Plasmids
The cDNA sequences of the NME6 short and long isoform (186 aa and 194 aa, respectively) were extracted and cloned from Origene plasmids products RG200541 and RC200541, into pcDNA3.1, pET28b and pEGFP-N1 plasmids, using primers and restriction enzymes listed in the supplementary table (Additional File 1: Table. S1). Resulting proteins are tagged either with FLAG (DYKDDDDK), His (HHHHHH), or enhanced green fluorescent protein (EGFP) tags to comply with the design of individual experiments. The full-length NME4 cDNA sequence (187 aa) was extracted and cloned from pET28a( +)-NME4-FL [29] into pcDNA3.1 using primers and restriction enzymes listed in the supplementary table (Additional File 1: Table. S1). The full-length NME3 cDNA sequence (169 aa) was extracted and cloned from pCMVTag3-NME3-tetra-cys-tag (a kind donation of Prof. Thomas Wieland, Heidelberg University, Germany) into pcDNA3.1 using primers and restriction enzymes listed in the supplementary table (Additional File 1: Table. S1). The obtained pcDNA3.1-NME4-FL-FLAG and pcDNA3.1-NME3-FLAG plasmids were used for immunoprecipitation experiments. The pCFPmito plasmid was used in immunofluorescence to label mitochondria.
Cell lines
Melanoma cell lines Mel 224, Mel 501, Mel 505, A375, A375M, WM793B, WM983B, LM6, CHL1, RPMI7951, were a kind donation of Dr. Bergamaschi (Barts and The London School of Medicine and Dentistry, London, UK) while MDA-MB-435 was purchased from ATCC® CCL-2™. The sarcoma cell lines HTB 82 (rhabdomyosarcoma), HTB92 (liposarcoma), HTB93 (synovial sarcoma) and WT (2fTGH cells, variant of HT 1080 fibrosarcoma) as well as H1299 (lung carcinoma) were a kind donation of Dr. Neda Slade (Laboratory for Protein Dynamics, Ruđer Bošković Institute, Zagreb, Croatia). Detroit 562 (pleural effusion of pharyngeal carcinoma) and Cal 165 (spinocellular pharyngeal carcinoma), were a kind donation of Dr. Jeannine Gioanni, (Centre Antoine Lacasagne, Nice, France). MDA-MB-231T (pleural effusion of breast adenocarcinoma) was donated by Dr. Patricia S. Steeg (Center for Cancer Research, National Cancer Institute, USA; [40]). H460 (lung carcinoma) was a kind donation of Dr. Marijeta Kralj (Laboratory of Experimental Therapy, Ruđer Bošković Institute, Zagreb, Croatia). Other cell lines MDA-MB-436 (pleural effusion of breast adenocarcinoma), C33 (cervix carcinoma), LNCaP (prostate adenocarcinoma metastatic), PC3 (prostate adenocarcinoma metastatic), Du145 (prostate carcinoma, metastatic), MCF7 (pleural effusion of breast adenocarcinoma), SKOV 3 (ovary adenocarcinoma), RD (rhabdomyosarcoma), HCT 116 (colorectal carcinoma), SW620 (colorectal carcinoma, lymph node metastasis) and HeLa (cervix adenocarcinoma) were purchased from ATCC® CCL-2™. Non-cancerous cell lines HEK293 (human embryonic kidney), HACAT (Human keratinocytes), WPMY-1 (human prostate fibroblast) were purchased from ATCC® CCL-2™ while MJ90 (HCA2) (human skin fibroblasts) cells were isolated previously from neonatal foreskin in the Pereira-Smith laboratory. With exception of MDA-MB-231T clones, cells were grown in DMEM (52100-021, Gibco) or RPMI (RPMI-XA, Capricorn Scientific) supplemented with 10% fetal bovine serum (FBS), 1% streptomycin-penicillin, 1 mM sodium pyruvate and 2 mM l-glutamine in a humidified chamber at 37 °C and 5% CO2. The MDA-MB-231T KI clones (KI-194-2, KI-186-1, KI-186-7, KI-186-18 and KI-186-26) were maintained in DMEM supplemented with 10% FBS, 1 mM sodium pyruvate, 2 mM L-glutamine and 1 mM G418. Cell lines were tested mycoplasma-free.
Antibodies
For immunoprecipitation, NME6 (HPA017909 Sigma-Aldrich), NME1 (OP48 Calbiochem), RCC1L (SAB1401860 Sigma-Aldrich), irrelevant IgG-M (556648, BD Biosciences) and irrelevant IgG-R (2729P, Cell Signaling Technology) were used in combination with Dynabeads protein G (10003D, Invitrogen). For phospho-Histidine (pHis) immunoblotting, pHis antibodies (Rabbit hybridomas 1-pHis-SC1-1 and 3-pHis-SC44-1, kind donation of Dr. Tony Hunter, Salk Institute, La Jolla, CA, USA), NME6 (HPA017909 Sigma-Aldrich), and NME1 (UM800025 Origene) were used with HRP-anti-rabbit (7074 Cell Signaling Technology) and HRP-anti-mouse (7076 Cell Signaling Technology) secondary, all diluted in specific BSA blocking buffer (5% BSA (w/v) in TBST, pH 8.5). For other Western blots, antibody cocktail (Cytochrome c, GAPDH, PDH-E1 α, ATP synthase α, ab110415 Abcam), antibody cocktail OXPHOS (against complexes C-I to C-V, ab110412 Abcam), AK2 (AP8134B Abgent), β-actin (60008-1-Ig Proteintech), Calreticulin (C41720 BD Biosciences), FLAG (F1804 Sigma-Aldrich), Histone H3 (ab1791 Abcam; or #14269 Cell Signaling Technology), Lamp1 (15665 Cell Signaling Technology), NME1/2 (Nm23A&B kindly provided by Dr. I. Lascu and Dr. S. Volarević), NME6 (HPA017909 Sigma Aldrich), OPA1 (612607 BD Biosciences), RCC1L (SAB1401860 Sigma-Aldrich), RCC1L (ab247142 Abcam), Na+K+ ATPase (05-369 Merck Millipore), β-tubulin (2128S Cell Signaling Technology) and VDAC (a kind donation of Dr Marco Colombini) primary antibodies were used with HRP-anti-rabbit (7074 Cell Signaling Technology) and HRP-anti-mouse (7076 Cell Signaling Technology) secondary, all diluted in MILK blocking buffer (5% non-fat milk (w/v) in TBST). For PLA assays and immunofluorescence: NME6 (HPA017909 Sigma-Aldrich), NME4 (Milon et al. [29]), FLAG-tag (8146S Cell Signaling Technology), ANT (ab110322 Abcam), Drp1 (8570S Cell Signaling Technology), Mfn1/2 (ab56889/ab57602 Abcam), OPA1 (612607 BD Biosciences) and RCC1L (SAB1401860 Sigma-Aldrich) were used with IgG-Cy5 anti-mouse (ab2338714 Jackson), Dylight 488 anti-rabbit (ab96899 Jackson) and Alexa-Fluor488-anti-rabbit (A11008 Invitrogen) secondary antibodies.
Recombinant protein expression, purification and thrombin cleavage
Recombinant proteins tagged with six histidine residues at the N-terminus were produced in E. coli strain BL21. Cells transformed with pET28b-NME6-186-His and pET28b-NME6-194-His were grown at 37 °C in LB medium until OD600 of 0.8, induced with 0.1 mM IPTG and grown overnight at 16 °C. Cells were washed and incubated on ice for 15 min in lysis buffer (50 mM HEPES, pH 7.4, 400 mM NaCl, 5 mM MgCl2, 10% glycerol (v/v), 10 mM imidazole, 1 mg/ml lysozyme). After sonication (8 × 30 s, 4 °C) and centrifugation (12,000 g, 1 h, 10 °C), the supernatants were applied onto metal affinity column (635502, Takara). After the washing step (washing buffer: 5 mM MgCl2, 50 mM HEPES pH 7.4, 400 mM NaCl, 10% glycerol, 10 mM imidazole), histidine tagged proteins were eluted (elution buffer: 50 mM HEPES pH 7.4, 300 mM NaCl and 150 mM imidazole). Amicon filters 10 kDa cutoff (UFC901024, Merck) were used to concentrate samples in storage buffer (25 mM HEPES, pH 7.4, 300 mM NaCl and 5 mM DTT). His-tag was removed from the NME6-194-His and NME6-186-His proteins using thrombin sepharose beads (7925-1, Biovision) according to manufacturers’ instruction. Briefly, 0.5 mg of recombinant proteins were incubated overnight at 4 °C with 7.5 µL of slurry in 50 mM Tris pH 8.0, 0.1 M NaCl. Protein concentration was assayed by Bradford method (500-0006, Bio-Rad). Samples were analyzed by Western blot.
Recombinant protein crosslinking with glutaraldehyde
Recombinant protein NME6-186-His or NME6-194-His (9.5 µg) was mixed with 0.075% glutaraldehyde in crosslinking buffer (25 mM HEPES, pH 7.4, 300 mM NaCl, 5 mM DTT and 5 mM MgCl2) and incubated at 37 °C for 15 min. The reaction products as well as untreated proteins (control) were boiled 5 min at 95 °C and subjected to 12% SDS-PAGE, transferred to a PDVF membrane and visualized by protein staining with naphthol blue.
Size-exclusion chromatography of recombinant proteins
Size-exclusion chromatography was performed at Biocentar d.o.o., Zagreb, Croatia on Akta avant 25 chromatography system (GE Healthcare). Recombinant proteins (NME6-186-His and NME6-194-His) were gel filtrated on Superdex 200 Increase 10/300 GL (GE Healthcare). The column was equilibrated with a mobile phase flow (10 mM NaH2PO4, 140 mM NaCl, pH 7.4) of one column volume (24 mL) followed by the injection of the sample. The sample was eluted with 1.5 volume of the column (36 mL). Fractions of 1 mL were taken at 2 min intervals. The chromatographic column was calibrated with the following Bio-Rad Gel filtration standards: thyroglobulin (670 kDa), γ-globulin (158 kDa), ovalbumin (44 kDa), myoglobin (17 kDa), and vitamin B12 (1.35 kDa).
NDP kinase activity
NDPK activity was assayed on purified NME6-186-His and NME6-194-His recombinant proteins, and NME1-His [65] as control, using the standard pyruvate kinase-lactate dehydrogenase coupled assay, described by Agarwal and colleagues with minor modifications [2]. Experiments were performed at room temperature in 1 cm quartz cuvette containing 500 µL of reaction mixture composed of 50 mM Tris–HCl pH 7.5, 75 mM KCl, 5 mM MgCl2, 1 mg/mL bovine serum albumin, 1 mM phosphoenolpyruvate, 0.45 mM NADH, 1 mM ATP, 0.2 mM dTDP, 2 U of pyruvate kinase, 2.5 U of lactate dehydrogenase and 200 ng of recombinant NME protein. The reaction was started by addition of dTDP and absorbance was recorded every 10 s at 340 nm. The experiment was repeated six times (n = 6).
Western blot
Proteins were extracted from mammalian cells in phosphate buffer saline (PBS) supplemented with protease inhibitors (11836170001 Roche). Pellets were sonicated (2 × 10 s, 4 °C) and protein concentration was determined by the BCA Protein Assay Kit (23227, Pierce). Proteins were boiled 5 min at 95 °C, separated on 10 or 12% SDS-PAGE (Tris–glycine based) and transferred to a nitrocellulose membrane. Membranes were stained either with ponceau red or naphthol blue. Primary antibody incubations were performed overnight at 4 °C, secondary antibody incubations were performed for 1 h at room temperature. Proteins were visualized with chemiluminescent reagent (NEL104001EA, Perkin-Elmer; 34096 and 34580, Thermo Scientific) using Alliance 4.7 imaging system (UVItec, Cambridge, UK). Phospho-His immunoblotting was performed according to the protocol published by Adam and coworkers, with minor modifications [23, 68]. All procedures were performed at 4 °C to preserve histidine phosphorylation. A fresh 12% SDS-PAGE (Tris–glycine based, stacking pH 7.4, resolving pH 8.8) was prepared. Gel, running buffer, transfer buffer, TBST and PBS were cooled down at 4 °C and adjusted to pH 8.2. The protein loading buffer (5 × LB = 10% SDS, 250 mM Tris–HCl, 0.02% bromophenol blue, 50% glycerol, 50 mM EDTA, 500 mM DTT) was cooled down, adjusted to pH 8.8 and diluted in PBS to 2 × LB on the day of experiment. MDA-MB-231T cells were grown until 90% confluency in a 10 cm dish, washed twice with PBS and scrapped in 500 µL of 2 × LB. The sample was incubated 10 min on ice before being sonicated (3 × 10 s, 4 °C) and clarified by centrifugation (14,000g, 10 min, 4 °C). The supernatant was carefully collected and equally divided in two parts, one incubated on ice to preserve histidine phosphorylation (4 °C), the other boiled (95 °C) for 10 min just before loading to lose histidine phosphorylation. After transfer, the nitrocellulose membrane was blocked (5% BSA (w/v) in TBST, pH 8.5), incubated with primary antibodies overnight at 4 °C and with secondary antibodies for 2 h at 4 °C. Proteins were visualized with chemiluminescent reagent (NEL104001EA, Perkin-Elmer; 34096 and 34580, Thermo scientific) using Alliance 4.7 imaging system (UVItec, Cambridge, UK).
Fluorescence staining
For NME6 immunofluorescent staining associated to localization, HeLa cells were seeded in 8 well chambers (Ibidi, Gräfelfing) and transfected with pCFPmito using Lipofectamine 2000 (11668019, Invitrogen). Twenty-four hours post transfection the cells were rinsed with ice-cold PBS and fixed with 2% formaldehyde for 10 min at room temperature, followed by permeabilization with 0,1% Triton X-100. The cells were incubated with primary NME6 antibody overnight at 4 °C. The next day cells were washed with ice-cold PBS and incubated with Alexa-Fluor488-anti-rabbit secondary antibody (1 h, room temperature, obscurity). The cells were mounted in mounting medium (DAKO, Glostrup, Denmark). For immunofluorescent staining associated to PLA, MDA-MB-231T cells were fixed with 3.2% paraformaldehyde, permeabilized with 0.2% Triton X-100 in PBS, and blocked with PBS containing 3% BSA and 0.1% Tween20 (blocking buffer) before incubation (1.5 h at 37 °C) with one or two primary antibodies freshly diluted in blocking buffer. After washing with PBS, cells were incubated with secondary antibodies (1 h, room temperature, obscurity). Slides were mounted with antifading medium (Vectashield, Eurobio) before image acquisition. For live cell imaging and time-lapse imaging, MDA-MB-231T were seeded in a 4 chamber glass bottom dish and transfected with pCFPmito, pNME6-194-GFP or pEGFPN1-NME6-186-GFP using Turbofect (R0531, Thermo Scientific). Before acquisition the cells were washed with PBS and stained with 5 nM of MitoTracker™ Deep Red FM (M22426, Invitrogen) for 20 min in incubator (37 °C, 5% CO2). Cells were analyzed 48 h post transfection for live cell imaging. Cells were analyzed 24 h post transfection for time-lapse recording, for a 24 h period.
Confocal imaging of live and fixed cells
For immunofluorescent staining and live-cell imaging related to localization, confocal microscopy was performed using Leica TCS SP8 X FLIM or a Leica TCS CSU SP8 confocal microscope equipped with an HC PL APO CS2 63 × /1.40 oil objective, 405-nm diode laser, argon-gas laser and a supercontinuum excitation laser (Leica Microsystems, Wetzlar, Germany). The stage-top environmental control system was used for live-cell imaging to maintain the temperature at 37 °C and Leibovitz’s L-15 medium (21083-027, Gibco) was used to support cell growth in the environment without CO2 equilibration. For time-lapse imaging z-stacks of 10 planes were taken every 15 min for a period of 24 h. For confocal imaging of live and fixed cells the excitation wavelengths and detection ranges were as follows: 488 nm and 500–550 nm for EGFP and Alexa488; 644 nm and 655–705 nm for MitoTracker™ Deep Red FM; 405 nm and 430–500 nm for DAPI and 458 nm and 470–520 nm for CFP. The hybrid (HyD) detectors were operated in the gated mode in order to suppress parasite reflection from the bottom glass surface of the cell-culture dish. Imaging was performed in a sequential scanning mode. For PLA assays and parallel (co-)immunofluorescence, excitation wavelengths and detection ranges were as follows: 488 nm and 500–545 nm for DyLight 488 and Syto 13; 522 nm and 600–659 nm for PLA red probes; 638 nm and 650–690 nm for Cy 5. Fluorescence emissions were precisely collected by dichroic filters and spectral detectors. The images were acquired through the entire cells by the mean of z-stacks with a z-step of 1 μm, a confocal pinhole of 1 (Airy units) for all channels, with at least 3 randomly chosen fields per condition.
Proximity ligation assay (PLA)
In situ PLA was performed using a Duolink kit (Sigma-Aldrich, France). Cells grown on chamber microscopy slides were fixed with 3.2% paraformaldehyde, permeabilized with 0.2% Triton X-100 in PBS, blocked with a Duolink blocking agent and incubated with primary antibodies. PLA probes (secondary antibodies tagged with DNA oligonucleotides) were added, and hybridization, ligation, amplification, and detection (using Duolink Detection Reagents Red) were carried out according to the manufacturer’s protocol. Briefly, incubation with PLA probes was performed in a preheated humidified chamber for 1 h at 37 °C followed by ligation (30 min at 37 °C) and amplification (1 h 40 min at 37 °C). Nuclei were stained with 2.5 µM Syto13 (ThermoFisher, France) for 15 min, and image acquired by confocal microscopy.
Stable clones overexpressing NME6-186-FLAG and NME6-194-FLAG
MDA-MB-231T cells were transfected with pcDNA3.1, pcDNA3.1-NME6-194-FLAG and pcDNA3.1-NME6-186-FLAG using Turbofect transfection reagent (R0531, Thermo Scientific) according to manufacturers’ instruction. The stable clone selection started 24 h post transfection in DMEM supplemented with G418 for 14 days. Selected cells were diluted and seeded at one cell per well in a 96-well plate. Single-cell colonies were selected under the microscope and allowed to grow in DMEM with G418. Viable colonies were saved and the plasmid’s random integration was confirmed by PCR for “empty” vector clones or by Western blot using anti-FLAG antibody for KI clones. Stable clones KI-186-7, KI-186-18 and KI-186–26 were used for analysis of mitochondrial mass, membrane potential and respiration, as well as PLA experiments. Stable clones KI-194-2 and KI-186-1 were used for evaluating NME6 protein expression in stable clones (Fig. 2b).
Silencing of NME6 by siRNA
Silencing was performed using DharmaFECT 4 Transfection Reagent (T-2004-02, Dharmacon) and ON-TARGETplus Human NME6 siRNA—SMARTpool (L-006755-00-0005, Dharmacon) to silence NME6, or scramble siRNA (D-001810-01, Dharmacon) as a negative control, according to the manufacturers’ instruction. Briefly cells were grown to reach 30% confluency the day of transfection. Six hours post transfection the medium was changed and cells were allowed to grow until analysis, 72 h post transfection.
Cell fractionation using a commercial kit
Cellular fractionation assay was performed using MDA-MB-231T cells and Cell Fractionation Kit (ab109719, Abcam), according to manufacturers’ protocol. Crude “Cytosolic”, “Mitochondrial” and “Nuclear” fractions were quantified using BCA Protein Assay Kit (23227, Pierce), and an equal amount of proteins from each fraction was analyzed by Western blot.
Isolation and purification of mitochondria
MDA-MB-231T cells were grown to 80% confluency, and 350 × 106 cells were used for the experiment. The whole procedure of mitochondrial isolation and purification was performed on ice as described earlier [30, 76] with minor modification. Cells were scrapped in PBS and centrifuged 5 min at 750 g. Collected cells were resuspended and combined in 10 mL of BufferA (210 mM mannitol, 70 mM sucrose, 0.2 mM EDTA, 10 mM HEPES pH 7.5). Cells were homogenized by 10 passages through a 25G needle. Homogenate (H) was centrifuged 5 min at 2,000 g. The supernatant (S1) was kept on ice and pellet, resuspended again in 10 mL BufferA, underwent 6 additional passages through a 25G needle and centrifugation (5 min, 2000 g). Pellet enriched in nuclei (P) was resuspended in 5 mL of BufferA and kept on ice until analysis. The supernatant was combined with S1 and centrifuged 10 min at 13,000 g. The resulting supernatant enriched in cytosol (C) was kept for analysis. The pellet was resuspended in 1.5 mL of BufferB (210 mM mannitol, 70 mM sucrose, 0.1 mM EGTA, 10 mM HEPES pH 7.5) and centrifuged 5 min at 500 g. The supernatant was transferred in a clean tube and centrifuged 10 min at 10,000 g. The resulting pellet enriched in crude-mitochondria (MC) was resuspended in 1 mL of BufferB and purified on 25% Percoll gradient by ultra-centrifugation (35 min, 100,000 g, Beckman 60Ti fixed angle). Two bands were collected from bottom to top (MP, percoll-pure mitochondria; CON, contaminants) and washed in 10 mL of BufferB (10 min, 7,000 g). Resulting pellets were resuspended in 0.5 mL BufferB. Protein concentration was measured using Bradford method (500-0006, Bio-Rad) and 2 µg from each fraction was analyzed by Western blot.
Mitochondrial subfractionation
The whole procedure of mitochondrial subfractionation was performed on ice using MC obtained from MDA-MB-231T cells as described above, and swelling-shrinking procedure as described earlier [77] with minor modification. After washing in BufferB (10,000 g, 10 min), MC pellet was resuspended in 200 µL of swelling buffer (SW1, 10 mM KH2PO4 pH 7.4) and incubated 20 min on the rotator. Then 200 µL of shrinking buffer (SW2,10 mM KH2PO4 pH 7.4, 30% sucrose, 30% glycerol, 10 mm MgCl2, 4 mM ATP) was added and the suspension was incubated for further 1 h on the rotator. The sample was gently sonicated (2 × 15 s) in a water bath sonicator and centrifuged at 12,000 g for 10 min. The resulting low-speed supernatant (MCss_ls) was kept on ice while the mitoplast-containing low-speed pellet (MCss_lp) was washed two times in BufferA (10 min, 12,000 g) before being resuspended in 400 µL of BufferSW1 and sonicated using ultrasonic homogenizer with a metal tip (Bandelin, Germany; 3 × 15 s, 60 s cooling interval). Both MCss_ls and MCss_lp were centrifuged 1 h at 160,000 g (Beckman ultracentrifuge; 70.1Ti fixed angle rotor). Resulting high-speed pellets (respectively MCss_ls_hp and MCss_lp_hp) were resuspended in 100 µL BufferB, while resulting high-speed supernatants (respectively MCss_ls_hs and MCss_lp_hs) were separately concentrated using microconcentrator YM-3 (Millipore, 42,403) according to manufacturer’s instruction. Protein concentration was determined by Bradford method (500-0006, Bio-Rad), and 2 µg of protein were analyzed by Western blot.
Multiple reaction monitoring mass spectrometry
The liquid chromatography-mass spectrometry (LC–MS) analysis was performed (Biocentar d.o.o., Zagreb, Croatia) on 1290 Infinity LC System (Agilent Technologies, USA) coupled with 6460 Triple Quad Mass Spectrometer (Agilent Technologies, USA). In silico digestion of NME6-194 and NME6-186 sequences by trypsin was performed using Skyline software (v. 3.7.0.10940.). The difference in isoforms was obtained within the following sequences: MTQNLGSEMASILR for the NME6-194 isoform and MASILR for the NME6-186 isoform. Total HeLa and MDA-MB-231T cell lysates were digested with trypsin (γ = 1 mg/mL) for 18 h at 37 °C and 600 rpm (Digital Shaking drybath, Thermo Scientific, USA). Acquity UPLC BEH separation column C18 1.7 µm, 2.1 × 150 mm (Waters, USA) was used for chromatographic peptide separation. Mobile phase A (0.1% (v/v) aqueous formic acid solution) and mobile phase B (acetonitrile) were both degassed in an ultrasonic bath. The separation was performed at 40 °C column temperature, 15 µL of sample was injected with a gradient flow of 0.3 mL/min starting at 95% A and decreasing to 60% A over 16 min. Mass spectra were recorded in a positive resolution mode with the capillary voltage set at 3.5 kV, at a gas temperature of 300 °C, and at a gas pressure of 40 psi. All measurements were performed in duplicate.
Immunoprecipitation
MDA-MB-231T cells were transfected with pcDNA3.1-NME3-FLAG, pcDNA3.1-NME4-FLAG, pcDNA3.1-NME6-186-FLAG, pcDNA3.1-NME6-194-FLAG using Turbofect transfection reagent (R0531, Thermo Scientific). Cells were collected 48 h post transfection, lysed by sonication (2 × 10 s, 4 °C) in TEEN buffer (50 mM Tris pH 7.4, 0.5% NP40, 150 mM NaCl, 5 mM EDTA) supplemented with protease inhibitor (11836170001, Roche) and clarified by centrifugation (16,000 g, 20 min, 4 °C). The supernatant was recovered and used as input (I). Pull-down using FLAG-agarose (A2220, Sigma) was performed according to the manufacturers’ instruction. Briefly, 40 µL of slurry was washed and incubated overnight with 200 µg of input protein (I), on the rotator at 4 °C. The next day, supernatant (Sn) was saved while FLAG-agarose-Ag complexes were washed and eluted (IP). Immunoprecipitation using Dynabeads™ Protein G (10003D, Invitrogen) was performed according to the manufacturers’ instruction. Briefly, 50 µL of beads were washed and incubated 40 min with 2 µg of antibody, on the rotator at room temperature. NME1, NME6 and RCC1L antibodies were used to immunoprecipitate endogenous proteins while irrelevant IgG-M and IgG-R were used as negative controls. Beads-Ab complexes were washed and incubated with 200 µg of input protein (I) on the rotator, overnight at 4 °C. The next day, supernatant (Sn) was saved while beads-Ab-Ag complexes were washed and eluted (IP). In both immunoprecipitation experiments, the whole elution volume (IP) was loaded on SDS-PAGE, while 30 µg of input (I) and supernatant (Sn) were loaded and analyzed by Western blot.
Mitochondrial membrane potential, mass, network characteristics and respiration
MDA-MB-231T cells (Ctrl, stable clones overexpressing NME6-186-FLAG) were grown until 90% confluence, detached with trypsin, and resuspended. Each suspension was distributed into two tubes and either incubated for 15 min with 50 nM Mitotracker GreenFM (Life Technologies, ThermoFisher Scientific, USA) or 50 nM TMRM (tetramethylrhodamine methyl ester, Life Technologies, ThermoFisher Scientific, USA) in a 5% CO2 humidified atmosphere at 37 °C and protected from light. Cell suspensions were immediately analyzed by FACS (BD LSR FORTESSA, Becton Dickinson, France) with excitation at 488 nm or 532 nm and emission band-pass filters 530/30 nm or 585/15 nm for Mitotracker GreenFM or TMRM, respectively. Mitochondrial mass was estimated by quantification of Mitotracker labeling. TMRM-labelled cells were further incubated for 15 min with 250 µM CCCP (carbonylcyanide m-chlorophenyl hydrazone, Sigma-Aldrich, France), and the mitochondrial membrane potential calculated as a difference of TMRM fluorescence before and after CCCP addition, normalized to mitochondrial mass. Confocal images of Mitotracker GreenFM-stained cells were used for quantification of mitochondrial shape and network parameters as described [43].
Oxygen consumption was measured in a thermostatically controlled Clark electrode oxygraph at 37 °C (Strathkelvin MS200A system). Detached cells were counted and resuspended at 100 × 106 cells/mL on ice. An aliquot of 5 × 106 cells was added in the oxygraph chamber containing KET buffer (150 mM KCl, 1 mM EGTA, 20 mM Tris pH 7, 2) and inorganic phosphate (5 mM) to give a final volume of 500 μl. Then digitonin (50 µg/mL) was added and incubated for two minutes to allow permeabilization of the plasma membrane. Oxygen consumption of cells was measured with glutamate (5 mM) and malate (2.5 mM), or with succinate (5 mM) as substrates (LEAK state [78]), as well as after addition of ADP (0,5 mM; OXPHOS state [78]) and after the addition of cytochrome c (10 μM; control for intact mitochondrial membranes). Results are expressed as nmol O2 consumed per minute and per 5 × 106 cells.