MB cell lines, D341 Med (HTB-187™), CHLA-01-MED (CRL-3021™), and CHLA-01R-MED (CRL-3034™) were purchased from the American Type Culture Collection (ATCC) and cultured according to the supplier’s recommendation. D341 cells were cultured in Minimum Essential Medium Eagle (EMEM- M0643, Life Technologies, CA, USA) supplemented with 20% foetal bovine serum (FBS). CHLA-01-MED and CHLA-01R-MED were cultured in Dulbecco’s Modified Eagle’s Medium/Nutrient Mixture F-12 Ham (D8900, Sigma-Aldrich, MO, USA) supplemented with B-27™ Supplement (Sigma-Aldrich, Ryde, NSW, Australia), 20 ng/ml basic FGF (Sigma-Aldrich), and 20 ng/ml EGF (Sigma-Aldrich). Cells were maintained at 37 °C in a humidified atmosphere of 5% CO2.
Metabolic activity of cells as a marker of cell viability and proliferation was assessed by WST-1 Cell Proliferation Assay Kit (10008883, Cayman Chemical, MICH, USA) according to the manufacture’s recommendation. Three MB cell lines, D341, CHLA-01, and CHLA-01R, were used in this assay. Briefly, cells were seeded at a density of 30,000 cells/well in 96 well plates. 24 h later, treatment was added as shown in the results section. Assay reaction was assessed 24 or 72 h after treatment. For that, 10 μL of WST-1 reagent was added to each well for 4 h, absorption was measured using a plate reader (Multiskan Go, Thermo Fisher Scientific, Scoresby, VIC, Australia). Drugs used, including mibefradil dihydrochloride hydrate (M5441), NNC55-0396 hydrate (N0287), verapamil hydrochloride (V4629), nifedipine (N7634), vincristine (V0400000), and lomustine (L5918), were purchased from Sigma-Aldrich.
ATP level and protein content
For the assessment of cellular ATP levels, a luminescent ATP assay was employed. 3 × 104 cells were seeded in 100 µL per well in transparent 96-well plates and media and left to settle overnight. Subsequently, drug treatments were added to cells for 72 h. Cells were then transferred to Eppendorf tubes and centrifuged for 3 min at 0.2 g. Following media removal, cells were washed with sterile phosphate-buffered saline (PBS) followed by another centrifugation to obtain cell pellet. For cell permeabilization, 40 μL of 0.5% Triton X-100/PBS was added to each tube for 10 min at room temperature. 10 μL of cell lysate was mixed with 90 μL assay buffer (300 μM d-luciferin, 5 μg/mL luciferase, 625 μM EDTA, 75 μM DTT, 6.25 mM MgCl2, 25 mM HEPES, 1 mg/mL BSA in PBS, pH 7.4) in white 96-well plates, followed by immediate measurement of luminescence using a plate reader (Fluoroskan Ascent, Thermo Fisher Scientific). Protein content from cell lysates (10 μL) was quantified using the DC Protein Assay (500-0116; Bio-Rad, CA, USA) as recommended by the manufacturer. Absorbance at 750 nm was measured using Multiskan Go Microplate Spectrophotometer (Thermo Fisher Scientific) and used for normalisation of ATP content in different treatments.
For immunoblotting, 5 µg of total protein samples were resolved on a 10% Bis-Glycine-polyacrylamide gel and transferred onto AmershamTM ProtranTM 0.2 μm Nitrocellulose (NC) blotting membrane (10600001, GE Healthcare). NC membranes were blocked in Tris-buffered saline with 0.1% Tween-20 (TBST) solution containing 5% (w/v) non-fat dry milk (NFDM) powder. Proteins were detected using primary antibodies rabbit polyclonal PARP (9542S, Sigma-Aldrich) at a 1:1000 dilution, rabbit monoclonal caspase-3 (ab32042, Abcam, Cambridge, UK) at a 1:1000 dilution, and mouse monoclonal glyceraldehyde 3-phosphate dehydrogenase (GAPDH) (G8795, Sigma-Aldrich) at a dilution of 1:10,000 as loading control. Secondary antibodies included horseradish peroxidase (HRP)–conjugated goat anti-rabbit (170-6515, Bio-Rad, 1:3000 dilution) and goat anti-mouse IgG (170-6516, Bio-Rad, 1:3000 dilution). Antibodies were prepared in 5% (w/v) NFDM powder dissolved in TBST solution. Anti-PARP and caspase-3 primary antibodies were incubated with membrane at 4 ̊ C overnight, while GAPDH and secondary antibodies were incubated at room temperature for 1 h. PageRuler™ Plus Prestained Protein Ladder (26620, Thermo Fisher Scientific) was used to ease detection of the molecular sizes (in kDa) of protein bands. AmershamTM ECLTM Prime Western Blotting Detection Reagent (RPN2236, GE Healthcare) was used to detect proteins. Digital images were recorded using the Amersham™ Imager 600 (29083461, GE Healthcare). Density of bands were quantified using Image LabTM Software (version 6.0.1, Bio-Rad) and normalized to GAPDH.
Spheroid generation was carried out by seeding CHLA-01R cells at 1 × 104 cells per well in 200 μL volume in round bottom 96 Ultra-Low Attachment Microplate (Product number 7007, Corning, MA, USA). Spheroid formation was initiated by centrifugation of the plate at 1000 g for 10 min and the plate was incubated at 37 °C and 5% CO2 for five days until spheres were formed.
Propidium iodide (PI) incorporation and dual live/dead viability assays
To assess cell membrane integrity, as a marker of cell death, PI fluorescent staining was performed using PI dye. PI is impermeable to the intact plasma membrane and therefore will only bind to DNA of cells with compromised cell membrane. To stain MB spheres, 100 μL of media were removed from each well and PI at a concentration of 40 μg/mL in PBS (100 μL) was added to the treated spheres and analysed under fluorescent microscope (IN Cell Analyzer 2000, GE Healthcare, IL, USA). ImageJ 1.49q software (NIH, Bethesda, MD, USA, website: https://imagej.nih.gov/ij/) was used to analyse PI incorporation by measuring the signal intensity of the sphere from the images. For live/dead dual staining, treated spheres were dissociated into single-cell suspension using Accutase solution (A6964, Sigma-Aldrich). Subsequently, cells were washed with PBS and stained with 3 μM calcein-AM (Thermo Fisher Scientific) and propidium iodide (5 μM) followed by incubation for 30 min. 100 µl of cell suspension was transferred to black plates and imaged with fluorescent microscope (IN Cell Analyzer 2000) using excitation of 475 nm /emission of 511 nm for calcein-AM and excitation of 542 nm /emission of 620 nm for PI. Generated images were automatically analysed using IN Carta Image Analysis Software (GE Healthcare).
Assessment of mitochondrial membrane potential
Detection of altered mitochondrial membrane potential was performed using 5,5′,6,6′-tetrachloro-1,1′,3,3′-tetraethylbenzim-idazolylcarbocyanine iodide (JC-1) Mitochondrial Membrane Potential Assay Kit (ab113850, Abcam) according to the manufacturer’s instructions. Briefly, MB cells were washed twice and incubated with the assay solution containing JC-1 dye (10 μM) for 30 min. Cells were then washed, suspended in assay buffer (supplemented with 5% FBS) and seeded at 6 × 104 in 50 μL in a black 96-well plate. 50 µl of 2× concentration of treatments, including different concentrations of mibefradil, NNC, Carbonyl cyanide 4-trifluoromethoxy phenylhydrazone (FCCP) as a positive control, or no treatment control, were added to cells followed by incubation at 37 °C for 6 h. Cells were imaged using IN Cell Analyzer 2000 (excitation wavelength used was 475 nm and emission wavelengths was 511 nm and 587 nm for the monomer and the aggregates of JC-1 molecules respectively). Images were analysed using IN Carta Image Analysis Software (GE Healthcare).
Measurement of cytosolic free Ca2+
Cytosolic free Ca2+ levels were measured using Ca2+ indicator Fluo-4 (Fluo-4 NW Calcium Assay Kit, F36206, Invitrogen) according to the manufacturer’s protocol. Briefly, MB cells D341 and CHLA-01 were plated at 7.5 × 104 in 100 µL per well in a black plate 96-well plate (µClear®, Greiner, Germany) coated with poly-L-lysine (P4832, Sigma-Aldrich, Australia). After overnight incubation, cells were washed twice with the assay buffer (1.26 mM calcium chloride, 0.49 mM magnesium chloride, 0.40 mM magnesium sulfate, 5.33 mM potassium chloride, 0.44 mM potassium phosphate monobasic, 4.16 mM sodium bicarbonate, 137.9 mM sodium chloride, 0.33 mM sodium phosphate dibasic anhydrous) and loaded with the dye solution for 50 min at 37 °C and 10 min at room temperature. Dye solution was removed, and cells were washed with the assay buffer (same as above excluding calcium chloride). For treatment, NNC (3.5-20 µM) in 0.5 mM BAPTA (B1214, Thermo Fisher Scientific) was added for 10 min in Ca+2 free buffer. Fluorescence (excitation at 475 nm and emission at 525 nm) was measured at 20°C using a plate reader (Tecan Infinite M Nano, Tecan Austria GmbH).
MB cell line, CHLA-01, was seeded at 3 × 105 cells per well in 12-well plate. After overnight incubation, cells were treated with different concentration of mibefradil (0, 3.5, and 7.5 μM) for 72 h. Cells were washed with PBS and lysed using chilled lysis buffer, 100 mM NaCl, 1% IGEPAL®, 0.5% sodium deoxycholate, 50 mM Tris (pH 8.0), 1× protease inhibitor (Roche Diagnostic GmbH, Germany), 1× phosphatase inhibitor (Roche Diagnostic GmbH, Germany). Protein concentrations were determined using Bio-Rad DC Protein Assay as described above.
Aliquots of 30 mg protein were sequentially reduced using 10 mM DTT overnight at 4 °C, alkylated using 50 mM iodoacetamide for 2 h at ambient temperature and then digested with 1.2 mg proteomics-grade trypsin/LysC (Promega) according to the SP3 protocol (8). Digests were halted by the addition of TFA to 0.1% and peptides collected by centrifugation at 21,000× g for 20 minutes. Samples were desalted using ZipTips (Merck) according to manufacturer’s instructions. Proteomic analysis deployed a combination of data-dependent acquisition (DDA) and data independent acquisition (DIA) methods on a Q-Exactive HF and Ultimate 3000 RSLCnano LC/MS system (Thermo Scientific, Massachusetts, USA).
First, a project-specific spectral library was generated using off-line high-pH reversed phase HPLC peptide fractionation. A pooled digest (~180 mg peptide) was desalted using Pierce desalting spin columns (Thermo Scientific), evaporated to dryness and finally resuspended in 25 ml HPLC loading buffer (2 % acetonitrile containing 0.05 % TFA). Peptides were separated on a 100 × 1 mm Hypersil GOLD (particle size 1.9 mm) HPLC column (Thermo Scientific) using an Ultimate 3000 RSLCnano system with microfractionation and automated sample concatenation enabled. The HPLC was operated at 30 mL/min using a 40 min linear gradient of 96% mobile phase A (water containing 1% triethylamine, adjusted to pH 9.6 using acetic acid) to 50% mobile phase B (80% acetonitrile containing 1% triethylamine), followed by 6 min washing in 90% B and re-equilibration in 96% A for 8 min. Sixteen concatenated fractions were collected into 0.5 mL Protein lo-bind Eppendorf tubes, evaporated to dryness then reconstituted in 12 mL HPLC loading buffer.
Peptide fractions were analysed using 90 min nanoflow HPLC gradient and a Top15 DDA-MS method as previously described (9). Individual peptide samples (~1 mg peptide) were analysed by DIA-MS using 26 × 25 amu sequential MS2 scans over the range of 397.5-1027.5 m/z, with 1 amu overlap between windows between MS1 spectra (390 - 1240 m/z) acquired at 120k resolution. MS2 spectra were acquired at a resolution of 30,000 using an AGC target of 1e6, maximum IT of 55 ms and normalized collision energy of 27.
DDA- and DIA-MS raw files were processed using Spectronaut software (version 13.12, Biognosys AB, Switzerland). The project-specific library was generated using the Pulsar search engine to search DDA MS2 spectra against the UniProt Homo sapiens protein sequence database using Biognosys (BGS) software factory settings, including N-terminal acetylation and methionine oxidation as variable modifications and cysteine carbamidomethylation as a fixed modification, up to two missed cleavages allowed and peptide, protein and PSM thresholds set to 0.01. Retention time alignment was based on the high precision iRT concept (10).With the exception that single-hit proteins were excluded, BGS factory settings were also used for relative quantitation between samples using the MaxLFQ algorithm (in Spectronaut version 15) for protein label-free quantitation and global normalization based on median MS2 intensity values.
Statistics and bioinformatics analysis
Proteins identified as candidates in Spectronaut (Supplemental Table S1) were imported into Perseus software (http://coxdocs.org/doku.php?id=perseus:start) for further processing. Label-free quantitation (LFQ) values were first log2 transformed and proteins with missing values were removed before using an unpaired t-test for statistical analysis. Proteins with altered expression of ±1.5 fold-change and false discovery rate (FDR)-adjusted p values below 0.05 compared to the control (Supplemental Table S2) were considered significant and selected for the enrichment analysis. Statistical overrepresentation test of significantly altered proteins was conducted using PANTHER online tool (11) with GO Biological Process annotation. Statistical analysis was performed using GraphPad Prism (version 8.0, GraphPad Software, Inc., San Diego, CA). Details of statistical analyses are provided in the corresponding figure legends.