Reagents and antibodies
Streptonigrin (SN), chloroquine (CQ), bafilomycin A1 (BA1), rapamycin (RAP), doxycycline (Dox), compound C, anti-GAPDH and anti-actin antibodies were obtained from Sigma-Aldrich (St. Louis, MO, USA). G418 and hygromycin B were purchased from Calbiochem (San Diego, CA, USA). Primary antibodies against GAPDH, β-tubulin, β-actin, LC3, Atg4, Atg5, Atg7, cleaved-caspase3, cleaved-PARP, AMPK/p-AMPK, and mTOR/p-mTOR were purchased from Cell Signalling Technology (Danvers, MA, USA). Anti-Cx32 antibody was obtained from Santa Cruz (Dallas, TX, USA). SiRNA targeting Cx32, Atg5 and AMPK were constructed by RiboBio (Guangzhou, China). EGFP-LC3 plasmids were constructed by Genecopoeia (Rockville, MD, USA). The BCA protein assay kit was purchased from Bio-Rad (Hercules, CA, USA). An Annexin V-FITC apoptosis detection kit was purchased from Biotool (Houston, TX, USA). A Chemiluminescent HRP substrate kit was obtained from Millipore Corporation (Billerica, MA, USA). Secondary antibody, Lipofectamine™ 2000, DEME and Eagle's minimum essential medium (EMEM) media were purchased from Invitrogen (Carlsbad, CA, USA).
Human cervical specimens and clinical data
The study was approved by the Research Committee of Ethics of the Affiliated Cancer Hospital of Xinjiang Medical University (13, 14). Human cervical tissue samples were collected from patients from 2012 to 2014 at the Affiliated Cancer Hospital of Xinjiang Medical University, Xinjiang, China. None of the patients had received any chemoradiotherapeutic agents preoperatively. We collected 50 specimens of CaCx from patients who underwent total hysterectomy and 30 specimens of benign multiple uterine fibroids as normal cervical controls. All the CaCx tissue specimens, corresponding peritumoural tissues (< 3 cm distance from the tumour tissue), and remote normal liver tissues (5 cm away from the tumour tissue) were collected within 10 min. After hysterectomy, cervical specimens were stored in liquid nitrogen for protein extraction. The expression of Cx32 and LC3 was detected by Western blotting.
Cell lines and low-density cultures
The humanCaCx cell line (C-33A) was purchased from American Type Culture Collection (Manassas, VA, USA) and were cultured in EMEM; HeLa-Cx32 cells (a gift from Dr. Andrew L. Harris) are a stable transgenic cell line expressing Cx32 under the control of a bidirectional tetracycline-inducible promoter that was previously described and characterized (14). The cells were grown as monolayer cultures in DMEM supplemented with 100 μg·ml−1 G418 sulfate and 200 μg·ml−1 hygromycin B. Cx32 expression was induced with 1 μg·ml−1 Dox for 48 h. All the cell lines were supplemented with 10% foetal bovine serum and were grown at 37°C in a 5% CO2 atmosphere. To physically inhibit gap junction formation, we adopted the low-density culture method, and 1×105 cells were seeded in a 150 mm dish to ensure that the cells were not in direct contact with each other (11, 13).
Tissue or cells were lysed with RIPA buffer (50 mM Tris-HCl pH 7.8, 150 mM NaCl, 2 mM EDTA, 1% Triton X-100, and 0.1% SDS) containing a protease inhibitor cocktail (Sigma-Aldrich). The proteins were quantified by using a Bio-Rad protein assay kit (Hercules, CA, USA), and equivalent amounts of protein (20 μg) were resolved on 12% or 9% SDS-PAGE gels and transferred onto 0.2 μm or 0.45 μm Immobilon-P transfer membranes (Millipore, Billerica, MA, USA). The membranes were subsequently incubated in blocking buffer (5% nonfat milk) for 1 h at room temperature and then incubated with appropriate primary antibodies in blocking buffer at 4°C overnight. The dilutions of the antibodies were as follows: anti-LC3 and anti-Cx32 were 1:1000; anti-cleaved-caspase3, anti-cleaved-PARP, anti-Atg4, anti-Atg5, anti-Atg7, anti-AMPK/p-AMPK and anti-mTOR/p-mTOR were 1:1500; and anti-β-tubulin, anti-β-actin and anti-GAPDH were 1:10000. The proteins were probed with the relevant secondary antibody, detected with an ECL reagent (Millipore) and quantified by ImageQuant LAS 4000TM and ImageJ software. Anti-β-tubulin, anti-β-actin and anti-GAPDH were used as loading controls.
Hoechst 33258 staining
Briefly, HeLa cells were fixed with 4% paraformaldehyde for 20 min and permeabilized with a solution containing 1% BSA and 0.5% Triton X-100 for 15 min at 37 °C. Then, the apoptotic cells were detected by staining with 0.1 μg·ml−1 Hoechst 33258 for 15 min in the dark and washed with PBS. Finally, morphological changes in apoptotic nuclei were observed under a fluorescence microscope (IX71; Olympus, Tokyo, Japan) with an ultraviolet filter.
Apoptosis was induced in cells by incubation with 1 μM SN for 7 h (11, 13). Apoptosis was assessed via flow cytometry using an annexin V-FITC apoptosis detection kit according to the manufacturer's protocol. After exposure to SN, the cells were trypsinized, washed and collected. Then, the cells were resuspended in annexin-V binding buffer and incubated with 5μl fluorescein isothiocyanate (FITC)-Annexin V and 2 μl PI for 15 min in the dark at 37°C. The apoptotic cellswere immediately analysed by flow cytometry using the Expo32 Software (Beckman XL, USA). The ratio of early apoptotic cells was compared to that of the controls for each experiment. Cell apoptosis was analysed with FlowJo 7.6 software.
Cells were fixed with 4% paraformaldehyde for 20 min, permeabilized with 0.5% Triton X-100 for 15 min, washed and blocked in 10% normal goat serum for 1 h. After blocking, the cells were incubated with a primary antibody against Cx32 (1:200) at 4°C overnight, washed and incubated with FITC-conjugated goat anti-mouse secondary antibody (1:400) in the dark at 37°C for 1 h. For identification of nuclear and cytoplasmic architecture, the cells were stained with 0.1 μg·ml−1 Hoechst 33342 for 10 min and Alexa Fluor 488 Phalloidin (1:20) for 20 min in the dark. The cells were observed under a confocal microscope.
Transmission electron microscopy
Cells were harvested by trypsinization, washed and fixed in 2.5% glutaraldehyde in 0.1 M phosphate buffer. After washing in phosphate buffer, the samples were post-fixed in 1% osmium tetroxide, washed again, and dehydrated in a graded series of ethanol. Then, the cells were embedded in spur resin for cutting into ultrathin sections. Transmission electron microscopy was used to observe all autophagosomal structures after staining the sections (a thickness of 50 nm) with uranyl acetate and lead citrate (29).
EGFP-LC3 plasmid transfection and siRNA interference
Cells were seeded in 6-well plates and grown to 80% confluence and then transfected with EGFP-LC3 plasmid using Lipofectamine 2000. For siRNA interference, the cells were grown to 30%-50% confluence, and then the cells were transfected for 6 h with the corresponding non-specific siRNA, targeted siRNA (50 nM) or scrambled control siRNA with Lipofectamine™ 2000. The cells were cultured in complete medium for 48h, and EGFP-LC3 plasmid transfection and siRNA interference were detected by immunofluorescence and Western blotting. The sequences for the synthetic DNA targeting EGFP-LC3 were as follows: forward primer: 5’ATGCCGTCGGAGAAGA
CCTTCAAG3’ and reverse primer: 5'TTACAC TGACAATTTCATCCCGAACGT3'. The sequences of the synthetic siRNAs targeting Cx32 (siCx32) were as follows: siCx32_1: 5’-CACCAACAACACATAGAAA-3’, siCx32_2: 5-GCATCTGCATTAT
CCTCAA-3’, and siCx32_3: 5’-GCCTCTCACCTGAATACAA-3’. The sequences of the synthetic siRNAs targeting Atg5 (siAtg5) were as follows: siAtg5_1: 5’-GGAATATCCTGCAGAAGAA-3’, siAtg5_2:5’-GGAACATCACAGTACATT
T-3’, and siAtg5_3:5’-GTGAGATATGGTTTGAATA-3’. The sequences of the synthetic siRNAs targeting AMPK (siAMPK) were as follows: siAMPK_1: 5′GAGGAGAGCTATTTGATTA_3′, siAMPK_2:5′-GCAGAAGTATGTAGAGCAA
-3′, and siAMPK_3: 5′-GATTGATGATGAAGCCTTA-3′.
All data are representative of at least three independent experiments and are presented as the mean ± standard error. Normal distribution test and homogeneity of variance test have been performed prior to statistical analysis. Analysis of variance (ANOVA), a Student’s t-test, and a Wilcoxon test were used to evaluate statistical significance. Pearson's correlation analysis was used toanalyse the correlation between Cx32 and LC3-II expression, and GraphPad Prism 6.0 software was used to create the histograms and scatter plots. A value of P<0.05 was considered to indicate a statistically significant difference.