Parental HCT116 cells were purchased from ATCC. HCT116 p53 KO clones and p53 KO clones stably expressing p53 mutants R175H, R248W, and R273H were created using CRISPR/Cas9 and subsequent infection with pWZL-p53mut-BLAST and pBabe-iRFP-PURO plasmids as previously described . Stock flasks of all cells were maintained in McCoy’s 5A (modified) medium (Gibco, 26600023) supplemented with 10% FBS and 1% penicillin-streptomycin. All cells were cultured at 37 °C in a humidified atmosphere of 5% CO2.
Serine and glycine deprivation
Serine and glycine deprivation experiments were conducted as previously described . Briefly, cells were seeded and left for 24 h in Dulbecco’s modified Eagle medium (DMEM) (Gibco, 21969) supplemented with 10% FBS and 2 mM L-glutamine. The medium was then changed daily either continuing with the seed DMEM (full medium conditions) or switching to starvation medium (− SG). The − SG medium consisted of MEM (21090) supplemented with additional 1× MEM vitamins (Gibco, 11120), 10% dialysed-FBS (Hyclone, Thermo Scientific), 2 mM L-glutamine, and additional D-glucose (to 25 mM).
For counting, cells were seeded in 24-well plates in full medium. The next day, cells were shifted to serine- and glycine-depleted medium or given fresh full medium. Medium was replaced every day. For counting, cells were trypsinized, re-suspended in PBS-EDTA, and counted with a CASY Model TT Cell Counter (Innovatis, Roche Applied Science) at the indicated time points in each experiment. Cell counts were conducted prior to any media change.
Measuring cell growth using iRFP was performed as previously described . Briefly, cells were seeded in 96-well CellBIND black microplates with clear flat bottom (Corning 3340) and allowed to settle. The next day, cells were shifted to serine- and glycine-depleted medium or given fresh full medium. Media were replaced daily. iRFP intensity was measured using an Odyssey Li-Cor. For quantification, plates were scanned at 169 μm resolution with a 3.5-mm offset and a low-intensity setting held constant throughout each experiment. Image Studio software (LI-COR, V5.2) was used to scan and subsequently quantify the plates.
Adherent cells were labelled with CellROX Green reagent (5 μM) for 15 min in serum-free and phenol-red-free DMEM (C10444, Life Technologies). Cells were then washed in phosphate buffered saline (PBS) and re-suspended in 1x PBS+ 2%FBS for analysis. 4′,6-Diamidino-2-phenylindole dihydrochloride (DAPI, Sigma Aldrich) was added to a final concentration of 1 μg/ml to each sample and was used to identify viable cells for analysis. Single cells were analysed on a BD Fortessa flow cytometer using unstained and cumene hydroperoxide-stained (100 μM) cells as controls. At least 10,000 events were collected for each sample. Data were analysed using FlowJo X 10.0.7r2 (FlowJo, LLC). Unless otherwise stated, median fluorescence intensity values were obtained and compared across samples.
The siRNA used to target human MDM2, p21 (CDKN1A), and ATF4, as well as the non-targeting siRNA control pool, were all purchased from Dharmacon (siGENOME SMART pool siRNA) and transfected at 20 nM concentration using Lullaby siRNA transfection reagent and the manufacturer’s recommended reverse transfection procedure (OZ Biosciences). Cells were left in the seeding/transfection medium for 24 h prior to any medium change.
Liquid chromatography-mass spectrometry
Liquid chromatography-mass spectrometry (LC-MS) sample preparation and analysis were performed broadly as described previously [18, 19]. Cells were incubated in fresh assay medium for 3 h. Metabolites were extracted by rapidly removing cell media, washing wells once with ice-cold PBS, and lysing cells in ice-cold methanol/acetonitrile/H2O (50:30:20) at volumes scaled based on cell counts of the counting plate to 2 × 106 cells per millilitre extraction buffer. Sample plates were shaken at 4 °C for 10 min before the extraction buffer was collected from each well, spun for 15 min at 16,000×g in a chilled (4 °C) centrifuge, and then analysed by LC-MS.
For metabolite analysis, a Q Exactive Orbitrap mass spectrometer (Thermo Scientific, Waltham, MA, USA) was used together with a Thermo Ultimate 3000 HPLC system. The HPLC setup consisted of a ZIC-pHILIC column (SeQuant, 150 × 2.1 mm, 5 μm, Merck KGaA, Darmstadt, Germany), with a ZIC-pHILIC guard column (SeQuant, 20 × 2.1 mm) and an initial mobile phase of 20% 20 mM ammonium carbonate, pH 9.4, and 80% acetonitrile. Cell and media extracts (5 μl) were injected, and metabolites were separated over a 15-min mobile phase gradient, decreasing the acetonitrile content to 20%, at a flow rate of 200 μl/min and a column temperature of 45 °C. The total analysis time was 23 mins. All metabolites were detected across a mass range of 75–1000 m/z using the Q Exactive mass spectrometer at a resolution of 35,000 (at 200 m/z), with electrospray (ESI) ionisation and polarity switching to enable both positive and negative ions to be determined in the same run. Lock masses were used, and the mass accuracy obtained for all metabolites was below 5 ppm. Data were acquired with Thermo Xcalibur software. The peak areas of different metabolites were determined using Thermo TraceFinder 4.0 software where metabolites were identified by the exact mass of the singly charged ion and by known retention time on the HPLC column. Commercial standards of all metabolites detected had been analysed previously on this LC-MS system with the pHILIC column.
For the analysis of p53 conformation, IP experiments were performed broadly as previously described . Adherent cells were washed once in ice-cold PBS. Protein lysates were then prepared using RIPA buffer (Millipore) supplemented with cOmplete ULTRA EDTA-free protease inhibitors (Roche) and PhosSTOP phosphatase inhibitors (Roche). Equivalent amounts of total protein (1.5–2 μg), determined using a Pierce BCA protein assay kit (ThermoFisher Scientific), were incubated overnight at 4 °C with either p53 Ab1620 (Abcam) or pAb240 (Santa Cruz Biotechnology) antibody (1:100 dilution) and 20 μl of Protein G Dynabeads (ThermoFisher Scientific). Beads were washed three times in RIPA and resuspended in buffer containing RIPA, NuPAGE LDS sample buffer, and NuPage Reducing Agent (both ThermoFisher Scientific). Protein was eluted from the beads by boiling at 95 °C for 10 min. The resulting samples were analysed by Western blotting.
For ATF4 IP experiments, samples were prepared as described above except they were incubated with ATF4 antibody D4B8 (Cell Signaling Technology) (1:100 dilution) instead of the p53 antibodies.
As with the IP experiments, protein lysates were prepared using RIPA buffer (Millipore) supplemented with cOmplete ULTRA EDTA-free protease inhibitors (Roche) and PhosSTOP phosphatase inhibitors (Roche). The resulting samples were separated using precast NuPAGE 4–12% Bis-Tris protein gels (ThermoFisher Scientific), transferred to nitrocellulose membranes using NuPAGE transfer buffer (ThermoFisher Scientific) with 20% methanol, and blocked in a PBS solution containing 5% BSA (Sigma Aldrich) and Tween-20 (Sigma Aldrich). Membranes were incubated overnight at 4 °C with primary antibodies (1:1000 dilution unless otherwise indicated). Membranes were washed in PBS-Tween20 and incubated with secondary antibodies (1:15000 dilution) for 45 min at room temperature prior to a final set of washes in PBS (no Tween 20) and detection. Proteins were detected using a Li-Cor Odyssey Infrared Scanner and LiCor Image Studio Software.
MDM2 (SMP-14) (sc-965), MDM2 (D-7)(sc-13161), CREB-2 (B-3)(sc-390063), p53 (DO-1) (sc-126), PSPH (H-11)(sc-365183), PSAT1 (L-24)(sc-133929), p53 (pAb240) (sc-99), and Actin (I-19)(sc-1616) were purchased from Santa Cruz Biotechnology.
The p53 (pAb1620) (ab16776) was purchased from Abcam.
COXIV (#11967), HSP90 (#4877), p21 (#2947), PHGDH (#66350), ATF4 (#11815), and HA-tag (#3724) were purchased from Cell Signaling Technology.
IRDye 800CW and 680LT all raised in Donkey (anti-Goat, anti-Rabbit, and anti-Mouse) were used for standard Western blots (LiCor).
Light chain-specific monoclonal antibodies with AF680 or AF790 probes were used for IP Western blots (Jackson ImmunoResearch).
Mouse procedures were carried out under UK Home Office licence number 60/4181 (Karen Blyth) and conducted in line with the Animals (Scientific Procedures) Act 1986 and the EU Directive 2010. Experiments were sanctioned by Local Ethical Review Process (University of Glasgow). Mice were housed on a 12/12 light/dark cycle and fed and watered ad libitum.
For HCT116 xenograft experiments, athymic female nude (nu/nu) mice (obtained from The Jackson Laboratory, 7–8 weeks old) were fed specified diets either containing or lacking serine and glycine as previously described [13, 21] for a period of 1 week prior to the start of the experiment. Mice in each group received bilateral subcutaneous injections of 100 μl of either HCT116 R175H or R248W cells (2 × 106 cells) suspended in phosphate buffered saline (PBS) (n = 5 mice per group). Each mouse was injected with only one cell line. Following injection, subcutaneous tumour growth was monitored by both iRFP imaging as previously described  and using callipers (both twice per week). Relative tumour growth was determined by comparing iRFP intensity to the baseline (day 3 post-injection) scan for each tumour. Tumour volume by callipers was calculated using the formula (length × width2/2).
Tissues were fixed in 10% neutral buffered formalin for 24 h before paraffin embedding and sectioning. Slides cut from paraffin blocks were de-paraffinised and rehydrated. Antigen retrieval was performed in citrate-based antigen unmasking solution (Vector H-3300) for 15 min in a microwave. Endogenous peroxidase activity was quenched by incubation with BLOXALL blocking solution (Vector SP-6000) according to the manufacturer’s instructions. For p21 and MDA staining, slides were blocked in 5% BSA with 5% rabbit serum in TBS-T for 1 h. Slides were incubated with primary antibody diluted to 1:1000 in blocking solution overnight at 4 °C. Secondary antibody incubation and downstream signal detection were performed using the Vectastain ABC elite kit (Vector PK-6010) and ImmPACT DAB (Vector SK-4105) following the manufacturer’s recommendations. For MDM2 IHC staining, slides were instead processed using the M.O.M. kit and protocol from Vector Labs (BMK-2202) with the primary antibody diluted to 1:1000. Slides were dehydrated, counterstained, and mounted with coverslips prior to analysis using Olympus BX51 microscope.
Seven random × 20 magnification images were taken from each IHC slide using an Olympus BX51 microscope with Zen Blue software (Zeiss). From these images, the positive staining per slide area was calculated using ImageJ software. Each image was expanded into an RGB stack with the green channel used for quantification as it offered the best separation for DAB staining. For each set of stained slides, a control slide was used to set a threshold for positive staining. This threshold was then applied in batch format to the rest of the images in the set (all images from all slides stained with a given antibody), and the resulting positive staining area for each was measured and collected by ImageJ. These measured areas were then averaged across the images of a given slide to give a staining percentage area value for each sample.
Human patient survival analysis
We obtained survival data by accessing the publicly available harmonised cancer datasets hosted on the National Cancer Institute GDC data portal. Through the portal, we compared survival between patients with R248 mutations (either R248W or R248Q) to those with the R175H mutation. Patient survival data arising from this search were imported into GraphPad Prism. The median survival of each group was compared using the Mantel-Cox Log-rank test.
Data plotting and statistical analysis
All data were plotted using Prism 7 (Graph Pad). Statistical analysis for each experiment was performed using the tools within Prism 7 and the indicated tests.