Generation ofATRX-depleted HeLa cell lines
HeLa and 293T cells were obtained from American Type Culture Collection and grown in Dulbecco's modified Eagle’s medium (Gibco, Grand Island, NY, USA). To obtain stable ATRX-depleted cell lines, 293T cells were transfected with shRNAs (sense sequences are shown in Table 1) using the lentiviral expression vector pGIZ (a gift of Dr. Shen from the Cancer Institute of New Jersey, Rutgers, USA). 293T cells were seeded in a 6-cm dish at a density of 1.2 × 106 , and 24 h later were transfected with 120 μl of transfection reagents (Shanghai YESEN Biotechnology Co., Ltd.), 3 μg shRNA plasmids, 1.5 μg pSPAX2 packaging plasmids, and 1.5 μg pMD2G enveloping plasmids according to the manufacturer’s instructions. Lentiviral supernatants were harvested at 48 and 72 h after transfection and filtered with a 0.45 μm filter (Merck Millipore, Billerica, MA, USA). HeLa cells were cultured in 6-well plates at a density of 2.0 × 105 and infected with lentiviral supernatants supplemented with 10 mg/ml Polybrene (Sigma-Aldrich; St. Louis, MO, USA). HeLa Cells were sub-cultured into 6-cm dishes and selected with 1 μg/ml Puromycin (Sigma-Aldrich; St. Louis, MO, USA). ATRX protein expression was measured using an anti-ATRX antibody (Santa Cruz, CA, USA) by immunofluorescence (IF) and western blotting (WB) to identify ATRX-depleted HeLa cell lines.
Colony formation assay
Cells were seeded into 6-cm dishes at densities of 8 × 101 (0 Gy), 2 × 102 (2 Gy), 8 × 102 (4 Gy), 4 × 103 (6 Gy), and 2 × 104 (8 Gy), and were irradiated by with an X-RAD 320iX machine (Precision X-ray, Inc, USA) at a dose rate of 1.0 Gy/min. After 10 days, cells were fixed with methanol and dyed with Giemsa, and the colonies were counted and calculated. The survival fraction (SF) was calculated as the ratio of treated group colony number to untreated group colony number [26].
CCK8 assay
Cells were seeded in 96-well plates at a density of 6 × 104 or 10-cm dishes at 2 × 106 cells, and irradiated with X-rays at 0, 2, and 8 Gy. In the 96-well plates, CCK-8 reagents were added into each well at 0, 6, 10, 24, and 48 h and A490 values were detected. In the 10-cm dishes, the supernatants were collected at 48 h, filtered through a 0.45 μm filter, and added to the HeLa cells cultured in 96-well plates, and A490 was detected as described above. b There were six replicate wells per group. The experiment was performed in triplicate.
Flow cytometry
Cell cycle distribution, polyploid cells, and apoptosis were detected by flow cytometry. For cell cycle and polyploid analysis, cells were collected after 24 h of irradiation with 2 and 8 Gy, centrifuged at 1000 rpm for 5 min, the supernatant was discarded, and cells were washed twice with PBS. Cells were re-suspended in 500 μl propidium iodide (BD, Franklin Lakes, NJ, USA) and stained for 30 min at room temperature (RT). For apoptosis detection, cells were stained with 10 μl Annexin V and 7-AAD (BD) for 15 min in the dark. Cells were collected and analyzed using FACSCanto II (BD).
Senescence analyses
Cells were seeded in 6-well plates at a density of 1 × 105 and irradiated with 4 Gy. After 0.5, 1, 2, 5, and 7 days, cell senescence was detected using senescence-associated β-galactosidase (SA-β-gal) kits (Beyotime® Biotechnology, Hangzhou, China). Cell number was quantified by β-galactosidase staining as a proportion of total cells [27].
Quantitative real time PCR(qRT-PCR)
RNA was extracted using the TRIzol reagent (Invitrogen, Carlsbad, CA, USA) after 48 h of irradiation at 0, 2, and 8 Gy, and the complementary DNA (cDNA) was synthesized from 1 μg of each RNA sample using a high-capacity reverse transcription kit (Takara Bio Inc. Japan). Primer sequences are shown in Table 2. The qRT-PCR reaction was performed using the CFX96 Touch Real-Time PCR Detection System (Bio-Rad, Hercules, CA, USA) and with the SYBR® Premix Ex Taq ™ II kit (Takara Bio Inc., Japan) protocol. The qRT-PCR data were analyzed using the 2―△△Ct method [28].
Immunofluorescence
The shCon-, shATRX1-, shATRX2-, and shATRX3-HeLa cells were grown on coverslips placed in 6-well plates for 12 h. Then, the coverslips were removed, fixed with 4% paraformaldehyde for 10 min at RT, permeabilized, and blocked with blocking buffer (0.3% Triton X-100 and 2% BSA in PBS) for 1 h at RT. The cells were incubated with anti-ATRX (1:500 in blocking buffer) overnight at 4°C, followed by incubation with secondary antibodies (1:1000, red fluorescence) for 1 h at 37°C. The coverslips were mounted onto microscope slides, fluorescence was observed, and images were processed.
γH2AX foci
Cells were irradiated with 4 Gy, and after 0, 0.5, 1, 3, and 6 h, the coverslips were removed and stained as described above. Anti-γH2AX (Cell Signaling Technology, Danvers, MA, USA) (1:500 in blocking buffer) and secondary antibodies (1:1000, red fluorescence) were used for the immune reaction. DAPI was used for nuclear staining. The coverslips were mounted on microscope slides, red fluorescence was observed, and γH2AX foci were quantified as a proportion of total cells [29].
Western blot
Total proteins were extracted and quantified, and 40 μg protein were separated by SDS-PAGE, followed by transfer to nitrocellulose membranes (200 mA, 1.5 h; Merck Millipore, Billerica, MA, USA). After blocking with 5% milk, the membranes were incubated with primary antibodies including anti-ATRX, anti-PARP1, anti-ATM, anti-p-ATM, and anti-GAPDH (Santa Cruz, CA, USA), anti-γH2AX, anti-Chk2, anti-p-Chk2, anti-Cyclin B1, anti-caspase-9 (cleaved), and anti-caspase-3 (cleaved) (Cell Signaling Technology), anti-Rad51 (Abcam, Cambridge, MA, USA), and anti-cdc2 (GeneTex, Alton Pkwy Irvine, CA, USA) overnight at 4°C. After washing with TBST, the membranes were incubated with IgG-HRP conjugated secondary antibody (Immunoway, Plano, TX, USA) for 1.5 h at RT. Finally, the membranes were detected using an enhanced chemiluminescence detection system (ECL detection kit, Santa Cruz, CA, USA). The films were scanned for quantification of protein bands.
Statistical analysis
All statistical analyses were performed using SPSS 24.0 (SPSS Inc., Chicago, IL, USA) or Graphpad Prism 5.0 (La Jolla, CA, USA). The results are presented as the mean ± SD and analyzed using the Student’s t test. All differences were considered statistically significant at P < 0.05.