Samples of human tumor tissue specimens and adjacent non-tumor tissues
Samples of paired HCC and adjacent non-tumor liver tissues from patients undergoing HCC resection were obtained from the Department of Medicine, General Surgery and Transplantation of the University of Udine, Udine, Italy. None of the patients had received any local or systemic anticancer treatments before the surgery. Both tumor and non-tumor tissues were histologically confirmed. This study was approved by the Regional Ethic Committee (CERU), August 24, 2019, Protocol number 18659, and informed consent was obtained from each patient.
In the registry of the Pathology Department of University Hospital of Udine, Udine, Italy, a total of 20 patients who had a pathological diagnosis of hepatocellular carcinoma between January 2015 and December 2017, were identified. For each case, a single pathologist looked at slides stained with hematoxylin-eosin to evaluate the histological grading of HCC according to the Edmondson and Steiner criteria. Another slide (not stained) was used for immunohistochemical analysis. Each slide included both the tumor and not neoplastic liver (cirrhosis or normal liver).
Immunoistochemical detection of APE1 was performed by immunohistochemistry using the anti-APE1 mouse monoclonal antibody as the primary antibody (Novus Biologicals, Cambridge, England). The slides were deparaffinized and rehydrated (Xylene: three washes for 5 minutes each; 100% Ethanol: two washes 10 minutes each; 95% Ethanol: two washes 10 minutes each; 70% Ethanol: two washes 10 minutes each; 50% Ethanol: two washes 10 minutes each; distilled water: two washes for 5 minutes). Using microwave, the tissue sections were brought to boil in 10 mM sodium citrate buffer (pH 6.0) and then maintained at a sub-boiling temperature for 10 minutes. The tissue sections were quenched with 3.0% hydrogen peroxide in methanol for at least 15 minutes to block endogenous peroxidase activity. To permeabilize the cells, the tissue sections were washed with 1% animal serum in PBS with 0.4% Triton X-100 (PBS-T). Then, the tissue sections were incubated with 5% animal serum in PBS-T for 30 minutes at room temperature to block any non-specific binding. The primary antibody (diluted 1:200; Novus Biologicals, Cambridge, England) was added and the tissue sections were incubated for 12 hours at room temperature and then at 4 °C overnight. A DAKO REAL EnVision Rabbit/Mouse (K5007) was used as a second antibody. Horseradish peroxidase activity was detected using DAKO REAL 3,3′-diaminobenzidine + chromogen (K5007) as substrate for 3 minutes in accordance with the manufacturer’s instructions. Sections were counter-stained with hematoxylin with a cover slip. We consider the reaction for APE1 positive when a dense, homogeneous brown staining is identified in the nucleus of hepatocyte and when a granular brown staining is identified in the cytoplasm of hepatocyte.
Stable HeLa clones for inducible silencing of endogenous APE1 and re-expression of recombinant shRNA resistant APE1 WT and mitochondrially targeted APE1 (MTS-APE1) were obtained as previously described [34, 35]. MTS-APE1 resistant sequence was designed substituting the N-terminal sequence involved in the nuclear localization of the protein with the well-characterized MTS sequence of manganese-superoxide dismutase (MLSRAVCGTSRQLAPALGYLGSRQ) [22, 23]. Expression and localization of the recombinant protein were confirmed by WB (Fig. 2A). APE1 silencing was induced by addition of doxycycline to the cell culture medium at a final concentration of 1 µg/mL for 10 days.
Nuclei And Mitochondria Isolation From Human Hcc Tissue Specimens
After collection, all procedures were carried out at 4 °C and in the presence of protease inhibitors to avoid proteins degradation. Fresh samples were finely minced, suspended in 5 mL of Isolation Buffer (IB) [10 mM Tris/MOPS, 1 mM EGTA/Tris, 200 mM Sucrose], and homogenized. Then, sample were centrifuged at 70 x g for 3 minutes to remove non-homogenized tissue. Supernatant was further centrifuged at 600 x g for 10 minutes to separate nuclear (pellet) and mitochondrial (supernatant) fractions. Nuclei were washed in T1 solution [10 mM HEPES pH 7.9, 0.1 mM EDTA pH 8.0, 10 mM KCl, 0.1 mM MgCl2] and then lysed in T2 solution [10 mM HEPES pH 7.9, 0.1 mM EDTA pH 8.0, 400 mM NaCl, 1.5 mM MgCl2, 5% glycerol] for 20 minutes on ice. After centrifugation at 14.000 x g for 20 minutes, the supernatant, accounting for nuclear protein extract, was collected. Mitochondria were centrifuged at 7.000 x g for 10 minutes, washed once in IB buffer, and then resuspended in IB buffer. Nuclear and mitochondria protein extracts were then quantified using Bio-Rad protein assay reagent (Bio-Rad).
Mtdna Damage Measurement By Quantitative Pcr In Patients’ Samples
mtDNA isolation and damage measurement were performed as previously described by Barchiesi et al. . Briefly, mtDNA was extracted by patients isolated mitochondria from non-tumor or HCC sample using a plasmid isolation kit  and quantified by Quant-iT™ PicoGreen™ dsDNA Reagent (Invitrogen). Q-PCR was performed on each sample to amplify a 16 ∼ Kbp fragment, using the following primers: FOR 5’-TCT AAG CCT CCT TAT TCG AGC CGA-3’ and REV 5’- CCA TCC AAC ATC TCC GCA TGA TGA AA-3’. Fluorescence readings of the Q-PCR reactions were quantified in triplicate with Quant-iT™ PicoGreen™ dsDNA Reagent (Invitrogen) and then averaged for each sample. Blank value was subtracted and the ratio of the fluorescence readings obtained for the tumor tissue to those of the distal section determined the relative amplification of the mtDNA for each patient sample. Relative mtDNA damage was then expressed as the inverse of this relative amplification.
Preparation Of Total Cell Extracts And Subcellular Fractionation
To prepare total protein extracts, cells were harvested by trypsinization and centrifuged at 250 x g for 5 minutes at 4 °C. The pellet was washed once with cold PBS and then resuspended in Lysis buffer [50 mM Tris-HCl pH 7.5, 150 mM NaCl, 1 mM ethylenediaminetetracetic acid (EDTA), 1% (vol/vol) Triton X-100, protease inhibitor cocktail (Sigma), 0.5 mM phenylmethylsulfonyl fluoride (PMSF)] at a cell density of 107 cells/mL, incubated on ice for 30 minutes, and centrifuged at 20.000 x g for 20 minutes at 4 °C. The supernatant was collected as total cell lysate (TCE). For subcellular fractionation, cells were scraped in PBS, collected, and centrifuged at 250 x g for 5 minutes. Then, the pellet was suspended at a cell density of 100 mg/mL in Mitochondrial Isolation Buffer (MIB) [20 mM HEPES pH 7.6, 1 mM EDTA, 220 mM Mannitol, 70 mM Sucrose] supplemented with 2 mg/mL Bovine Serum Albumin (BSA). Cells were mechanically broken using a 7 mL dounce homogenizer (Wheaton), centrifuged at 650 x g for 10 minutes at 4 °C. The pellet was conserved to prepare nuclear subfraction. Supernatant collected was centrifuged at 14.000 x g for 15 minutes at 4 °C. Isolated mitochondria were washed with MIB supplemented with 2 mg/mL BSA and 1M KCl and centrifuged as before. A last wash was performed using MIB without BSA, and then mitochondria were resuspended in MIB and considered as mitochondrial protein extract (MCE). In parallel, nuclei were resuspended in T1 solution [10 mM HEPES pH 7.9, 10 mM KCl, 0.1 mM MgCl2, 0.1 mM EDTA70, 2 mM PMSF] and centrifuged at 1.000 x g for 15 minutes at 4 °C. This step was performed twice followed by nuclei resuspension in T2 lysis buffer [20 mM HEPES pH 7.9, 420 mM NaCl, 1.5 mM MgCl2, 0.1 mM EDTA70, 5% glycerol, 2 mM PMSF]. Samples were incubated on ice for 20 minutes and centrifuged at 20.000 x g for 20 minutes at 4 °C. Supernatant represented the nuclear protein fraction (NCE).
Protein concentration was determined using Bio-Rad protein assay reagent (Bio-Rad). Subfractions purity was evaluated by Western blot analysis using LSD1 and ATP5A as nuclear and mitochondria markers, respectively, to exclude the presence of cross contaminations between the two organelles.
Western Blot Analysis
The reported amount of total (TCE), nuclear (NCE) or mitochondrial (MCE) protein extracts were separated onto 12% SDS-PAGE. Then, proteins were transferred into a nitrocellulose membrane (Sartourius Stedim Biotech S.A.). Saturation of the membranes was performed for 1 h at room temperature using 5% non-fat dry milk in TBS-T [1XTBS supplemented with 0.1% Tween 20], followed by primary antibody incubation overnight at 4 °C [anti-APE1: 1:1.000 monoclonal (Novus); anti-Mia40: 1:500 polyclonal (costumed produced by APS Antibody Production Services); anti-FLAG: 1:1.000 monoclonal (Sigma); anti-ATP5A: 1:2.000 monoclonal (Abcam); anti-LSD1: 10.000 polyclonal (Abcam)]. Membranes were washed three times for 5 minutes with TBS-T, incubated for 2 h with the secondary antibody, and washed again for three time. The signal was detected with the Odyssey CLx scanner (Li-Cor Bioscience) and densitometric analysis was performed with ImageStudio software (Li-Cor Bioscience). In accordance with our Digital Image Integrity Policy uncropped Western blot images have been included as Supplementary Materials.
Dna Extraction And Mtdna Damage Analysis In Cell Lines
DNA was extracted using Qiagen genomic-tip 20/G and following manufacturer’s indications. After isolation DNA was precipitated overnight with isopropanol, and then 10 µg were digested with Formamidopyrimidine DNA Glycosylase (Fpg) enzyme at 37 °C for 30 minutes to remove damaged bases leaving an abasic (AP) site. Fpg was inactivated at 60 °C for 10 minutes and DNA was precipitated overnight, resuspended in 50 µL of Tris-EDTA buffer pH 8.0. Quantification was determined with Quant-iT™ PicoGreen™ dsDNA Reagent (Invitrogen), according to manufacturer’s instructions and DNA concentration was adjusted to 3 ng/µL.
mtDNA lesions were quantified by Q-PCR, using the following primers: Mitolong Forward: 5’-TCT AAG CCT CCT TAT TCG AGC CGA-3’ and Mitolong Reverse: 5’-TTT CAT CAT GCG GAG ATG TTG GAT GG-3’ which amplified an 8.9 Kbp mitochondrial fragment; Mitoshort Forward: 5-CCC CAC AAA CCC CAT TAC TAA ACC CA-3’ and Mitoshort Reverse: 5’-TTT CAT CAT GCG GAG ATG TTG GAT GG-3’ which amplified a 221 bp mitochondrial fragment. DNA was amplified using Platinum™ SuperFi™ DNA Polymerase (Invitrogen) using the following protocol: 2 minutes at 94 °C, 18 cycles of denaturation for 15 sec at 94 °C, annealing for 10 seconds at 66 °C, extension for 5.30 minutes at 68 °C for the 8.9 Kb fragment or annealing 45 seconds at 60 °C and extension for 45 seconds at 72 °C for the 221 bp fragment. A final extension for 10 minutes at 68 or 72 °C was performed for each fragment. To ensure quantitative conditions a sample with the 50% of template amount was included in each amplification and, as negative control, a sample without the template were used. PCR products were quantified in triplicate by using Quant-iT™ PicoGreen™ dsDNA Reagent (Invitrogen). The Mitoshort fragment was used to calculate the relative amount of mtDNA copies and to normalize the lesions frequency calculated with the Mitolong fragment .
For the clonogenic assay, 500 cells were plated the day before the beginning of the silencing. After 10 days, cells were stained with 0.5% (wt/vol) methyl violet. Four biological replicates were preformed and for each replicate four 10 cm2 plates per clone were analyzed. Plates were imaged using a live scanner (GE Healthcare). The analysis was performed using a modified CellProfiler pipeline for colonies counting . Briefly, the pipeline used was based on four steps: background correction, identification of the single plate, colony detection, and measurement of colonies parameters. Colonies were identified using the module Identify Primary Object with three classes intensity threshold: foreground, middle and background. Middle class pixels were then categorized as background, to avoid overestimation of the colony area.
Oxygen Consumption Rate (ocr)
OCR was determined by direct measurement with a SeaHorse Extracellular Flux Analyzer XpE instrument (Agilent Technologies). OCR for the mitochondrial stress test was determined following the manufacturer’s instructions. OCR of HeLa stable clones was measured at baseline and after the addition of the stressors oligomycin to evaluate ATP production, FCCP to measure the maximal respiration and rotenone and antimycin A for the spare capacity calculation. Time and type of stressor administration are indicated in the graph (Fig. 3A). For statistical analyses, all OCR values were normalized with those of SCR.
Statistical analysis was performed using the Microsoft Excel. One-way ANOVA was used for three group comparisons and Student’s t-test was used for two group comparisons. p values of less than 0.05 were considered as significant, while values less than 0.01 or lower were considered as highly significant.