Reagents and antibodies
The anti-Lamin B1 rabbit polyclonal antibody, anti-Beclin1 rabbit polyclonal antibody, anti-STING (TMEM173, EPR13130) rabbit monoclonal antibody, and anti-DNase II rabbit monoclonal antibody were purchased from Abcam (Cambridge, UK). The Stat6 (D-1) mouse monoclonal antibody, Lamin B1 mouse monoclonal antibody and cGAS (D-9) were purchased from Santa Cruz Biotechnology, Inc. (CA, 95060, USA), and anti-phospho-histone γH2AX mouse monoclonal antibody (Ser139) and anti-RPA2 mouse monoclonal antibody were from Millipore (Billerica, MA, USA). The anti-Lamin A/C (R386) rabbit polyclonal antibody, anti-IRF3 rabbit polyclonal antibody and anti-LAMP2 rabbit polyclonal antibody were from Bioworld Technology, Inc. (MN, USA). Anti-SQSTM1 rabbit polyclonal antibody and anti-LC3 rabbit polyclonal antibody were purchased from MBL, Ltd. (Chiba, Japan). Anti-phospho-STING (Ser366) rabbit monoclonal antibody and phospho-IRF3 rabbit monoclonal antibody were purchased from Cell Signaling Technology (Danvers, MA, USA). Nuclear Fast Red Staining Solution (0.1%; G1320), LysoTracker and DAPI (C0060) were purchased from Solarbio (Beijing, China). The full-length expression plasmids Flag-cGAS, Flag-SQSTM1 and Flag-Beclin-1 were purchased from YouBio, Inc. (Beijing, China). Bafilomycin A1 and H-151 were purchased from Selleck (Shanghai, China), and chloroquine (CQ) and 2,3'-cGAMP were purchased from Sigma Aldrich (St. Louis, MO, USA).
Cell culture and treatment
Breast cancer cells: MDA-231, MCF-7, BT-549, and other cancer cells: 786-0, DU145, PC-3M, HCT-116, and HeLa were maintained in 1640 or Dulbecco’s modified Eagle’s medium with high glucose (Gibco, Life Technologies, Grand Island, NY, USA) supplemented with 10% fetal bovine serum. The cells were incubated in a humidified atmosphere with 5% CO2 at 37 ℃.
Small interfering RNA
RNAi was designed and synthesized by GenePharma (Suzhou, China). RNAi was performed by the transfection of siRNA oligos using Lipofectamine 2000 transfection reagent (Invitrogen) according to the manufacturer’s instructions. The sequences are as follows: si-cGAS-1: Forward: 5'-GGCCUCUGCUUUGAUAACUTT-3', Reverse: 5'-AGUUAUCAAAGCAGAGGCCTT-3'; si-cGAS-2: Forward: 5'-GGCUAUCCUUCUCUCACAUTT-3'; Reverse: 5' –AUGUGAGAGAAG GAUAGCCTT-3'. si-LC3-1: Forward: 5'-GCUACAAGGGUGAGAAGCATT-3'; Reverse: 5'-UGCUUCUCACCCUUGUAGCTT-3'; si-LC3-2: Forward: 5'-GCGAGUUGGUCAAGAUCAU TT-3'; Reverse: 5'-AUGAUCUUGACCAACUCGCTT-3'; si-LC3-3: Forward: 5' –GCUUCCUC UAUAUGGUCUATT-3'; Reverse: 5'-UAGACCAUAUAGAGGAAGCTT-3'. si-SQSTM1-1: Forward: 5'-AGAUUCGCCGCUUCAGCUUTT-3'; Reverse: 5'-AAGCUGAAGCGGCGAAUC UTT-3'; si-SQSTM1-2: Forward: 5'-CGCUCACCGUGAAGGCCUATT -3'; Reverse: 5'-UAGGC CUUCACGGUGAGCGTT-3'; si-SQSTM1-3: Forward: 5'-GCACUACCGCGAU GAGGACTT-3'; Reverse: 5'-GUCCUCAUCGCGGUAGUGCTT-3';si-DNase2-1: Forward: 5'-GGCAGCCU GUAGACUGGUUTT-3'; Reverse: 5' -AACCAGUCUACAGGCUGCCTT-3'; si-DNase2-2: Forward: 5'-GCAUACAGCUGGCCUCAUATT-3'; Reverse: 5'-UAUGAGGCCAGCUGUAUG CTT-3'. The control RNAi (siNC) was composed of scrambled sequences.
Western blotting
Total cell lysates were obtained by incubating the cells in 2× SDS for 30 minutes at 4°C. After centrifugation at 10,000×g for 10 minutes at 4°C, the supernatant was collected and stored at -20°C for subsequent analysis. For cell fractions, cytoplasmic and nuclear proteins were extracted using nuclear and cytoplasmic protein extraction kits (Sangon Biotech Co., Shanghai, China), respectively. Equal amounts of cell proteins (20-40 μg/lane) were separated by SDS-PAGE in 10% gels and transferred to PVDF membranes (Millipore, Billerica, MA, USA) using a semidry transfer cell (Bio-Rad,Hercules, CA, USA) at 25 V for 60 minutes. The membranes were then blocked for 1 hour with TBS-T (20 mM Tris-HCl pH 7.6, 137 mM NaCl and 0.1% Tween-20) containing 5% nonfat dry milk (Cell Signaling Technology, Beverly, MA, USA) or with 1% BSA (Sigma Aldrich) and incubated overnight with primary antibodies. After the membranes were washed, they were incubated for 1 hour with peroxidase-conjugated goat anti-rabbit IgG or peroxidase-conjugated goat anti-mouse IgG. The proteins were visualized using an enhanced chemiluminescence kit (Bio-Rad, CA, USA). Band images of three independent experiments were quantified by optical density using Lab-Works 4.6 software (Bio-Rad, CA, USA). β-actin was used as an internal control for each protein. The antibodies included anti-LC3 (1:1000), anti-β-actin (1:1000), anti-cGAS (D-9) (1:500), anti-SQSTM1 (1:1000), anti-DNase2 (1:1000), anti-lamin A/C (R386) (1:500), anti-IRF3 (1:500), anti-TMEM173 (1:1000), and anti-LAMP2 (1:1000).
Immunofluorescence
Immunofluorescence staining was performed as described previously [18]. The results were observed and recorded using a fluorescence microscope (Model CX51; Olympus, Tokyo, Japan), and Photoshop version 7.0 (Adobe Systems, Inc.) was used to analyze the results. The antibodies used included anti-LC3 (1:1000), anti-β-actin (1:1000), anti-cGAS (D-9) (1:500), anti-SQSTM1 (1:1000), anti-DNase II (1:1000), anti-lamin A/C (R386) (1:500), IRF3 (1:500), anti-STING (1:1000), and anti-LAMP2 (1:1000).
Senescence associated-β-galactosidase staining (SA-β-gal)
Cells were subjected to SA-β-gal staining using an SA-β-gal staining kit (GenMed, GenMed Scientifics, Inc., USA) according to the manufacturer's directions. One hundred cells were counted in random fields, and the percentage of SA-β-gal-positive cells was calculated. Cell counting was repeated three times, and the mean was calculated.
Electron microscopy
Cells were fixed with 0.1 M sodium phosphate buffer (4% paraformaldehyde and 0.1% glutaraldehyde) at 4°C overnight. The samples were embedded with Lowicryl K4M (BioChemika), and sections were prepared with the Leica EM UC7 ultramicrotome and then stained with saturated uranyl acetate. The sections were observed under a transmission electron microscope (JEOL-1230, Japan) and recorded.
Co-immunoprecipitation
A total of 1x107 transfected cells were washed twice with PBS, 600 μl of precooled hypo-osmic buffer (25 mM Tris, pH 7.4, 85 mM KCl) was added, and the samples were incubated on ice for 30 minutes and centrifuged at 4°C at 12000 rpm for 10 minutes. The supernatant was saved and incubated with Flag antibody-conjugated agarose beads (MBL, Chiba, Japan) and gently shaken on a turntable overnight at 4°C. The beads were washed with hypo-osmic buffer containing protease inhibitor cocktail for 10 minutes; this process was repeated 4 times. Finally, the beads were dissolved in 1.5×SDS loading buffer, and 30 μl of supernatant was analyzed by Western blotting. The primary whole cytoplasmic supernatant was used as input.
IP-PCR
A total of 2×107 transfected cells were washed with preheated PBS at 37°C 3 times, fixed with 1% formaldehyde in PBS in a 37°C incubator for 15 minutes, quickly washed with ice-cold PBS 5 times, scraped into an Ep tube, and centrifuged at 800 g at 4°C for 3 minutes. Then, the supernatant was discarded, and 500 μl of hypo-osmic buffer (25 mM Tris, pH 7.4, 85 mM KCl) was added to isolate the cytoplasmic protein. Part of the supernatant was saved as the input. The remaining part was used for IP experiments. An appropriate amount of 500 μl elution buffer (1% SDS, 0.1 M sodium bicarbonate was used to elute the protein-DNA complex on the beads for 10 minutes at room temperature. Then, RNase A was added, the samples were heated at 37°C and shaken for 2 h, Proteinase K was added, and the samples were heated at 55°C and shaken for 2~3 h. Then, the samples were heated at 65°C and shaken overnight to isolate the protein-DNA complexes. Finally, the IP DNA was extracted by the phenol and chloroform method, and the results were analyzed by PCR.
Sucrose density gradient centrifugation
Gradient concentrations of sucrose solution (5%-40%, the concentration interval was 5%) with protease inhibitor cocktail were established as described [19]. The cytoplasmic proteins, extracted by hypo-osmic buffer as described above, were carefully dropped on the top layer and centrifuged at 40,000 rpm (Beckman, Brea, CA, USA) for 4 hours at 4°C. After centrifugation, the samples were carefully collected from a 500 μl aliquot of each fraction, and the aliquot of each fraction was analyzed by Western blot. Cytoplasmic DNA was extracted from 100 μl of each fraction and analyzed by PCR.
Acid extraction of cytoplasmic histones
Cytoplasmic histones were isolated by acid extraction methods with some modifications [20]. Briefly, cytoplasmic proteins from 1×107 cells of each kind, extracted by hypo-osmic buffer as described above, were slowly added to 0.4 N H2SO4 (500 μl of H2SO4 to a 100 μl cytoplasmic solution) and incubated at 4°C with intermittent rotation for 2 hours. After centrifugation at 5500 rpm for 5 minutes, the supernatants were gently added to 150 μl of 100% TCA (final concentration of 20%) and kept on ice for at least 5 hours without agitation. After centrifugation, the pellets were washed with 500 μl of acetone+0.1% HCl, and the tubes were left open overnight to evaporate the acetone. The pelleted histones were resuspended in 20 μl of ddH2O and subsequently analyzed by SDS-PAGE and Western blotting.
PCR detection
DNA in nuclear or cytoplasmic were separately extracted. The cells were cultured in 10 cm dishes and counted. A total of 1×103 cells were treated with hypo-osmic buffer (25 mM Tris pH 7.4, 85 mM KCl) and centrifuged, and the supernatant (the cytoplasmic components) was saved. Then, the cytoplasmic DNA was extracted by the phenol and chloroform method. The sediment (nuclear DNA) was extracted by Tigen Kits (Beijing, China). PCR was performed with using Alu and rDNA primers: Alu: Forward: 5'-CCTGAGGTCAGGAGTTCGAG-3'; Reverse: 5'-CCCGA GTAGCTGGGATTACA-3' (115 bp); 5.8S rDNA: Forward: 5'- GAGGCAACCCC CTCTCCTC TT-3'; Reverse: 5'-GAGCCGAGTTCACCGCTA-3' (136 bp); 18S rDNA: Forward: 5'-CGCCG CGCTCTACCTTACCTA-3'; Reverse: 5' -TAGGAGAGGAGCGAGCGACCA-3' (159 bp); 28S rDNA: Forward: 5' -CTCCGAGACGCGACCTCAGAT-3'; Reverse: 5'-CGGGTCTT CCGTAC GCCACAT-3' (173 bp). Quantitative real-time RT-PCR was performed on a PCR system (Applied Biosystems, Inc., Carlsbad, CA, USA) using SYBR Premix. The results were evaluated as the ratio of cytoplasmic: nuclear DNA; cytoplasmic DNA was normalized to nuclear DNA. The results were analyzed by GraphPad Prism 8.0.
FISH
The Alu probes were as follows: Alu-1 :5'-CCTGAGGTCAGGAGTTCGAGACCAGCCT-3'; Alu-2: 5'-ACGCCTGTAATCCCAGCACTTTGGGAGG-3'; Alu-3:5' –TCGCGCCACTGCACTC CAGCCTGGGCGA-3'. They were synthesized and conjugated with a single Quasar 570 molecule at the 5' end (Sango Biotech, Shanghai, China). Cells grown on cover slides were fixed in 3% paraformaldehyde (pH 7.4) containing 0.1% Triton-X 100 for 30 minutes and permeabilized in 0.1% Triton X-100 for 20 minutes. After denaturation at 95°C for 5 minutes, hybridization was performed in a mixture of probes (20 ng/per slide) and 35% deionized formamide, 10% dextran sulfate, 1× Dehart's, and 2× SSC for 20 hours at 42°C. The slides were washed for 40 minutes in 2×SSC with several changes. Nuclei were stained with Hoechst 34580 (Sigma Aldrich) for 10 minutes, and the results were observed and recorded using a fluorescence microscope (Model CX51, Olympus, Tokyo, Japan), and Photoshop version 7.0 (Adobe Systems, Inc. San Jose, CA, USA) was used to observe and analyze the results. At least 500 cells were evaluated, and the results were evaluated as the ratio of the intensity in cytoplasmic and nuclear DNA; cytoplasmic signals were normalized to those of the nuclei.
Comet assay
The comet assay was performed using a comet assay kit according to the manufacturer's instructions. First, 1×105 cells were prepared. Neutral or alkaline electrophoresis was performed. The slides were viewed by epifluorescence microscopy using a FITC filter. The results were analyzed by Comet Score software (Version 2.0038).
The comet assay was performed using a comet assay kit (Abcam, Cambridge, UK) according to the manufacturer's instructions. First, 1×105 cells were prepared. Comet Agarose was pipetted onto the Comet Slide to form base layer cells, which were combined with Comet Agarose at 37°C. Cell samples were combined with Comet Agarose at a 1/10 ratio (v/v). We pipetted the agarose/cell mixture on top of the base layer. The cells were treated with lysis buffer and alkaline solution. Electrophoresis was performed under alkaline or neutral conditions. Voltage was applied to the chamber for 10-15 minutes at 1 volt/cm. The cells were stained with DNA dye. The slides were viewed by epifluorescence microscopy using a FITC filter. The results were analyzed by Comet Score software (Version 2.0038).
BrdU incorporation assay
BrdU incorporation assays were performed as described previously (18). Briefly, BrdU (10 µM) was added to the culture medium for 30 minutes before analysis, and then, the cells were fixed with 4% formaldehyde, permeabilized with 0.1% Triton X-100, and denatured with 20 mM HCl in 150 mM NaCl and 3 mM KCl for 20 minutes at 25°C. The cells were then incubated with a primary antibody mixture composed of primary antibodies (BrdU and MCM7 or Lamin B1).
Trypan blue exclusion assay
Cell growth was determined by Trypan blue exclusion assays with a Trypan Blue Staining Cell Viability Assay Kit (Beyotime, Shanghai, China). Cells (1 ×104 cells/per well) grown with or without treatment with CQ, bafilomycin A1, or H-151 in 96-well plates were harvested, and 50 μl of trypan blue was added to a 50 μl cell suspension according to the manufacturer’s protocol. Viable cells were counted under a microscope with a hemocytometer. The assays were performed in triplicate and repeated at least three times.
Live/dead viability assay
A live/dead assay was performed using a live/dead cell viability assay kit (Abcam, Cambridge, UK). A total of 1×105 cells were seeded in 12-well or 96-well plates and incubated for 24 hours. The cells were treated with CQ or bafilomycin A1 and incubated for the indicated times. Subsequently, the cells were rinsed twice with PBS before the fluorochromes were added and incubated for 45 minutes. Fluorescence images were then taken (Model CX51, Olympus, Tokyo, Japan), and live or dead cells were counted and calculated.
MTS assay
MTS assays were performed using an MTS assay kit (Bestbio, Shanghai, China). A total of 1×104 cells were seeded in 96-well plates and incubated for 24 hours. The cells were treated with CQ or bafilomycin A1 and incubated for the indicated times. Ten microliters of MTS solution were added to each well and incubated for 3 hours at 37°C. The absorbance was measured at 490 nm, and cell viability was analyzed.
Statistical analysis
All analyses were performed using GraphPad Prism 8 (CA, USA) and Excel 2010 (WA, USA). Relationships were analyzed using t-tests. A P-value of less than 0.05 was considered significant. All statistical tests and P values were 2-sided, and the level of significance was set at <0.05 (*), <0.01 (**), <0.001 (***), or<0.0001 (****); ns indicates no significance.