Reagents and antibodies.
Lipofectamine 3000 (L3000015), MitoSOX (M36008), carboxy-H2DCFDA (C400) and the anti-IFI6 antibody (1:1000, PA5-99824) were purchased from Invitrogen (CA, USA). RPMI-1640 (72400047), penicillin and streptomycin (10378016), and Fetal bovine serum (FBS) (16140071) were obtained from Gibco (CA, USA). N-acetyl-L-cysteine (NAC) (A7250), PEG-catalase (CAT) (C1345), dithiothreitol (DDT) (45839), MitoTEMPO (SML0737), ATP (A6559), BAPTA-AM (A4926), and Thapsigargin (Tg) (T9033) were purchased from Sigma-Aldrich (Munich, Germany). DS16570511 (HY-115595) was obtained from MedChemExpress (NJ, USA). Antibodies against GAPDH (1:10000, ab181602), β-Actin (1:5000, ab179467), NCLX (1:1000, ab136975), MCU (1:1000, ab121499), VDAC1 (1 µg/ml, ab15895), NDUFB8 (1:5000, ab192878), SDHA (1:5000, ab137040), RISP (1 µg/ml, ab14746), COXIV (1 µg/ml, ab33985), ATPB (1:10000, ab170947), ATF3 (1:2000, ab207434), ATF4 (1:1000, ab184909), ATF6 (1:1000, ab122897), XBP1 (1:5000, ab109221), PDI (1:1000, ab2792), BiP (1:1000, ab21685), NOX1 (0.5 µg/ml, ab131088), NOX2 (1 µg/ml, ab80508), NOX3 (1 µg/ml, ab81864), NOX4 (1:1000, ab133303), and NOX5 (1:1000, ab198213) were obtained from Abcam (Cambridge, UK).
Human study subjects and cell cultures.
A total of 23 fresh ESCC samples and paired adjacent normal tissues (PANTs) were collected from patients undergoing esophagectomy at Shanghai Tongji Hospital Affiliated with Tongji University. Immunohistochemistry (IHC) for IFI6 was conducted on 3-µm sections of formalin-fixed, paraffin-embedded tissue samples consisting of nonpathological esophageal tissues (8 specimens), esophageal hyperplasia tissues (12 specimens) and ESCC tissues (83 specimens). The clinicopathological characteristics of the subjects are summarized in Tables S1 and S3. Informed consent was obtained from each patient before enrollment in this study. This study was approved by the Medical Ethics Committee of Shanghai Tongji Hospital.
The human ESCC cell lines Eca109, TE-1, Ec9706, Kyse150, and Kyse410 and the normal esophageal squamous epithelial cell line Het-1a were obtained from the Cell Bank of the Chinese Academy of Sciences (Shanghai, China). All cells were cultivated in RPMI 1640 medium supplemented with 10% FBS and 1% penicillin and streptomycin at 37 °C in 5% CO2. All cell lines were authenticated by the STR method and tested for mycoplasma contamination.
In vivo study.
Four- to five-week-old female BALB/c nude mice were purchased from Jiesijie Laboratory Animal (Shanghai, China). All animal experiments were approved by the Animal Care and Use Committee of Shanghai Tongji Hospital and conducted in accordance with ethical standards. Nude mice were randomly divided into four groups as follows (n = 5 mice/group): (i) ShControl; (ii) IFI6 KD; (iii) OEControl; and (iv) IFI6 OE. The indicated Eca109 cells (2 × 106 cells per 0.1 ml of phosphate-buffered saline) were suspended in Matrigel (Biosciences, CA, USA) and implanted subcutaneously into the right flanks of the nude mice. The tumor volume was measured using a caliper every 5 days. Four weeks after implantation, the nude mice were sacrificed, and tumor samples were harvested.
RNA extraction and qRT-PCR.
One microgram of total RNA was extracted using TRIzol obtained from Invitrogen (CA, USA) and was reverse transcribed to cDNA with SuperScript II (Invitrogen, CA, USA). qRT-PCR was conducted using SYBR Green technology (Takara Bio, Beijing, China). β-Actin was used as the housekeeping gene. Data were analyzed through the 2−ΔCT method. The primers used for detection are shown in Table S4.
Cells and tissues were collected and lysed in cell/tissue lysis buffer (Solarbio, Beijing, China). Proteins were extracted, and the protein concentration was measured by the BCA method (Solarbio, Beijing, China). Proteins were separated by 10% sodium dodecyl sulfate (SDS)-PAGE and transferred to 0.45-µm polyvinylidene difluoride membranes (Invitrogen, CA, USA), which were blocked with blocking buffer (EpiZyme, Shanghai, China), incubated overnight with primary antibodies, and washed and incubated with the HRP-conjugated anti-rabbit secondary antibody (Abcam, UK) for 1 h. Peroxidase activity was visualized via the ECL method (EpiZyme, Shanghai, China).
Paraffin-embedded sections were deparaffinized and rehydrated and were then subjected to antigen retrieval for 20 min in citrate buffer (pH 6.0). After the sections were cooled, 3% H2O2 was used to block endogenous peroxidases. The sections were blocked with 10% goat serum and incubated with the anti-IFI6 primary antibody (Invitrogen, Carlsbad, CA, USA) at a 1:1000 dilution overnight at 4 °C. After incubation with the secondary antibody, immunodetection was performed with DAB staining (Zhongshan Goldenbridge Biotechnology Company, Beijing, China). For evaluation, three staining fields in each section were randomly selected and evaluated by two independent pathologists who were blinded to patient information. The (Staining index) SI of IFI6 was determined by combining the staining intensity score (1, negative; 2, weak staining; 3, moderate staining; 4, strong staining) and the proportion of positively stained tumor cells (0, no positive cells; 1, < 10%; 2, 10–35%; 3, 35–75%; 4, > 75%). Samples with SI ≥ 8 were considered to exhibit high expression, while samples with SI < 8 were deemed to be low expression samples .
Mitochondria were isolated from cells with a mitochondria extraction kit (Solarbio, Beijing, China) according to the manufacturer’s instructions. BN-PAGE was performed with the NativePAGE™ system (Invitrogen, CA, USA). In brief, mitochondria were solubilized with digitonin (digitonin/protein ratio of 4 g g− 1), incubated on ice for 30 min, and centrifuged at 20000 × g for 30 min. The solubilized protein was collected from the supernatant, and the protein concentration was determined by the BCA (Solarbio, Beijing, China) method. Protein samples were mixed with Coomassie blue G-250 (Invitrogen, CA, USA) to obtain a dye/detergent mass ratio of 1/4 and were then loaded into a 4–16% Bis-Tris gel. After electrophoresis, proteins were transferred to 0.45-µm polyvinylidene difluoride membranes (Invitrogen, CA, USA) and were then sequentially probed with antibodies against complex I (NDUFB8), complex III (RISP), complex IV (COX IV), complex II (SDHA) and complex V (ATPB). The NDUFB8, RISP and COX IV immunoblot bands with high molecular weights were used to identify the complex I-, complex III-, or complex IV-containing respiratory supercomplexes (RSCs). Signal intensities were normalized to their corresponding ATPB signals.
Cell proliferation assay.
To assess cell proliferation, the indicated Eca109 and TE-1 cells were seeded in 96-well plates and incubated under standard conditions in complete medium. Twenty-four hours after seeding, cells were incubated with 50 µM EdU (RiboBio, Guangzhou, China) for 6 h and subsequently subjected to fixation, permeabilization and EdU staining, which were conducted according to the manufacturer’s instructions. Nuclei were counterstained with Hoechst 33342 (Invitrogen, CA, USA) for 10 min. The proportion of cells stained with EdU was determined via fluorescence microscopy.
Detection of the mitochondrial membrane potential and apoptosis induction.
The indicated Eca109 and TE-1 cells were seeded in 6-well plates and treated as indicated. Then, the cells were detached, washed, double-stained with Annexin V-FITC and PI (BD Biosciences, CA, USA) based on the manufacturer’s recommendation, and analyzed by flow cytometry (Beckman Coulter, CA, USA). FlowJo (Tree Star, OR, USA) was used for data presentation and analysis.
The mitochondrial membrane potential was assessed via the JC-1 method. In brief, the indicated cells were washed and incubated with JC-1 (Beyotime, Beijing, China) working solutions for 20 min at 37 °C in the dark. After incubation, the cells were washed with JC-1 wash buffer and observed under a fluorescence microscope. In healthy cells with a Δψm indicating membrane depolarization, JC-1 forms JC-1 aggregates exhibiting red fluorescence. In apoptotic cells, JC-1 remains monomeric, exhibiting green fluorescence.
Measurement of ATP levels.
To assess cellular ATP production, an ATP luminescence assay (Invitrogen, CA, USA) was used for quantitative determination of ATP levels with recombinant firefly luciferase and its substrate D-luciferin according to the manufacturer’s recommendation. After a standard curve for a series of ATP concentrations was generated, the ATP content in the samples was calculated from the standard curve.
Analysis of cellular respiration.
The Seahorse XFe96 Bioanalyzer (Seahorse Bioscience, MA, USA) was used to determine the OCR and ECAR according to the manufacturer’s instructions. ECAR and OCR were determined using the Seahorse XF Glycolysis Stress Test Kit (Seahorse Bioscience, MA, USA) and the Seahorse XF Cell Mito Stress Test Kit (Seahorse Bioscience, MA, USA), respectively. In brief, for OCR determination, 20000 of the indicated ESCC cells were seeded in a Seahorse 96-well plate, and 1 h before measurement, the culture medium was replaced with Seahorse XF medium supplemented with 1 mM sodium pyruvate, 2 mM glutamate, and 25 mM glucose. Periodic measurements of the OCR were performed at baseline and after sequential administration of oligomycin (Oligo, 1 µM), trifluoromethoxy carbonylcyanide phenylhydrazone (FCCP, 0.5 µM), and rotenone (Rot, 0.5 µM) and antimycin A (AMA, 0.5 µM) (Rot&AMA). For ECAR measurement, the culture medium was replaced with XF assay medium supplemented with L-glutamine (300 µg/ml). Glucose (10 mM), Oligo (1 µM) and 2-deoxy-D-glucose (2-DG) (100 mM) were sequentially added to the cell culture, and real-time ECAR was determined with the Seahorse XFe96 Bioanalyzer. For complex-specific OCR measurement, cells were permeabilized with a solution of digitonin (500 µg per 105 cells) supplemented with 1 mM MgCl2, 1 mM ADP, 5 mM KH2PO4, 150 mM KCl and 5 mM Tris (pH 7.2). The substrates and inhibitors used for complex-specific OCR measurement were malate (5 mM), Rot (1 µM), AMA (1 µM), ascorbate (5 mM), TMPD (500 µM), pyruvate (5 mM), succinate (5 mM), and G3P (5 mM). All reagents and compounds were purchased from Sigma-Aldrich (Munich, Germany).
For lentiviral-mediated transduction of shRNA, cells were initially treated with polybrene (Sigma-Aldrich, Munich, Germany) and selected with puromycin (Sigma-Aldrich, Munich, Germany). ShRNA-IFI6, shRNA-ATF3, shRNA-NOX4, and negative control shRNAs were purchased from Genechem (Shanghai, China). The oligonucleotide sequences used to silence target genes are shown in Supplementary Table S5. IFI6, ATF3, and NOX4 lentiviruses were purchased from Genechem (Shanghai, China).
For construction of overexpression plasmids, the pcDNA3.1 (V79020) expression plasmid was obtained from Invitrogen (CA, USA). Human IFI6, ATF3, and NOX4 cDNA from Eca109 cells were cloned by PCR and subcloned into the pcDNA3.1 expression vector to generate IFI6-pcDNA3.1, ATF3-pcDNA3.1, and NOX4-pcDNA3.1. After construction, the IFI6-, ATF3-, or NOX4-pcDNA3.1 plasmid was transfected into Eca109 and TE-1 cells using Lipofectamine 3000 and selected with G418.
Measurement of mitochondrial Ca 2+ .
To monitor mitochondrial calcium dynamics, the indicated ESCC cells were incubated with the calcium-specific fluorescent probe Rhod-2 AM (Invitrogen, CA, USA) in Hanks' balanced salt solution at room temperature for 30 min. After washout, fluorescence was recorded with an integrated spectrofluorometer (Photon Technology International, NJ, USA) at excitation and emission wavelengths of 550 nm and 580 nm, respectively. After 1 min of baseline recording (basal signal, F0), cells were promptly treated with the agonist Tg, and fluorescence intensities were monitored at 3-s intervals for another 10 min. The relative fluorescence intensity (F/F0), where F represents the fluorescence intensity at a given time and F0 represents the initial fluorescence intensity, was used to indicate [Ca2+].
Detection of cellular and mitochondria-specific ROS determination.
To detect mitochondrial ROS, the indicated ESCC cells were seeded in 24-well plates and incubated with the mitochondria-specific ROS probe MitoSOX (Invitrogen, CA, USA) for 20 min at 37 °C. For cellular ROS determination, the indicated ESCC cells were seeded in 24-well plates, loaded with the cellular ROS indicator carboxy-H2DCFDA and further incubated at 37 °C for 20 min. Nuclei were counterstained with Hoechst 33342, and the fluorescence intensity was measured by fluorescence microscopy or flow cytometry.
Construction of reporter plasmids and luciferase assay.
Different fragments (containing bp -3000 to + 1, -2000 to + 1, or -1000 to + 1) of the human NOX4 promoter were synthesized and subcloned into the pGL3 vector (Promega, WI, USA). Another set of mutant plasmids was constructed; in these plasmids, one of the potential ATF3-binding motifs (AAGGACTCACT, ACTAATGTCATG, TATGAAGACATTT, or AATTGCATCACC) was deleted from the NOX4 promoter.
The above promoter reporters were transfected into Eca109, TE-1, or HEK293T cells or were cotransfected with the ATF3-pcDNA3.1 expression vector. The indicated cells were harvested, and the promoter activity of NOX4 was measured with a dual luciferase reporter assay kit (Promega, WI, USA) according to the manufacturer’s instructions. In brief, the indicated cells were seeded in 24-well plates, transfected with reporter plasmids using Lipofectamine 3000 (Invitrogen, CA, USA) and incubated for 24 h. After transfection, cells were collected with passive buffer, and firefly and Renilla luciferase activities were measured with a Dual Luciferase Reporter Assay System (Promega, WI, USA) and an illuminometer.
RNA-seq expression data (quantified by count and fragments per kilobase per million mapped reads (FPKM)) in the TCGA-ESCA dataset were downloaded from the Genomic Data Commons (GDC) portal (https://portal.gdc.cancer.gov/). TCGA dataset manipulation and analysis as well as GSEA were implemented in R 3.6.0.
For microarray data analysis, normalized data (GEO accession no. GSE20347, no. GSE23400, no. GSE45670 and no. GSE75241) and the corresponding probe annotation data were downloaded from the NCBI GEO database (https://www.ncbi.nlm.nih.gov/gds). Microarray data manipulation and analysis were performed in R 3.6.0.
IFI6 coexpression analysis was based on calculation of the Pearson correlation coefficient (rPCC) values of the correlations between the expression levels of IFI6 and potential mRNAs in the four above GEO datasets. mRNAs for which |rPCC|>0.5 and P < 0.05 were recommended for further analysis (Table S6).
Transcription factor-promoter binding predictions were performed with JASPAR (http://jaspar.genereg.net).