No. Quantity. Minimum Essential Medium (supplemented with. Earl's salts, glutamine, and non-essential amino acids). Gibco 41500-034 (powder). 4.8 g. HEPES.
Supplementary Materials Suppl. Tables 1 & 2 Suppl. Methods 1 to 5 Suppl. Figures 3, 4 A & B, 5 A to C, 6 and 10.
pH, Lactate and Hypoxia: Reciprocity in Regulating High-Affinity Monocarboxylate Transporter Expression in Glioblastoma
James P. Caruso, Brandon J. Koch, Philip D. Benson, Elsa Varughese, Michael D. Monterey, Amy E. Lee, Ajal M. Dave, Sam Kiousis, Andrew E. Sloan, Saroj P. Mathupala. Department of Neurosurgery and Karmanos Cancer Institute Wayne State University School of Medicine, Detroit, Michigan 48201
Suppl. Table 1: Formulation of low serum (2% w/v) modified Minimum Essential Media (mMEM) for routine monolayer culture of U-87MG and U-251MG.
Media components (per 500 ml)
Supplier / Cat. No.
Quantity
Minimum Essential Medium (supplemented with Gibco 41500-034 (powder) Earl's salts, glutamine, and non-essential amino acids)
4.8 g
HEPES
Sigma H3375
1.3 g
NaHCO3
Sigma S6014
1.1 g
BSA Fraction V
Sigma A2153
2.0 g
ZnSO4 (1.94 mg/ml stock solution)*
Sigma Z0251
50 µl
Hydrocortisone (0.36 mg/ml stock solution)*
Sigma H4001
05 µl
Vitamin B12 (0.7 mg/ml)*
Sigma V6629
05 µl
Insulin/transferrin/Se (x 100 stock solution)
CellGro 25-800-CR
5.0 ml
Gentamicin (50 mg/ml stock solution)*
RPI G38000
0.5 ml
Fetal bovine serum
Gibco 26140-079
10 ml
The components were added to 400 ml deionized water while stirring. Once dissolved, final media volume was brought up to 500 ml. The pH was adjusted to 7.4 and the medium filter sterilized prior to storage (4° C) or use. *Detailed formulation of these stock solutions are provided in the following publication: Monterey MD, Szerlip NJ, Mathupala SP (2013) Low-cost media formulation for culture of brain tumor spheroids (neurospheres), Biotechniques 55, 83-88. Note: The low-serum mMEM was formulated off the above reference, where EGF and b-FGF were replaced with 2% (v/v) FBS and 4% (w/v) BSA Fraction V for anchorage-dependent growth of both U87MG and U-251MG cells, at rates similar to that in standard MEM supplemented with 10% FBS.
Suppl. Table 2: Formulation of serum free Minimum Essential Media (MEM) for routine spheroid (neurosphere) culture of TS667 cell-line.
Media components (per 500 ml)
Supplier / Cat. No.
Quantity
Minimum Essential Medium (supplemented with Gibco 41500-034 (powder) Earl's salts, glutamine, and non-essential amino acids)
4.8 g
HEPES
Sigma H3375
1.3 g
NaHCO3
Sigma S6014
1.1 g
Glucose
Sigma G5400
1.75 g
BSA Fraction V
Sigma A2153
2.0 g
Biotin (0.2 mg/ml stock solution)*
Sigma B1595
2.5 ml
ZnSO4 (1.94 mg/ml stock solution)*
Sigma Z0251
50 µl
Hydrocortisone (0.36 mg/ml stock solution)*
Sigma H4001
05 µl
Vitamin B12 (0.7 mg/ml)*
Sigma V6629
05 µl
Insulin/transferrin/Se (x 100 stock solution)
CellGro 25-800-CR
05 ml
Gentamicin (50 mg/ml stock solution)*
RPI G38000
0.5 ml
EGF (10 µg/ml stock solution)
Miltenyi Biotec 130-093-825
1.0 ml
b-FGF (FGF-2; 10 µg/ml stock solution)
Miltenyi Biotec 130-093-838
0.5 ml
The components were added to 500 ml deionized water while stirring. Once dissolved, media pH was adjusted to 7.4 and filter sterilized prior to storage (4° C) or use. *Detailed formulation of these stock solutions are provided in the following publication: Monterey MD, Szerlip NJ, Mathupala SP (2013) Low-cost media formulation for culture of brain tumor spheroids (neurospheres), Biotechniques 55, 83-88. Note: The MEM-based serum-free media formulation was developed off the above article, where MEM (supplemented with Earl's salts) was substituted for DMEM.
1
Suppl. Method 1: Sequential steps undertaken to clone the 4.2 kbp proximal promoter region into pGL2-luciferase reporter gene vector. First, the MCS of pBluescript II SK (Agilent Technol., Wilmington, DE) vector was re-engineered to KpnI-SpeI-AflII-SnaBI-HaeII-MluI-SacI using a pair of annealed oligonucleotides. The oligonucleotides (50 µM each) were suspended in an annealing buffer (10 mM Tris-HCl, 1 mM EDTA, 0.1 M NaCl, pH 8.0), denatured at 94 °C for 4 min and then allowed to anneal by slow cooling to room temperature over 5-10 min (page 21, Block–it H1 RNAi Inducible Entry Vector Manual, 2010; Invitrogen). In brief, the MCS of the vector was removed by double digestion with KpnI-SacI and the synthetic dsDNA fragment generated by annealing the oligonucleotides 5'- CAC-TAG-TCT-TAA-GTA-CGT-AAG-CGC-CACGCG-TGA-GCT and 5'- CAC-GCG-TGG-CGC-TTA-CGT-ACT-TAA-GAC-TAG-TGG-TAC was cloned in position. The AflII-MluI MCT2 promoter-exon-I fragment was cloned into the same sites of the reengineered vector. To sub-clone the promoter-exon I fragment into pGL2, with the exon in frame with luciferase reporter gene, an HaeII site in exon I was utilized. For this stage, a second pBluescipt II SK vector was engineered with the MCS KpnI-SpeI-BbeI-HindIII-SacI using a similar strategy as described above. The oligonucleotides used were 5'- CAC-TAG-TGG-CGC-CAA-GCTTGA-GCT and 5'- CAA-GCT-TGG-CGC-CAC-TAG-TGG-TAC. (Note: BbeI is an isoschizomer of HaeII that has no sites in pBluescript II SK vector). The promoter-Exon I fragment in the first pBluescript II SK clone was double digested with KpnI and HaeII and cloned into the second pBluescipt II SK vector at the KpnI and BbeI sites. To create the MCT2 proximal promoter-exon I-luciferase in-frame fusion, this final subclone was double-digested with KpnI and HinDIII and ligated into the same sites of the MCS in pGL2 luciferase reporter vector.
Suppl. Method 2: Transfection conditions used for reporter gene assays. A 24-well format was used for all assays. The media volume in each well was reduced to 0.5 ml at the time of transfection. Fugene-6 transfection conditions: (3:1 ratio; Fugene: DNA) 0.5 ml media/well + 20 µl transfection complex (1.2 µl Fugene-6 + 0.4 µg plasmid DNA) Fugene-HD transfections: (3:2 ratio; Fugene: DNA) 0.5 ml media/well + 25 µl transfection complex (0.75 µl Fugene-HD + 0.5 µg plasmid DNA) Fugene-HD transfections: (5:2 ratio; Fugene: DNA) 0.5 ml media/well + 25 µl transfection complex (1.25 µl Fugene-HD + 0.5 µg plasmid DNA)
Cell dissociation protocol: 1] Aspirate spent media (10 ml) from 10 mm diameter tissue culture plates with cells at 75-80% confluency. 2] Rinse plates with 10 ml PBS. 3] Add 10 ml Accutase (10 ml/ 75 cm2 surface area). 4] Incubate at r.t. for 5-10 min until cells become detached. Tap the plate to disperse the cells as a single-cell suspension. Add 10 ml plating media (based on downstream experiment). 5] Pellet cells for 5 min at 100 x g at 4 °C (700 rpm; Sorvall RT6000D centrifuge). Re-suspend cells in 10 ml plating media. 6] Enumerate the cells. We used a Beckman-Coulter Z1 particle counter (Beckman-Counter, Indianapolis, IN). 7] Plate at required density in 24-well plates per experimental parameters.
Suppl. Method 3: Preparation of pH-balanced media (pH 7.3 ± 0.1) for reporter gene assays. Optimem-I media supplemented with 4% FBS (pH 7.5; antibiotic-free) was freshly prepared. Two, 35 ml aliquots were placed in 50 ml disposable polystyrene tubes. KCl and nigericin were added to one tube to a final concentration of 5 mM KCl and 0.5 µg/ml nigericin (175 µl 1.0 M KCl and 17.5 µl 1.0 mg/ml nigericin per 35 ml media). While one 10 ml aliquot of pH 7.5 media from each tube was retained as-is, two other 10 ml aliquots of media from each tube were pH adjusted to 7.0 or 6.5 by use of a 10 ml beaker Initial and final pH of media after overnight equipped with a mini-stir-bar, using a semiequilibration in 5% CO2 at 37 °C (n=7). micro calibrated pH electrode (Accumet #13620-293, Fisher Scientific, Chicago, IL) placed in the beaker. pH adjustments were done as follows; a 200 µl aliquot of 1.0 M HCl was withdrawn into a 0.2 ml micro-pipette (equipped with a 200 µl pipette tip). The pipet tip was placed in the medium and HCl slowly introduced by turning the volume adjustment knob of the micro-pipet clockwise, while rapidly stirring the media. Approximately 60 µl of 1.0 M HCl is needed to bring pH down to 7.0 (from 7.5) and approximately 140 µl of 1.0 M HCl is needed to bring the pH down to 6.5 (from 7.5). Then, each 10 ml aliquot of media (six aliquots; two sets of pH 6.5, 7.0, and 7.5, +/- K+ and nigericin) was filter sterilized directly into individual wells of a 6-well plate, and the plate placed in a tissue culture incubator (37 °C, 5% CO2) for 4 hrs to equilibrate pH. Then, the media in reporter gene-transfected 24-well plate was replaced with the pH adjusted, CO2 equilibrated media. In brief, media in wells (of columns 1, 3 and 5) of the 24-well plate was replaced with 1.0 ml aliquots of pH 6.5, 7.0 or 7.5 media (- KCl, - nigericin) respectively. Media in wells of columns 2, 4 and 6 of the 24-well plate was replaced with 1.0 ml aliquots of pH 6.5, 7.0 or 7.5 media (+ KCl, + nigericin) respectively. Note: After 4 hrs in the CO2 incubator, the pH of the freshly-pH-adjusted media will collapse to within 0.2 units of final pH (pH 7.3 ± 0.2). Within 8-12 hrs of being placed in incubator the pH of media in the 24-well plate will have adjusted to the final required pH of 7.3 ± 0.1. Note: The raw reporter assay readings of the wells containing nigericin and KCl will be 20-25% of the raw reporter readings obtained for the control wells that did not contain nigericin and KCl.
1
Suppl. Method 4: Isolation of membrane protein fraction from glioma cells for western blot analysis. The protcol was modified from the folowing reference: Holden P, Horton WA (2009) Crude subcellular fractionation of cultured mammalian cell lines, BMC Res Notes 2, 243. Reagents: (all solutions were prepared in autoclaved or disposable sterile plastic-ware, and autoclaved water was used to prepare reagents to minimize protease contamination). 5.0 M NaCl solution (autoclaved) 1.0 M HEPES buffer solution, pH 7.4 (prepared in autoclaved water, then stored at 4 °C; do not autoclave HEPES buffer solutions) 2.5 mg/ml Digitonin solution in H2O. Mix 25 mg digitonin (Sigma #D5628) with 10 ml autoclaved H2O in a disposable (sterile) 15 ml polypropylene tube. Digitonin will form a white suspension. Bring a 500 ml beaker half-full of water to boil. Remove the beaker from heat source and place the capped tube in it. As the tube warms, mix occasionally. Digitonin will go into solution and form a clear, transparent solution. Remove the tube to room temperature. Digitonin solution is now ready for use. The solution can be stored at -20 °C and reused by warming again. Note: Potency of digitonin (#D5628 is 50%). Use as is. 10% (v/v) Igepal CA-630 in H2O (prepared in autoclaved water, then stored at 4 °C). Note: Igepal CA-630 (#19656, United States Biochemicals; I-8896 Sigma) is a substitute for Nonidet P-40, a non-ionic, nondenaturing detergent that solubilizes plasma membrane and other organelle membranes. The above reference uses NP-40 (Tergitol), a stronger non-ionic surfactant, which may break-open both cytoplasmic and nuclear membranes. x100 Halt protease inhibitor cocktail (#87785, Thermo Scientific) 0.5 M EDTA, pH 8.0 (autoclaved)
Digitonin Buffer (for cytosolic protein extraction) Final volume = 10 ml Chemical (final concentration)
Volume (stock solution)
Stock solution
150 mM NaCl
0.3 ml
5.0 M NaCl
50 mM HEPES, pH 7.4
0.5 ml
1.0 M HEPES, pH 7.4
250 µg/ml digitonin
1.0 ml
2.5 mg/ml digitonin
x1 Halt Inhibitor mix
0.1 ml
x100 Halt Inhibitor mix
5 mM EDTA, pH 8.0
0.1 ml
0.5 M EDTA, pH 8.0
H2O
8.0 ml
--
Store at -20 °C. Thaw to room temperature prior to use.
1
Igepal Buffer (for membrane protein extraction) Final volume = 10 ml Chemical (final concentration)
Volume (stock solution)
Stock solution
150 mM NaCl
0.3 ml
5.0 M NaCl
50 mM HEPES, pH 7.4
0.5 ml
1.0 M HEPES, pH 7.4
1% (v/v) Igepal CA-630
1.0 ml
10% (v/v) Igepal CA-630
x1 Halt Inhibitor mix
0.1 ml
x100 Halt Inhibitor mix
5 mM EDTA, pH 8.0
0.1 ml
0.5 M EDTA, pH 8.0
H2O
8.0 ml
--
Store at -20 °C. Thaw to room temperature prior to use.
Protocol: This is designed for monolayer cultures of glioma cells in 100 mm diameter cell-culture plates or for spheroid/suspension cultures in 10 ml volumes. 1. Aspirate spent media. Rinse plates once with 10 ml cold PBS. For spheroids/suspension cultures, rinse by centrifugation (100 xg, 5 min (700 rpm, Sorvall RT-6000D centrifuge) and resuspension in 10 ml PBS. 2. To plate or cell pellet, add 2.5 ml Accutase. Incubate r.t. for 3-5 min. until cells detach. In case of suspension/spheroid culture, gently vortex occasionally until a uniform suspension is obtained. 3. Add 7.5 ml cold PBS to plates or tubes to a final volume of 10 ml. Centrifuge the cell suspensions at 378 xg (1500 rpm, Sorvall RT-6000D centrifuge) 4. Add 0.5 ml digitonin buffer to each cell pellet. Gently mix by pipette. Transfer to 1.5 ml microcentrifuge tube on ice. Keep 10 min with occasional mixing. 5. Centrifuge at 2000 xg at 4 °C (5000 rpm in a typical microcentrifuge). 6. Recover supernatant – this will be the cytoplasmic fraction. 7. Add 0.5 ml Igepal buffer to pellet. Make a suspension by repeated pipetting or vortexing. Keep on ice for 30 min. 8. Centrifuge at 7000 xg at 4 °C (9000 rpm in a typical microcentrifuge). 9. Recover the supernatant – this will be the membrane fraction. 10. The residual pellet will contain the nuclear and insoluble proteins. Store sample fractions from steps 6, 9 and 10 at -80 °C until use.
2
Test of membrane fractionation method by immunoblotting for MCT2.
The lung cancer cell line A549 (ATCC CCL-185) that highly expresses MCT2 and routinely used as a positive control for MCT2 antibody screening studies, was used to test the protocol. 20 µg aliquots of each fraction (digitonin = cytoplasmic; Igepal = membrane; residual pellet = nuclear) were resolved in a 10% SDS-PAGE gel, under the same conditions as described under Methods for western blotting in the main manuscript. The resolved proteins were transferred to PVDF membrane, and immunoblotted as described under methods. The primary band at approximately 50 kDa in the lane for membrane fraction corresponds to MCT2 protein (mwcalculated = 52 kDa; mwapp = 48 kDa)
3
Suppl. Method 5: Western blot analysis. Protein concentrations of membrane fractions from previous step were quantitated using a dye-binding assay at 1/250 dilution (Bradford assay, #500-0006, Bio-Rad, Hercules, CA). 10 µg aliquots of samples (unheated) were resolved in 1.5 mm thick 10% SDS-PAGE gels (#165-800x, Mini-Protean Tetra Gel apparatus, Bio-Rad) using the standard Laemmli Tris-glycine-SDS buffer system (Bio-Rad). Sample concentration in tracking gel was achieved at 60V, while resolving gel was run at 120 V until completion. In brief, protein samples were mixed with x4 sample loading dye (0.25 M Tris-HCl, pH 6.8, 8% SDS, 0.3 M DTT, 30% glycerol, 0.02% BPB) in a final volume adjusted to 50 µl with the Igepal-saline-HEPES buffer (Suppl. Method 4). 0.5 µl of a molecular weight marker ladder (SuperSignal Protein ladder, #84785, Pierce, Rockford, IL) was resolved on an end-lane to size molecular weight distribution in the gel. Following separation, the proteins were transferred to a PVDF membrane (#162-0177, Bio-Rad) in a Mini-Trans Blot apparatus (#170-3930, Bio-Rad) at 100V for 1 hr using CAPS buffer (10 mM CAPS, 10% methanol, pH 11). Each membrane was blocked for 1 hr in 5% (w/v) non-fat milk (Carnation Instant Nonfat Dry Milk, Nestlé-USA, Glendale, CA) in 40 ml TBS-Tween buffer (10 mM Tris-HCl, pH 7.5, 0.15 M NaCl, 0.05% Tween-20) at room temperature. Then, the membrane was incubated overnight (16 hrs) at 4 °C with a rabbit anti-human MCT2 polyclonal antibody (1 mg/ml; #M4470-12, United States Biological, Swampscott, MA), 1/500 diluted in 5 ml of 5% milk-TBS-Tween. The membrane was washed x3 in TBSTween buffer at r.t., followed by incubation for 1 hr at r.t. with secondary antibody (goat anti-rabbit IgG, #0031460, Pierce) 1/5000 diluted in 10 ml of 1% (w/v) milk in TBS-Tween. The membrane was washed as before and incubated with 7 ml of ECL mix (#170-5060, Clarity substrate, Bio-Rad) for 5 min. the membranes were exposed wet (covered in plastic-wrap) for 30 consecutive frames (30 min) in a chemiluminescence detection apparatus (Omega 12iC, Ultra Lum, Claremont, CA). The membranes were then washed briefly under distilled water, and incubated in a 0.1% CBB dye (Coomassie Brilliant Blue-R250, Bio-Rad), 10% acetic acid, 40% methanol staining solution for 10 minutes. The membrane was washed briefly under distilled water until clear of dye runoff, briefly placed in a 10% acetic acid, 40% methanol solution and both surfaces of membrane gently wiped with wetted Kim-Wipes to remove precipitated dye film, briefly washed in distilled water again and air-dried. The membranes were imaged for profiling of protein loading. Western blot and protein blot scans were digitized and quantitated using Un-Scan-It software (ver. 6.1, Silk Scientific, Orem, UT) with background subtraction. Note: all western blotting and protein staining incubations were carried out on a rocker platform.
1
Suppl. Fig. 3: Outline of strategy for de novo cloning of the MCT2 proximal promoter from a Fosmid gene library.
A 161 kbp Fosmid clone harboring the putative exon I region was identified by mapping chromosome 12 human genome clone registry (NCBI). The DNA sequence was mapped in silico and sequentially dissected with restriction enzymes to obtain a 4.2 kbp DNA fragment that harbored the promoter-Exon I region of human MCT2 gene.
Suppl. Fig. 4A: MCT2 promoter responds to changes in extracellular glucose or pyruvate levels by a similar magnitude.
Response of 4.2 kbp MCT2 promoter to extracellular pyruvate (5 mM) or glucose (25 mM) was evaluated in basal DMEM medium (supplemented with 2 mM glutamine as a minimal energy substrate; see Materials and Methods section). The 0.6 kbp minimal promoter (in 25 mM glucose) was used as the control (n=4). The 4.2 kbp promoter showed a 3.7 to 4.4 fold-induction to pyruvate and glucose respectively (**; p
