The DLD-1 colorectal cancer cell line was obtained from Fox Chase Cancer Centre (FCCC). The cell line was cultured in Roswell Park Memorial Institute (RPMI) 1640 culture medium, supplemented with 10% (v/v) fetal bovine serum (FBS), 1% (v/v) non-essential amino acids and 1% (v/v) Penicillin/Streptomycin/ in 75 cm3 culture flasks. Cells were sub-cultured twice a week by discarding the media and washing the cells in PBS to remove any residual media. This was followed by an incubation in 1 X Trypsin/EDTA at 37 °C with 5% CO2 in a humidified atmosphere for 5 minutes. The Trypsin/EDTA was inactivated with culture medium and cells were seeded at appropriate concentrations and dilution factors when necessary.
Before transfections could take place, cells were seeded on 6-well or 24-well tissue culture plates at specific concentrations, depending on the experiment being performed. Cells were allowed to reach 50-70% confluency prior to transfection. Thereafter, serum-free Opti-MEM® media was used to dilute the experimental Dharmacon™ ON-TARGETplus SMARTpool Human-RPSA siRNA (targeted to LRP/LR) as well as the negative control MISSION® esiRNA-RLUC siRNA. In order for the transfection procedure to take place, appropriate amounts of DharmaFECT® transfection reagent were also added to the cells. This was followed by incubation at 37°C for 72 hours in order to allow the transfection to take place.
SDS-PAGE and Western blotting
In order to determine the levels of several proteins upon siRNA-mediated down-regulation of LRP/LR, western blotting was performed. The Trans-Blot® Turbo™ Transfer System was used for electroblotting with set parameters at 25V for 30 minutes. Following this, the membranes were blocked for an hour using 3% (w/v) BSA (Bovine Serum Albumin) in 1 X PBS-Tween (0.1% (v/v) Tween 20 and PBS). Thereafter, the membranes were incubated overnight in the appropriate primary antibody diluted in blocking buffer [IgG1-iS18 (1:1000), anti-β actin peroxidase, (1:1000), anti-rabbit TRF-1 (1:1000), anti-rabbit TRF-2 (1:1000), anti-rabbit pTERT (1:1000) and anti-mouse hTERT (1:1000)]. The membranes then underwent three 10-minute washes in 1X PBS-Tween. Thereafter, the membranes were incubated for 1 hour at room temperature in the appropriate anti-mouse or anti-rabbit secondary antibody with a horseradish peroxidase (HRP) conjugate which was diluted in 3% (w/v) BSA in 1X PBS-Tween. This was followed by three more 10-minute washes in 1X PBS-Tween before the chemiluminescent substrate was added and proteins were detected using the ChemiDoc™ imaging machine. In addition, the 42 kDa β-actin antibody was used for the detection of β-actin which served as a loading control. The Bio-Rad Image Lab 5.1 Image acquisition and analysis software was used to analyse the blot features and capture the image data.
Cell cycle analysis – Flow cytometry
A total of 100 000 DLD-1 colorectal cancer cells were seeded per well in 6-well tissue culture plates prior to transfection for 72 hours. The cells were then harvested and washed with 1X PBS followed by a centrifugation at 420 x g for 10 minutes. Thereafter, cells were fixed in cold 70% ethanol in a dropwise manner while vortexing, in order to ensure all cells were fixed. Fixation took place for at least 24 hours at 4°C. Following this, cells were washed with 1 X PBS after which they were centrifuged twice at 420 x g for 10 minutes in order to rid the cells of the ethanol. Following the supernatant being discarded, cells were incubated with 200 µl of Guava® cell cycle reagent containing Propidium Iodide for 30 minutes in the dark. These resulting suspensions were evaluated using the BD Accuri C6 flow cytometer and software. Pulse processing was employed for the exclusion of cell doublets, using the pulse area vs. pulse width/height. Since the maximum emission of PI is in the orange range of the spectrum, the FL2 laser using a 562-588 nm band pass filter was utilized to detect the dye. Thereafter, the percentage of cells within each cell cycle was determined using markers set within the analysis program.
Telomerase activity assay - Quantitative polymerase chain reaction (qPCR)
A total of 750 000 DLD-1 cells were seeded per well in 6-well tissue culture plates prior to transfection for 72 hours. The cells were then harvested and centrifuged at 1500 x g for 10 minutes. The resultant pellets were resuspended in 250 μl of CHAPS ((3-((3-cholamidopropyl) dimethylammonio)-1-propanesulfonate)) lysis buffer) followed by 30-minute incubation on ice. Thereafter, samples were centrifuged for 20 minutes at 15000 x g at 4°C in a microcentrifuge where the resulting supernatant was snap frozen on dry ice. The protein concentration was then quantified using Nanodrop spectroscopy (Nanodrop® ND-1000) and the proteins were diluted to 500 ng/μl.
Telomerase activity detection
Relative telomerase activity was quantified by the TRAPeze® RT1 Telomerase detection Kit (Merck Millipore), following the manufacturers protocol. All samples were then subjected to experimental analysis by qPCR accompanied by a positive control (human embryonic kidney (HEK293) cell extracts with confirmed telomerase activity). Three negative controls were used: a minus telomerase control, consisting of only CHAPS Lysis Buffer to ensure the buffer had not been contaminated and had no telomerase activity; a no template control (water) consisting of only nuclease free/PCR Grade Water so as to normalise against primer dimer formation of the Amplifluor primers in the absence of telomerase activity, and lastly, since telomerase is a heat-sensitive enzyme, a heat-treated telomerase negative control was also included so as to evaluate each sample for heat sensitivity. Thereafter, 10 µl of each 500 ng/µl sample was incubated at 85 ⁰C for 10 minutes prior to detection, in order to inactivate telomerase. All reactions were performed in triplicate in 96-well qPCRplates. The mastermix contained the following: OneTaq® HotStart Taq Polymerase (5 U/μl), nuclease-free water and the TRAPeze RT reaction mix. All samples were analysed via qPCR with the CFX Maestro™ thermo cycler with the following cycling parameters applied: One cycle of 37°C for 30 minutes, 95°C for 2 minutes and 45 cycles of 95°C for 15 seconds, 59°C for 60 seconds and 45°C for 10 seconds. Telomerase activity was thereafter calculated from the standard curve generated by 1:10 serial dilutions (20–0.0002 amoles) of the provided TSR8 control template as per Merck Millipore instructions. Negative controls were included. The data was then analysed with CFX Maestro™ software version 1.0. All values were normalised against the negative controls, whereby, all negative control values (including signals for CHAPS only, no template control and heat-treated, respectively) were subtracted from the signal of each sample and thereafter, the mean value calculated for all biological repeats.
Proteome Profiler Antibody Arrays™
In order to determine whether proteins involved in the apoptotic and MEK/ERK signalling pathways are affected post siRNA-mediated down-regulation of LRP, Proteome profiler antibody arrays were performed as per the supplier’s instructions (R&D systems). A total of 750 000 DLD-1 cells were seeded per well in 6-well tissue culture plates prior to transfection for 72 hours. The cells were harvested, and cell counts were performed. Thereafter, 300-600 µl of cold lysis buffer (the lysis buffer for the apoptotic array contained a protease inhibitor cocktail) was added to each sample, followed by a 30-minute incubation at 4 °C. Each cell suspension was then centrifuged at 10 000 x g for 5 minutes where the resultant supernatant was used as the experimental protein lysate. A BCA assay was performed, each lysate was diluted, and 200-600 µg of protein was loaded on each membrane. Following this, each membrane was placed into the 8-well tray provided with each kit, followed by incubation with 1 ml of 1X Array buffer at room temperature for 1 hour, in order to ensure no non-specific binding to the membrane surface. The 1X Array buffer was then removed, and the membranes were incubated with 1 ml of protein sample overnight at 4ºC with gentle shaking. Thereafter, each membrane was placed into separate containers where they were washed three times with 20 ml 1X Wash Buffer for 10 minutes. Arrays were incubated with the detection antibody cocktail solution for 1 hour at room temperature and then washed three times with 1 X Wash buffer. This was followed by incubation with 1X Streptavidin-HRP at room temperature for 30 minutes. The arrays were washed three times with washing buffer, and protein spots were visualized using the chemiluminescence detection reagents supplied in the Array Kits. The intensity score of each duplicated array spot was measured with the ImageLab (version 5.1) software and the average intensity was calculated by subtracting the averaged background signal and PBS spots (negative control). The identity and the respective coordinates of all the antibodies on the arrays can be found in the Supplementary Data section.
Quantification of mRNA expression levels – Quantitative reverse transcriptase polymerase chain reaction (RT-qPCR)
A total of 750 000 DLD-1 cells were seeded per well in 6-well tissue culture plates prior to transfection for 72 hours. To determine mRNA expression levels, RNA was first extracted from the DLD-1 samples. The extraction procedure followed the Quick-RNA™ MiniPrep kit (manufacturer’s protocol. Briefly, cells were trypsinized and collected by centrifugation. The cell pellets were then resuspended and lysed in the provided lysis buffer, followed by centrifugation. Thereafter, the supernatant containing the RNA was further purified by incubating the samples in DNase buffer for 15 minutes in order to eliminate any trace contaminants of DNA. The samples were then loaded onto spin columns specific for RNA extraction and washed twice using 400 µl wash buffer. Lastly, the purified RNA samples were eluted using 80 µl nuclease-free water. The resultant sample concentrations were determined using Nanodrop spectroscopy (Nanodrop® ND-1000).
Thereafter, a 1% (w/v) agarose gel containing a nucleic acid gel stain was used for gel electrophoresis (agarose, in 1 x TBE buffer consisting of Tris, boric acid and EDTA) to evaluate the RNA integrity. The samples were loaded with 6 x loading dye and Low range DNA ladder was used as a molecular weight marker. The DNA was separated at 100 V for approximately 30 min in TBE buffer. The RNA was visualized using ChemiDoc™ system in order to view total/intact RNA as well as any DNA contamination (Refer to Supplementary data section).
Once the absence of DNA contamination was confirmed, the samples were converted into cDNA. The SensiFAST™ cDNA synthesis kit was used as per the manufacturer’s instructions whereby a mastermix was prepared containing appropriate volumes of: 5X TransAmp buffer, reverse transcriptase and nuclease-free water. Thereafter, 1 µg of mRNA was added to each PCR tube prepared. A no reverse transcriptase control was also prepared for each sample, containing each reagent except reverse transcriptase. Thereafter, the samples were anyalzed via qPCR using the CFX Maestro™ thermo cycler with the following parameters: One cycle of 25°C (primer annealing) for 10 minutes, one cycle of 42°C (reverse transcription) for 15 minutes, one cycle of 48°C (additional reverse transcription for highly structured RNA) for 15 minutes and one cycle of 85°C (inactivation) for 5 minutes. The resultant samples were stored at -20°C for mRNA quantification.
mRNA expression level quantification - Quantitative polymerase chain reaction (qPCR)
In order to quantify mRNA expression levels, qPCR was performed using the SensiFAST SYBR™ No-ROX kit as per the manufacturers protocol whereby mastermix was prepared containing: 2X SensiFAST SYBR™ No-ROX mix, 10 µM of the forward primer (refer to the Supplementary data section), 10 µM of the reverse primer (refer to the Supplementary data section), nuclease-free water and lastly, 1 µg of cDNA. This mixture was pipetted into the necessary wells of a 96-well qPCR plate, ensuring that the mixture was thoroughly combined. Thereafter, the samples were placed in the CFX Maestro™ thermo cycler with the following parameters: One cycle at 95°C for3 minutes, 45 cycles at 95°C for 15 seconds, one cycle Tm of gene for 30 seconds, melt curve: one cycle at 95°C for 10 seconds, 65°C for 1 minute and one cycle at 72°C for 30 seconds. Refer to the the Supplementary data section for the melt peaks of each gene.
Thereafter, the average Cq value from technical repeats was used for calculations. In addition, to acquire reliable comparisons of gene expression levels between samples, corresponding qPCR reactions must be performed for reference genes known have invariant expression. The reference genes used in this study included GAPDH (Glyceraldehyde 3-phosphate dehydrogenase) and ACTB (β-actin). These reference genes were used since these genes are present in all cells. Moreover, the reference genes were used because when tested, the mRNA levels of these genes were not affected between each treatment (refer to supplementary data section). Normalisation with stably expressed reference genes as internal controls, known as the comparative Cq or the ΔΔCq method, was used for the normalisation of mRNA data. In addition, reverse transcriptase controls (RTC’s) were subtracted from all samples for further normalisation. The comparative Cq method normalises the Cq value of a target gene to internal reference genes before comparisons are made between samples. First, the difference between Cq values (ΔCq) of the target gene and the mean of two reference genes was calculated for each sample, and then the difference in the ΔCq (ΔΔCq) was calculated between samples. The fold-change in expression of the two samples was calculated as 2-ΔΔCq, where 2 derives from 1 + efficiency and efficiency is assumed to be 1 (i.e., 100% efficiency) . ). Refer to the Supplementary data section for primer sequences, qPCR melt curves and peaks for each gene.
Sequential Window Acquisition of All Theoretical Mass Spectra (SWATH MS)
A total of 750 000 DLD-1 cells were seeded per well in 6-well tissue culture plates prior to transfection for 72 hours. The cells were then harvested and centrifuged at 1500 x g for 10 minutes. Cell pellets were resuspended in 200 ul of lysis buffer [1% SDS, 100 mM Tris-HCl, pH 8.0, MS grade H2O] per pellet. Thereafter, cells were sonicated on ice for 9 pulses (10 sec per pulse) followed by centrifugation at 15 000 x g for 10 minutes to clear cell debris. Cell lysates were then incubated with 25 units, (1ul of stock –2500 units in 100ul) of benzonase, per 0.5 million cells and at 2mM MgCl2 at 37°C for 30 minutes. This was followed by another centrifugation at 15 000 x g for 10 minutes. The supernatant was collected, and the concentration determined using a BCA assay. Protein solutions were then reduced using 10 mM DTT for 30 minutes at 37°C and alkylated using 40 mM IAA for 30 minutes in the dark.
Sample clean-up and digestion
All experiments were performed with a KingFisher™ Flex magnetic particle processing robot. The Fisher™ Flex system was configured for automated HILIC-protein clean-up and on-bead trypsin digest. Deep-well 96-plates were loaded in each carousel position with each plate filled as follows: 1) 96 well tip heads; 2) 10 µl, 20mg/ml hyper porous magnetic HILIC micro spheres in 20% ethanol and 180 µl equilibration buffer (100 mM NH4Ac, 15% ACN pH 4.5); 3) equilibration Buffer (500 µl); 4) Protein extract mixed 1:1 with bind buffer (200 mM NH4Ac, 30% ACN pH 4.5), final volume of 100 µl; 5) 500 µl 95% ACN (wash 1); 6) 500 ul 95% ACN (wash 2); 8) 200 µl 50 mM ammonium formate pH 8.2 and Promega sequencing grade trypsin for an enzyme:protein ratio of 1:10. The Bindit programme was then run with the magnetic pins transferring the magnetic HILIC beads from position 2 to 8 and in the process binding proteins, washing off SDS and other contaminants and finally generating peptides ready for LC-MSMS analysis post the on-bead trypsin digest.
Spectral library building (Data-dependent analysis)
Post HILIC peptide samples were vacuum dried, resuspended in 2% ACN/0.2%FA and spiked with iRT peptide standards. Three injections were then performed per sample for each of the conditions. Analysis was performed using a Dionex Ultimate 3000 RSLC system coupled to an AB Sciex 6600 TipleTOF mass spectrometer. Peptides were first de-salted on an Acclaim PepMap C18 trap column (75 µm × 2 cm) for 5.5 min at 5 µl/min using 2% acetonitrile/0.2% formic acid, than separated on Acclain PepMap C18 nanoRSLC column (75 μm × 15 cm, 2 µm particle size) using a 60 min linear gradient at a flow-rate of 0.5 l/min (A: 0.1% formic acid; B: 80% acetonitrile/0.1% formic acid). An electrospray voltage of 2.5 kV was applied to the fused silica emitter (New Objective: 20 μm ID x 5 cm, 10 μm tip). The 6600 TipleTOF mass spectrometer was operated in Data Dependant Acquisition mode. Precursor MS scans were acquired from m/z 400-1500 using an accumulation time of 250 ms followed by 80 MSMS scans, acquired from m/z 100-1800 at 25 ms each, for a total scan time of 2.3 sec. Multiply charge ions (2+ - 5+, 400 -1500 m/z) were automatically fragmented in Q2 collision cells using nitrogen as the collision gas.
SWATH –MS analysis
Three injections, representing each of the technical replicates, were performed per sample for each of the conditions. For SWATH–MS, the LC gradient used for spectral library building was applied. The SWATH-MS method consisted of the acquisition of 100 MS2 scans of overlapping sequential precursor isolation windows (variable m/z isolation width, 1 m/z overlap, high sensitivity mode) covering the 400 to 900 m/z mass range, as well as a single MS1 scan. The accumulation time was 300 ms for the MS1 scan and 25 ms for the MS2 scans for a total of 2.3 s total cycle time.
Protein identification and spectral library building
Raw data were searched against the human UNIPROT sequence database (reviewed entries, downloaded on 2 June 2017) supplemented with a list of common contaminating proteins as well as the sequences of the Biognosys iRT peptide retention time standards. Thereafter, data processing was performed using Protein Pilot (v 5.0.1). The following search settings were applied: trypsin as the proteolytic enzyme, IAA based alkylation, thorough search effort. False discovery rate (FDR) analysis was then performed with 1% global FDR cut-off applied at PSM, peptide and protein levels. A spectral library was constructed by importing the .group Protein Pilot output into the Skyline (v 184.108.40.20679) spectral library builder. A cut-off of 0.995, corresponding to 1% peptide FDR, was applied during import. The Biognosys iRT peptides were appended to the library in order to normalize the peptide retention time. The following filters were applied for peptide and protein import into Skyline: Tryptic peptide, size 5-36 amino acids, with up-to 1miss-cleavage and one matched cleavage site allowed; Structural modifications: Carbomedomethyl (Cys), Oxidation (Met), Acetylation (N-terminal); Precursor charge states 2-4, product charge states 1-2, product ions: b and y; Ion match tolerance of 0.1 m/z. Post protein, peptide and transition import into Skyline a decoy peptide list was generated by shuffling the sequences of all imported peptides.
SWATH data files were converted to mzML format as well as centroided using the Proteo Wizard MS Convert tool. The converted SWATH mzML files were imported into Skyline (v 220.127.116.1179). The following filters were applied for SWATH mzML import: Precursor charge states 2-4, product charge states 1-2, product ions: b and y; 3-6 transitions per peptide; Product m/z range 100-1800; MS1 filtering: Isotope peaks included (Count), Precursor mass analyzer (Centroid), Peaks (3), Mass accuracy (20 ppm); MS2 filtering: Acquisition method (DIA), Product mass analyzer (Centroid), Mass Accuracy (20 ppm) and an isolation scheme of 80 variable windows as per the SWATH method run in Analyst; For retention time filtering only scans within 10 min of the predicted iRT retention times were selected. Once all SWATH mzML files were imported all repeated peptides and proteins were removed and a peak scoring model was trained using mProphet and the decoy peptides generated during spectral library building. All peaks were then re-integrated using this model and only peptides with q-values of less than or equal to 0.01 were used for further processing.
Selection of differentially expressed proteins
Differentially expressed proteins were detected via the Skyline external tool, MS Stats. Thereafter, the MS Stats output list of differentially expressed proteins was further filtered so that only entries fitting the following criteria remained: Minimum fold change ≥ 2 and Maximum adjusted p-value ≤ 0.01.
A one-way ANOVA followed by a two-tailed student’s t-test with a confidence interval of 95% was used as a means of analysing and confirming the data. To further validate the data, the Bonferroni post-hoc test was applied, with p-values of less than 0.05 considered to be significant. The statistical analysis was performed using the Microsoft® Excel 365 statistical programme.