 × 105 MSCs were incubated with 1% BSA for 30 minutes. The following antibodies were used: CD29-PE, CD34-APC, CD44-FITC, CD45-FITC, CD73-PE, CD90-PE. CD105-FITC, CD133-PE, CD146-PE, and HLA-DR-PE (BD Biosciences).
HEK293T cells and Hela cells were maintained in our lab. HEK 293T cells and Hela cells were cultured in DMEM medium supplemented with 10% FBS (PAN, ST30-3306), 100 U/mL Penicillin-Streptomycin at 37 °C in 5% CO2. Cells were negative for mycoplasma.
Wild type / LmnaR527C/R527C bone-marrow derived macrophages (BMDMs), peritoneal macrophages and LmnaWT/R527C /LmnaR527C/R527C mouse embryonic fibroblasts (MEFs) was isolated from mice.
Peritoneal macrophages were obtained from lavage of the peritoneal cavity with RPMI 1640 medium and were centrifuged and resuspended and cultured in indicated culture medium. For BMDMs, bone marrow cells were isolated from femurs and tibiae of 8-12 weeks old mice. Cells were cultured with 20 ng/mL recombinant murine GM-CSF (Peprotech, AF-315-03) in a 10-cm dish for 7 d before experiments. Mature macrophages were harvested by incubating with PBS-EDTA for 10 min and cultured in RPMI 1640 medium supplemented with 10% FBS, 100 U/mL Penicillin-Streptomycin at 37 °C in 5% CO2.
MEFs were generated from embryonic day 13.5 (E13.5) embryos of LmnaWT/R527C and LmnaR527C/R527C mice under the normal culture condition, and were cultured in DMEM medium supplemented with 10% FBS, 100 U/mL Penicillin-Streptomycin at 37 °C in 5% CO2.
LMNA KO, BANF1 KO, LAP2 KO Hela cells and Cgas KO, Aim2 KO MEF cells were constructed by CRISPR-Cas9 system. Specific guide RNAs (Table S2) were ligated into the BsmB1 restriction site of the inducible lentiviral vector (lentiCRISPR v2). Lentivirus particles were produced by co-transfected HEK293 cells with guide RNA plasmids (2mg), packaging plasmids pCMV-VSV-G (800ng) and psPAX2 (800ng). The medium was changed to fresh DMEM containing 10% FBS at 6 h post transfection and viral supernatant was collected at 48 h. Then, target cells were infected with 1 mL viral supernatant or directly transfected with guide RNA plasmid. Possible knockout cells were screened by puromycin (Gibco, A1113803) and each monoclone was confirmed by sequencing if necessary. The specific guide RNA sequences were as listed in Key Resource. HEK293 cells were transfected by standard polyethylenimine (Polysciences, 02371) precipitation method.
Generation of stable cell lines
LMNA KO Hela cells stably expressing GFP-Lamin A, GFP-Lamin A(R527C), Flag-Lamin A, Flag-Lamin A(R527C), Dam-Lamin A, Dam-Lamin A(R527C), and Hela cells stably expressing Flag-progerin were generated as follows. Specific protein coding sequences were ligated into the inducible lentiviral vector (pCDH-CMV-MCS-EF1α-puro). Lentivirus particles were produced by co-transfected HEK293 cells with reconstructed plasmids (2mg), packaging plasmids pCMV-VSV-G (800ng) and psPAX2 (800ng). The medium was changed to fresh DMEM containing 10% FBS at 6 h post transfection and viral supernatant was collected at 48 h. Then, target cells were infected with 1 mL viral supernatant and incubated for 48 h. Possible knockout cells were screened by puromycin and each monoclone was confirmed by sequencing if necessary. HEK293 cells were transfected by standard polyethylenimine precipitation method. All stable cell lines were used at early passages in experiments.
Mice and In Vivo experiments
All animals care and use adhered to the Guide for the Care and Use of Laboratory Animals of the Chinese Association for Laboratory Animal Science. All procedures of animal handling were approved by the Animal Care Committee of Peking University Health Science Center.
Wild type C57BL/6J mice were purchased from Department of Laboratory Animal Science of Peking University Health Science Center, Beijing. Cgas-/- mice and Aim2-/- mice on a C57BL/6J background were gift from Pro. Zhengfan Jiang (Peking University). The Lmna R527C mutant mice (C57BL/6) were generated by Cyagen Biosciences using the CRISPR/Cas9 system. Cgas-/-LmnaR527C/R527C mice and Aim2-/-LmnaR527C/R527C mice were generated by LmnaR527C/R527C mice bred with Cgas-/- mice and Aim2-/- mice. Mice were kept and bred in specific pathogen-free conditions under controlled temperature (23 ± 1oC) and exposed to a constant 12 h light-dark cycle. All animals are guaranteed adequate clean water and nutritious feed. The primers for genotyping were as listed in Table S2
Age- and sex-matched C57BL/6 littermates were produced and used in all the in vivo experiments. For irradiation mice model, Wild type and LmnaR527C/R527C 6-week-old male mice were treated by 4 GY irradiation. Blood was collected at day -1and day 7 post-irradiation for hemanalysis, genome DNA was collected at day -1and day 300 post-irradiation for telemore length analysis. Mice were sacrificed at day -1and day 7 post-irradiation and spleen was harvested for RT-PCR analysis and RNAseq, or mice were monitered survival and body weight change daily. For high-fat-diet mice model, wild type, LmnaR527C/R527C, and Aim2-/-LmnaR527C/R527C 6-week-old male mice were fed with normal chow or high-fat diet (Research Diets, D12492). Blood and genome DNA were collected at month 0, month 1, month 3, month 5, and month 7 after normal chow or high-fat diet for hemanalysis and telemore length analysis. Mice were monitered survival and body weight change daily. Echocardiographic analysis was performed at month 8, and then mice were sacrificed and tissues were harvested for HE staining analysis.
Expression constructs generated for this study were prepared by standard molecular biology techniques and coding sequences entirely verified. All the truncations deletions and mutants were constructed by standard molecular biology technique. Each truncation, deletions and mutants were confirmed by sequencing. The constructs used were as listed in Table S3.
Protein expression and purification
The gene coding for wild type or R527C mutant Lamin A were cloned into the pET21d vector. Plasmids were transformed into E. coli BL21(DE3) for expression. Strains carrying the different plasmids were grown to an OD600nm of 0.6–0.7 at 37 °C, expression of wild type or mutant Lamin A protein was subsequently induced by the addition of 0.5 mM IPTG, and further incubated overnight at 20°C. Bacterial cells were pelleted by centrifugation, and resuspended in lysis buffer (20mM Tris-HCl, pH8.0 and 150 mM NaCl) supplemented with 1mM PMSF, 100μg/ml Dnase (New England Biolabs, M0303) and 100μg/ml lysozyme. After cells were lysed using a high-pressure cell disrupter and the pellets were collected by centrifugation at 17,000 × g for 20min. The pellets were incubated in a buffer containing 20mM Tris-HCl, pH8.0, 150mM NaCl, 8M Urea at room temperature for 30min, the supernatants collected after centrifugation (17000 × g, 20min) were further purified by HiTrap Q HP ion-exchange and Superdex 200 gel filtration chromatography under denature condition (with 8M Urea always in the buffer). The purified protein was dialysis against suitable buffer for refolding as needed before use.
0.8 mg/mL wild type or R527C mutant Lamin A protein were mixed in 150 mM NaCl, 1mM EDTA, 1mM DTT and 25mM Tris-HCl/MES with various PH (PH=6.0/6.5/7.0/7.4). Sample were incubated at room temperature for 30 minutes prior to the absorption (turbidity) measurement at 600nm by FlexStation 3. Readings were recorded in triplicate for each protein sample. All assays were performed in triplicate.
0.8 mg/mL wild type or R527C mutant Lamin A protein were mixed in 150 mM NaCl, 1mM EDTA, 1mM DTT and 25mM Tris-HCl/MES with PH=6.5. Samples were deposited on 8-well glass bottom Ibidi slides, then samples were incubated at room temperature and were imaged on microscope. Droplet and fiber formation were observed over time.
Antibodies and Beads.
Commercially available antibodies against the following proteins were used in this study. Mouse antibodies: Lamin A/C (Abcam, ab8980), DYKDDDDK tag (Proteintech, 66008-3-Ig), and GAPDH (Proteintech, 60004-1-Ig). Rabbit antibody: Lamin A/C (Proteintech, 10298-1-A), HA tag (Proteintech, 51064-2-AP), DYKDDDDK tag (Proteintech, 20543-1-AP), cGAS (Cell Signaling Technology, 15102), beta Tubulin (Proteintech, 10094-1-AP), Lamin B (Proteintech, 12987-1-AP), GFP tag (Proteintech, 50430-2-AP), and γH2A.x (Cell Signaling Technology, 9817). HPR-conjugated secondary antibodies (goat anti-mouse IgG and goat anti-rabbit IgG) were purchased from Proteintech. Alexa Fluor 488 or 555-conjugated secondary antibodies (donkey anti-mouse IgG and donkey anti-rabbit IgG) were purchased from Invitrogen.
Western blot analysis
Whole-cell extracts were prepared using RIPA lysis buffer (50 mM Tris, pH 7.4, 150 mM NaCl, 1% NP-40, 0.1% SDS, and 0.5% deoxycholate, supplemented with a protease inhibitor (TargetMol, B14001) and centrifuged at 10,000 g for 10 min at 4 °C. Proteins were resolved on 10% SDS-PAGE and transferred onto nitrocellulose membranes. Blocked membranes were incubated with the primary antibodies for 1 h at 37 °C. Secondary antibodies conjugated to HRP and Western Chemiluminescent HRP Substrate were used for detection.
Different treatments and genotypes of HeLa cells, MSCs, BMDMs and peritoneal macrophages on coverslips were washed twice with pre-warmed phosphate buffered saline (PBS) and fixed in 4% (wt/vol) paraformaldehyde for 10 min. After three washes in PBS, cells were permeabilized with 0.5% (vol/vol) Triton X-100 for 5 min. After three washes in PBS, cells were blocked in PBS containing 1% (wt/vol) bovine serum albumin (BSA) for 60 min, and incubated with indicated antibodies in PBS containing 1%(wt/vol) BSA for 1 h at 37 °C. After three washes, cells were incubated with Alexa Fluor 488-conjugated secondary antibodies or Alexa Fluor 555-conjugated secondary antibodies for 1 h at 37 °C, and then with DAPI (40,6-Diamidino-2-phenylindole; Invitrogen, P36961) for 15 min. The coverslips were washed extensively and mounted onto slides. Imaging of the cellswas carried out using N-STORM 5.0 microscope.
Quantitative RT-PCR (qRT-PCR) analysis
Total RNA was isolated from the tissues and cells by TRNzol reagent (TIANGEN Biotech, DP424). Then, cDNA was prepared using HiScript III 1st Strand cDNA Synthesis Kit (Vazyme Biotech, R312-02). qRT-PCR was performed using the Applied Biosystems 7500 Real-Time PCR Systems with SYBR qPCR Master mix (Vazyme Biotech, Q331-02). The data of qRT-PCR were analyzed by the Livak method (2−ΔΔCt). Beta-actin was used as a reference gene. The primers for qRT-PCR were as listed in Table S2.
RNA sequencing (RNA-seq)
Whole RNA of tissues and cells with specific treatments were purified using TRNzol reagent. The transcriptome library for sequencing was generated using VAHTSTM mRNA-seq v2 Library Prep Kit for Illumina (Vazyme Biotech, R312-02) following the manufacturer’s recommendations. After clustering, the libraries were sequenced on Illumina Hiseq X Ten platform using (2 × 150 bp) paired-end module. The raw data were transformed into raw reads by base calling using CASAVA.
Luciferase Reporter Assay
HEK293T cells (2×105) were plated in 24-well plates and transfected using polyethylenimine, with pGL3-mIfnb-promoter plasmids (firefly luciferase; 10 ng) and pRL-TK (renilla luciferase plasmid; 10 ng) together with 100 ng pCMV-3×FLAG-7.1 or pCMV-3×Flag-STING plasmid. The medium was changed to fresh DMEM containing 10% FBS with DMSO or 100nM C176 (TargetMol, T5154) at 6 h post transfection. After transfection for 24 h, cells were lysed and luciferase activity was measured with the Dual-Luciferase Assay System (Promega, E1910) according to the manufacturer’s instructions. Reporter gene activity was determined by normalization of the firefly luciferase activity to renilla luciferase activity.
HEK293 cells (1×107) were seeded on 10 cm2 dishes and were transfected with a total of 10 ug of empty plasmid or various expression plasmids. The medium was changed to fresh DMEM containing 10% FBS at 6 h post transfection. After transfection for 24 h, cells were lysed in lysis buffer (0.5% Triton X-100, 20 mM HEPES (PH 7.4), 150 mM NaCl, 12.5 mM b-glycerolphosphate, 1.5 mM MgCl2, 2 mM EDTA, 10 mM NaF, 1 mM Na3VO4, 2 mM DTT) containing protease inhibitors. Lysates were centrifuged and incubated with anti-Flag antibodies at 4 °C overnight. The next day, prewashed protein A/G beads (Thermo, 20422) were added and incubated at 4 °C for 4 h. The beads were washed with cold PBS 4 times and eluted with DTT-containing SDS sample buffer by boiling for 10 min for western blotting.
Histology and immunohistochemical staining
For H&E staining, mice tissues were quickly placed in cold saline solution and rinsed after they were collected. Then, tissues were fixed in 4% paraformaldehyde, dehydrated and embedded in paraffin prior to sectioning at 5 mm, and sections were stained with hematoxylin and eosin. Several H&E staining images were randomly selected for pathological scoring in a blinded fashion.
For immunohistochemical staining, the human epidermal tissues paraffin sections were dewaxed and rehydrated through xylene and an alcohol gradient. Antigen retrieval was performed by heating the sections to 100 °C for 4 min in 0.01 M citrate buffer (pH 6.0) and repeated 4 times. The operations were performed according to the instructions of the two-step detection kit. The samples were treated by endogenous peroxidase blockers for 10 min at room temperature followed by incubation with Lamin A antibodies at 37 °C for 1 h. Then after washed with PBS, the samples were incubated with reaction enhancer for 20 min at room temperature and secondary antibodies at 37 °C for 30 min, and finally sections were visualized by 3,30-diaminobenzidine tetrahydrochloride (DAB) and counterstained with hematoxylin.
Telomere length detection
Total DNA was isolated from the tissues by Genomic DNA Extraction Kit (TIANGEN Biotech, DP304). Then, qRT-PCR was performed using the Applied Biosystems 7500 Real-Time PCR Systems with SYBR qPCR Master mix. The data of qRT-PCR were analyzed by the Livak method (2−ΔΔCt). 36B4 was used as a reference gene. The primers for qRT-PCR were as listed in Table S2.
Control and patient-derived MSCs were washed twice in PBS, fixed for 3–5 min at room temperature in 3% formaldehyde, and washed with PBS again. Then cells were incubated overnight at 37 °C (without CO2) with freshly prepared SA-β-gal stain solution (1 mg/ml X-gal, 40 mM citric acid/sodium phosphate, pH 6.0, 5 mM potassium ferrocyanide, 5 mM potassium ferricyanide, 150 mM NaCl, 2 mM MgCl2) according to Senescence β-Galactosidase Staining Kit (Cell Signaling Technology, 9860). Positive staining appeared after 2–4 h and was evaluated after 12–16 h incubation at 37 °C in a CO2-free atmosphere. The blue stained cells were monitered under the microscope.
Slides were prepared with 85 μl of 0.5% normal melting agarose. LMNA KO Hela cells stably expressing Flag-Lamin A, Flag-Lamin A (R527C), and Hela cells stably expressing Flag-progerin were then mixed with 75 μl of 0.5% low melting agarose and applied to the prepared slides. Alkaline lysis (10% DMSO, 1% Triton-X in alkaline lysis buffer: 2.5 M NaCl, 10 mM Tris, 100 mM Na2EDTA, pH 10) followed for 1 h. Then slides were placed in a horizontal gel electrophoresis chamber containing alkaline buffer solution with NaOH (10 mM) and Na2EDTA (200 mM) at pH 13.2. After a 20-min DNA “unwinding” period, electrophoresis was performed at 25 V and 300 mA for 20 min. Following neutralization (Tris–HCl, pH 7.5), the cells were stained with ethidium bromide (Invitrogen, 15585011). The slides were examined and the tail length (diameter of the nucleus plus migrated DNA) was measured with microscope. The DNA migration of 100 randomly selected cells was examined for each sample.
Fluorescence Recovery after Photobleaching (FRAP)
FRAP was performed with a N-STORM 5.0 microscope using the semiconfluent monolayer of live cells over slides covered with LMNA KO Hela cells stably expressing GFP-Lamin A (wild type/R527C/1-390/1-425). The laser intensity and photobleaching duration were standardized to eradicate at least 80% of fluorescence. After two low laser intensity scans, circle areas in different cell regions were bleached using a 488 laser at high intensity. Next, the bleached areas were scanned for 20 min using a low intensity laser. The fluorescence intensity in the obtained images was measured in the regions of interest (ROIs) using the ImageJ software. To calculate the ratio (R) between the time of half recovery in the cell–cell contacts and the free membrane, we used the formula: R = (Fe – F0)/(Fi – F0), where Fe is the fluorescence at the end of the experiment, Fi is the fluorescence at the beginning, and F0 is the fluorescence just after bleaching.
MEF Immortalization assay was conducted as described 48. MEFs were generated from embryonic day 13.5 (E13.5) embryos of LmnaWT/R527C and LmnaR527C/R527C mice under the normal culture condition that includes 20% oxygen and 5% CO2. For spontaneous immortalization, we followed a modified 3T3 protocol by seeding 1×106 cells in a 10-cm dish every 3.5 d. Aliquots of the cells at indicated passages were expanded for continued passages, frozen in liquid nitrogen.
Mice blood was obtained at indicated day after irradiation or high-fat diet by a tail snip. 50 μL blood was collected to heparin-coated tube and then was analyzed with HEMAVET 950 Analyzer.
Two-dimensional M-mode echocardiography was performed on anesthetized mice using a Vevo2100 high-resolution imaging system with a 30-MHz high-frequency transducer to evaluate the cardiac function.
Cell viability assay
Wild type/LmnaR527C/R527C BMDMs and peritoneal macrophages (1×107) were seeded into 10 cm2 dishes and treated with or without 10 nM C176 for 1 month. Cell viability was examined using the Cell Counting Kit-8 assay (Bimake, B34302) according to the manufacturer's instructions. The Optical density (OD) values were measured at 450 nm by FlexStation 3. Each experiment was repeated at least three times.
Assay for Transposase-Accessible Chromatin sequencing (ATAC-seq)
Pellet 50,000 viable control and patient-derived MSCs at 500 RCF at 4°C for 5 min. Aspirate all supernatant. Add 50 μL cold ATAC-Resuspension Buffer (RSB) containing 0.1% NP40, 0.1% Tween-20, and 0.01% Digitonin into the cell pellet and pipette up and down 3 times. Incubate on ice for 3 minutes. Wash out lysis with 1 mL cold ATAC-RSB containing 0.1% Tween-20 but no NP40 or digitonin and invert tube 3 times to mix. Pellet nuclei at 500 RCF for 10 min at 4°C. Aspirate all supernatant. Resuspend cell pellet in 50 μL of transposition mixture (25 μL 2x TD buffer, 2.5 μL transposase (100 nM final), 16.5 μL PBS, 0.5 μL 1% digitonin, 0.5 μL 10% Tween-20, 5 μL H2O) by pipetting up and down 6 times. Incubate reaction at 37 °C for 30 minutes. Afterward, the DNA was purified with QIAEX II Gel Extraction Kit (Qiagen, 20021) and amplified with primers containing barcodes by using the TruePrep DNA Library Prep Kit (Vazyme Biotech, TD501-01). All libraries were adapted for sequencing.
DamID library preparation and sequencing
The gDNA of Dam-Lamin A (wild type/R527C) stably expression LMNA KO Hela cells were ethanol-precipitated overnight at −20 °C and dissolved in Tris-EDTA (pH 7.5) to a concentration of 1 mg/ml. gDNA (2.5 ml) was digested with Dpn I (New England Biolabs, R0176) at 37°C overnight, and Dpn I was inactivated by heating to 80 °C for 20 min. Dpn I–digested gDNA was ligated to the adaptor AdR by T4 Ligase. After enzyme was inactivated by heating to 65 °C for 10 min, samples were digested by Dpn II (New England Biolabs, R0543) at 37 °C for 1 hour. PCR reaction was performed to amplify the regions flanked by adaptors. The PCR products were cleaned with the QIAEX II Gel Extraction Kit. All libraries were adapted for sequencing. The primers for AdR were as listed in Table S2.
Chromatin Immunoprecipitation (ChIP)
Chromatin Immunoprecipitation was conducted as described 75. Approximately 5 x 107 pretreated cells were crosslinked with 1% formaldehyde at room temperature for 10 min, and the reaction was quenched with 0.125M glycine for 5 min. The cells were washed twice with PBS, then scrapped and pelleted at 2500 rpm for 5 min at 4 °C. After lysis and sonicat, the majority of the sonicated DNA fragments were sheared to a size of around 200–600 bp. The sonicated chromatin was spun down at 12,000 rpm for 10 min at 4 °C to collect the chromatin. Then the soluble chromatin was incubated with 3 mg antibody and the mixture were rotated at 4 °C overnight. After incubation, pre-washed Protein G Dynabeads (Invitrogrn, 10004d) were added and incubated for 4 h at 4 °C in a rotator. Then the magnetic Dynabeads were pelleted by placing the tubes in a magnetic rack and were washed for a total of five times: once with wash buffer A (20 mM Tris-HCl (pH 8.0), 500 mM NaCl, 2 mM EDTA, 0.1% SDS, 1% Triton X-100); once with wash buffer B(10 mM Tris-HCl (pH 8.0), 250 mM LiCl, 1 mM EDTA, 1%NP-40); three times with wash buffer C (1 mM EDTA, 10 mM Tris-HCl(pH 8.0)).
For sequencing, beads were resuspended in 100 mL elution buffer (50 mM Tris-HCl (pH 8.0), 10 mM EDTA, 1% SDS), followed by incubation at 65 °C overnight for reverse crosslink. Purify DNA of input and Lamin A antibody immunoprecipitation samples from control and patient-derived MSCs with QIAEX II Gel Extraction Kit. The extracted DNA was used for sequencing.
For western blot analysis, the Replication Proteion A antibody beads which combined Replication Proteion A antibody were resuspended in 50 μl 1×SDS loading buffer containing 100 mM DTT followed by incubation at 100 °C for 20 min. Input samples were added 4×SDS loading buffer containing 100 mM DTT followed by incubation at 100 °C for 20 min.
Whole-cell extracts of control and patient-derived MSCs (3×107) were prepared using RIPA lysis buffer (50 mM Tris, pH 7.4, 150 mM NaCl, 1% NP-40, 0.1% SDS, and 0.5% deoxycholate, supplemented with a protease inhibitor tablet) and centrifuged at 10,000 g for 10 min at 4 °C. Lysis were centrifuged and incubated with anti-Lamin A antibodies at 4 °C overnight. The next day, prewashed protein A/G beads were added and incubated at 4 °C for 4 h. The beads were washed with cold PBS 4 times and eluted with 50 mM Tris-HCl (pH=7.5) by incubation at 100 °C for 10 min for mass spectrometry.
CGAMP Quantification by Mass Spectrometry
CGAMP Quantification by Mass Spectrometry was conducted as described 76. Briefly, transfer the lysed cells in 300 μL extraction reagent with internal standard. Incubate samples and prepared cGAMP (Sigma Aldrich, 5.31889) calibration solutions to 95 °C for 15 min and cool down on ice. Precipitate proteins of the cell lysates by incubating at −20 °C overnight. Subsequently, centrifuge at 20,000 × g for 15 min to pellet down protein precipitates. Vaporize protein-free supernatants completely, and dissolve the pellet in 150 μL HPLC-grade water. Vortex samples and transfer 75 μL of the solution into the glass micro-inserts of the thread-bottles for HPLC-MS/MS measuring.
The FastQC and Trim Galore were used for raw data quality control, then the R package Rsubread 77 was used for mapping and counting the reads. The count matrix was normalized by FPKM. The differentially expressed genes were identified by the GFOLD, a Linux software 78. The GO annotations of DEGs were performed in the DAVID database (https://david.ncifcrf.gov/home.jsp).
ChIP-seq and DamID-seq Data Analysis
Reads from ChIP-seq and DamID-seq experiments were aligned to Human genome GRCh38 using annotated chromosomes and scaffolds. Alignment was performed using bowtie 2 79. Regions with an exceptionally high coverage of ChIP-Seq and DamID-seq reads were identified using MACS2 80. Wiggle files representing counts of ChIP-Seq and DamID-seq reads across the reference genome were created using MACS2. Resulting wiggle files were normalized for sequencing depth by dividing the read counts in each bin by the millions of mapped reads in each sample and were visualized in the UCSC genome browser (The human genome browser at UCSC, 2002) or WashU genome browser (The human epigenome browser at Washington University, 2011).
ATAC-seq Data Analysis
ATAC-seq reads were aligned using Bowtie 2 with the Human genome GRCh38. This was followed by peak calling on each replicate individually using MACS2 with the function “callpeak”. Peaks were classified (annotatePeaks.pl–annStats) as intronic, exonic, upstream or intergenic, according to the gene feature they intersected. Intersection is scored first considering the number of bases overlapped, and then the closeness in size between the peak and the feature. The height of the peaks, as well as any reads outside the peaks, were quantified using bedtools 81. The peak levels were divided by the background signal for normalization. The MACS2-generated files were visualized in the UCSC genome browser (The human genome browser at UCSC, 2002)
All analyses were repeated at least three times, and a representative experimental result was presented. Data were analyzed using GraphPad Prism version 8.0. Continuous variables with normal distribution are expressed as the mean ± standard deviation (SD). Comparisons between groups were all verified for normal distribution by D’Agostino-Pearson omnibus test. Student’s t-test (for pairwise comparisons) and one-way ANOVA (for comparisons among three or more groups) were used. The post hoc test with Bonferroni correction was performed for multiple comparisons following ANOVA. Asterisks denote statistical significance (*P < 0.05; **P < 0.01; *** P < 0.001). Data are shown as means ± SD (n ≥ 3).