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, Densitometric analyses of immunoblots were performed using ImageJ software. Actin was used as an internal control for protein loading and normalization
, Cells were seeded onto 24-mm glass coverslips (VWR #631-0161), allowed to grow to 50%-60% confluency and were fixed in 2.5% glutaraldehyde (Sigma Aldrich # G5882) and 2% paraformaldehyde (Sigma Aldrich # P6148). Tumor samples from PDX were directly fixed under same conditions, Mitochondrial Content and Structure Analysis Electron Microscopy on OCCL and PDX Tumor Samples
, The mean area of each group was determined on 8 independent images, evaluating 2000 mm 2 per cell line. Size of measured structures was expressed in mm 2 . Mitochondrial Staining Using MitoTracker Probe Cells were seeded onto six-well plates and grown up to 70% confluency. Mitochondrial content per cell line was estimated using MitoTracker Deep Red FM (Molecular Probes/Invitrogen #M22426). For assessment of mitochondrial membrane potential, cells were stained with MitoTracker Red CMXRos (Molecular Probes/Invitrogen #M7512) and tetramethylrhodamine, methyl ester (TMRM, Thermo Fisher #T668). Cells were stained with 250 nM MitoTracker Deep Red FM or 250 nM MitoTracker Red CMXRos or 100 nM TMRM for 30 min at 37 C. Cells were then washed with PBS solution, trypsinized, and resuspended in PBS, 5% potassium ferrocyanure (Electron Microscopy Science # 25154). Samples were embedded in EPON and ultrathin sections were contrasted with uranyl acetate and lead citrate
, Cells were incubated in a CO 2 free incubator at 37 C for 1 h. Cartridges equipped with oxygen-and pH-sensitive probes were preincubated with calibration solution (Agilent Technologies #100840-000) overnight at 37 C in an incubator without CO 2 . Prior to the rate measurements, the XF96 Analyzer (Seahorse biosciences, North Billerica, MA) automatically mixed the assay media in each well for 15 min to allow the oxygen partial pressure to reach equilibrium. Oxygen consumption rate (OCR) and extracellular acidification rate (ECAR) were evaluated in a time course before and after injection of the following compounds: OCR measurement (using Agilent Technologies #103015-100) (i) 1 mM Oligomycin; (ii) 0.5 mM FCCP [Carbonyl cyanide-4-(trifluoromethoxy)phenylhydrazone; (iii) 0.5 mM Antimycin A + Rotenone / ECAR measurement (using Agilent Technologies #103020-100): (i) 10 mM Glucose; (ii) 1 mM Oligomycin; (iii) 50 mM 2-deoxyglucose (2-DG). A volume of 25 mL of compound was added to each injection port, and 3 baseline measurements were taken prior to the addition of any compound. After a 3 min wait, 3 response measurements were taken after each addition. ECAR and OCR values were normalized to the total amount of protein per well. ECAR and OCR data points refer to the average rates during the measurement cycles and were reported as absolute rates (mpH / min for ECAR, pMoles / min for OCR). For experiment testing carbon source preference, cells were incubated overnight in DMEM ± 10 mM Glucose, ± 2 mM Glutamine. Basal ECAR/OCR measurement was performed the following day. For fatty acid oxidation or glutaminolysis inhibition experiments, cells were incubated overnight in DMEM ± 10 mM Glucose, ± 2 mM Glutamine. OCR was measured 30 min after Etomoxir treatment (40 mM, Seahorse Technology Cells were seeded (4 replicates) in XFe96 Cell Culture Microplates (Seahorse, Bioscience #101085-004) at 80%-90% confluency in DMEM supplemented with 10% FCS ± 10 mM Glucose, ± 2 mM Glutamine, ± 1 mM Pyruvate. Cells were incubated for 24 hours at 37 C in 5% CO 2 atmosphere. Before the experiment, the culture medium was removed from each well and replaced with 175 mL of serum-free unbuffered Seahorse XF Base Medium base pH, vol.7
, Cambridge Isotope Laboratories #CLM-1396) + 2 mM of glutamine or 2 mM 13 C-L-glutamine (Cambridge Isotope Laboratories #CLM-1822) + 10 mM of glucose for 24 hours. Intracellular metabolites were extracted at À20 C with 8 mL of acetonitrile/methanol/water+0.1% of formic acid (2:2:1) and cells were scraped from the cover glasses. The solution was sonicated for 30 s and incubated for 15 min on ice for the metabolite extraction. Subsequently, the sample was frozen with liquid nitrogen, freeze-dried and finally re-extracted with an aqueous solution before mixing with the appropriate solvent for LC-MS analysis. LC-MS Analysis Analysis of intracellular amino acids was performed by liquid chromatography (HPLC U3000, Isotope Profiling in OCCL Cultivation, Sampling and Metabolite Extraction 6 3 10 5 IGROV1 and OC314 cells were seeded onto 30-mm glass coverslips and were incubated the day after in no glucose, vol.29, pp.156-173, 2019.
, Isotopic cluster of each amino acids and central metabolites was determined by extracting and integrating the exact mass of all 13C-isotopologues with Tracefinder software (Thermo Fisher Scientific). Isotopic cluster of each amino acids and central metabolites was determined by extracting and integrating the exact mass of all 13C-isotopologues with Tracefinder software (Thermo Fisher Scientific). The correction was performed with IsoCor adapted for higt resolution mass spectrometry. Carbon isotopolog distributions were expressed relative to the sum of all analyzed isotopologs, the positive FTMS mode at a resolution of 60, p.0
1-derived vectors with two different shRNAs targeting human PML (TRCN0000003866 and TRCN0000003868 for shPML#1 and shPML#2, respectively), or expressing a scrambled shRNA (shCTRL #SHC001), were purchased from Sigma-Aldrich. Viruses were produced by co-transfection (with Lipofectamine 2000, Invitrogen #11668) of 293T cells with the vector plasmid, a vesicular stomatitis virus envelope expression plasmid (Vsvg) and a second-generation packaging plasmid (pPax2), M DCl 0.025 M before analysis. PML and PGC-1a Silenced Cell Lines For generation of PML-silenced stable cell lines from CAOV3 ,
, Stable cell lines were propagated in DMEM (GIBCO, Thermo Fisher Scientific #11995) with glucose
, 4 mM L-glutamine (Thermo Fisher Scientific # 25030081), 1 mM sodium pyruvate (Thermo Fisher Scientific # 11360070) supplemented with 10% fetal bovine serum (FBS, BioSera #FB-1003/500), penicillin (100 U / ml) and streptomycin (100 mg / ml
, Thermo Fisher Scientific # 15140122) and 1 mg ml À1 of puromycin (GIBCO# A11138-03)
, After 24 hr, cells were transfected with 20 nM of non-targeting siRNA (siCtrl, Dharmacon #D-001810-02) or PML-targeting siRNA (Dharmacon siPML#1: #J-019734-06; siPML#2: #J-019734-07; siPML#pool, a mix of 4 individual siPML
, ) using 4 mL of DharmaFECT 1 transfection reagent in 2 mL final volume according to the manufacturer's instructions, 2001.
, After 24 hr serum starvation, 5 3 10 4 cells were plated to the upper side of the Transwell device, in triplicates, in 100 mL of serum-free medium, whereas the lower well contained 600 ml of regular 10% FBS culture medium in order to create an FBS gradient. We ended the experiment after O.N. incubation. At the end of the experiment, the remaining cells in the upper side of the Transwell device were removed. Migrating cells at the bottom side of the Transwell device were fixed and stained with crystal violet for 20 min and then, Growth, Migration and Anchorage Independent Growth Growth Kinetics Cells were seeded at 2 3 10 4 cells per well in 24-well plates (Corning #353047) and at indicated time points counted using Vi-Cell analyzer
, Sigma Aldrich #A2576) and appropriate antibiotics and layered onto a 15 mL tubes (BD Biosciences, #352059) overlaid with medium without agarose. After two weeks, growth media was removed and viable colonies were stained with 2.5 mg / ml iodonitrotetrazolium chloride (Sigma Aldrich #I10406), scanned and finally quantified using the ImageJ software, Soft Agar Assays for Anchorage-Independent Growth 4 3 10 4 cells were passed 4-5 times through a 21G syringe
, PBS and blocked for 15 min in 3% BSA and 0,1% Triton. Cells were incubated with a specific antibody recognizing PML, p.500
, Cells were stained with DAPI (2 mL / ml, Invitrogen #D1306) for nuclei detection. Slides were examined using an Upright Epifluorescence Microscope with Apotome (Zeiss), and images were acquired with identical exposure times and settings using a digital camera. Fluorescence image analysis was performed using the ImageJ software. For antioxidant impact on PML-NB, cells were treated with N-acetyl-L-Cystein (NAC at 5 mM, Santa Cruz #SC-5621) for 45 min followed incubation with a goat polyclonal secondary antibody to rabbit IgG, vol.488, p.1000
, e8 Features of Oxidative Stress Briefly, cells were seeded onto six-well plates and grown up to 70% confluency, incubated directly with fluorescent probes for basal conditions. Then excess reagent was removed by washing the cells with PBS, trypsinized and resuspended in PBS solution containing 1% FBS for flow cytometric analysis. Flow cytometry data were acquired using an LSR FORTESSA analyzer (BD biosciences). For ROS quantification upon treatment, cells were treated for 24 hr with Carboplatin, Cell Metabolism, vol.29, pp.156-173, 2019.
6 mg / ml) or Ironomycin (at 6 mM, synthesis is described in Mai et al., 2017), and then processed, as described above. Cellular ROS Cells were incubated with 2 mM CellRox Reagent (Life Technologies, #C10422) for 30 min at 37 C in the dark ,
, RhoN OX -M lysosomal specificity for 60 min at 37 C in the dark. For normalization to lysosomal content that varies between cells, Lysosensor probe (1 mM, Life Technologies, #L7535) was used. The formula was applied: lysosomal Fe 2+ = RhoM speMFI / Lysotracker speMFI, Lysosomal Fe 2+ Content Cells were incubated with 5 mM RhoM probes
, Life Technologies # D3861) for 60 min at 37 C in the dark. For Bodipy C11 IF, 3 3 10 5 cells were seeded onto glass coverslips placed inside a six-well plate. 48 hr later cells were incubated with 2 mM Bodipy C11 Reagent for 60 min and then fixed in 4% paraformaldehyde for 20 min, rinsed in PBS. Slides were examined using an Upright Epifluorescence Microscope with Apotome (Zeiss) and images were acquired with identical exposure times and settings using a digital camera, Lipid Peroxide Product Cells were incubated with 2 mM Bodipy C11 Reagent
, 1% Triton X-100, 25 mM NaF, 1 mM Na3VO4, 10 mM b-glycerophosphate, 5 mM sodium pyrophosphate, 0.5 mM PMSF) supplemented with EDTA-free protease inhibitor cocktail tablet (Roche #1836170) and incubated on ice for 20 min with vortexing every 5 min. Cell extracts were centrifuged at 13,000 rpm for 10 min at 4 C and supernatants were transferred into fresh tubes. The protein concentration was determined using the BCA Protein Assay kit -Reducing Agent Compatible according to the manufacturer's instructions (Thermo Fisher Scientific # 23250). For immunoprecipitation, 300 mg of fresh protein extract were incubated overnight at 4 C with rotation, with 50 mL of PGC-1a (SantaCruz #sc-13067) coupled to magnetic beads (Dynabeads antibody coupling kit, Invitrogen #1143.11D) at 2 mg antibody per mg dynabeads. Beads were washed three times using IP lysis buffer. Lastly, 50 mL of samples buffer 2x (Biorad #1610737) were added on top of the beads and boiled for 5 min at 95 C. Western blot analysis of IP samples was performed as described above. Cell Treatments and Cell Viability Assays 10 4 cells were seeded per well in 96-well plates in DMEM medium with 10% FCS. Carboplatin (ACCORD, 10 mg / ml) and Paclitaxel (KABI, 6 mg / ml), or Ironomycin (in-house drug), Corning #353003). 24 hours later, cells were transiently silenced for PML (see PML and PGC-1a Silenced Cell Lines). 48 hr post transfection, cells were washed with cold PBS and scraped on ice. Cell suspensions were centrifuged at 13,000 rpm for 10 min at 4 C. Cell pellets were flash frozen in liquid nitrogen, resuspended in IP lysis buffer (50 mM HEPES pH 7.5, 150 mM NaCl, 1 mM EDTA, 1 mM EGTA, 10% glycerol, p.839
, Sigma Aldrich #R7017) was added to each well. Plates were incubated at 37 C for 2 hr and read in a Multi Detection plate reader (Fluostar
, For each sample, 1 mg of total RNA was reverse transcribed using an iScript Reverse Transcription Kit (Bio-Rad #1708840). qRT-PCR was performed using Power SYBR Green PCR Master Mix (Applied Biosystems, #4367659) on a Chromo4 Real-Time PCR detection System (Bio-Rad) with primers at 300 nM final concentration. Primers (forward and reverse) used for quantitative (q)RT-PCR amplification were, from Cell Lines For gene expression analysis, total RNA isolation was performed using miRNEasy kit (QIAGEN, #217004) according to the manufacturer's instructions
, Expression levels were normalized to CYCLOPHILIN-B and represented as fold change compared to the control (2^(-DDCt)). For evaluation of siRNA or drug impacts on gene expression, cells were incubated 48 hours with specific siRNA or with N-acetyl-L-Cystein (NAC at 5 mM, Sigma Aldrich #A7250) or Rosiglitazone
, Cell Metabolism, vol.29, pp.156-173, 2019.