Cell culture. The human CRC cell lines HCT116 and LoVo were obtained from the ATCC (the American Type Culture Collection, Manassas, VA, USA) and maintained at 37°C in a humidified incubator containing 5% CO2, in McCoy’s 5A (Gibco BRL, Grand Island, NY, USA) supplemented with 10% Fetal Bovine Serum (FBS; Gibco BRL) and a Penicillin-Streptomycin Solution. The cell lines were free of Mycoplasma. The following chemicals were also added into the culture media used in this study: Campathecin(catalog #CSN16581) and the TKT inhibitor, Oxythiamine (catalog #136-16-3) were purchased from CSNpharm (Chicago, USA) while Adenosine (catalog # 58-61-7); Guanosine (catalog #118-00-3); Cytidine (catalog #65-46-3) and Uridine (catalog #58-96-8) were bought from Sangon Biotech (Shanghai, China).
SiRNA transfection. The siRNAs specifically targeting SIRT5 has been described previously. The SIRT5 siRNAs comprised of the following sequences: siRNA-1, 5′- GCUGGAGGUUAUUGGAGAATT − 3′, siRNA-2, 5′- GUGGCUGAGAAUUACAAGATT − 3′. In addition, the siRNA specifically targeting TKT was purchased from GenePharma (Shanghai, China). The TKT siRNA included: 1) 5′- CCAGCCAACAGCCAUCAUUTT − 3′, 2) 5′- CCGGCAAAUACUUCGACAATT − 3′.
These siRNAs were transfected into subconfluent cells using the Dharma FECT 1 transfection reagent (Dharmacon, Lafayette, CO, USA) according to the manufacturer’s instructions.
Each transfection was conducted in six-well plates where 30% confluent CRC cells were transfected with 1µg of small interfering RNA (siRNA) and 5µL of Dharma FECT 1 in 100 µL of the Opti-MEM medium (Invitrogen). The negative control was a nonspecific siRNA (NC-siRNA).
Western blotting. Cells or tissues were collected and lysed using the Radioimmunoprecipitation Assay (RIPA) lysis buffer supplemented with a protease inhibitor cocktail (Kangcheng, Shanghai, China). Proteins were then separated through SDS-polyacrylamide Gel Electrophoresis (SDS-PAGE) before being immunoblotted. The following primary antibodies were used: anti-SIRT5 (catalog #HPA022002, 1:2000, Sigma-Aldrich), anti-γ-H2A.X (catalog #9718, 1:1000), anti-p-ATM (catalog #5883, 1:1000), anti-p-ATR (catalog #2853, 1:1000), anti-p-CHK1 (catalog #2348, 1:1000), anti-p-CHK2 (catalog #2197, 1:1000), anti-cleaved caspase 3 (catalog #9664, 1:1000), anti-cleaved caspase 8 (catalog #9496, 1:1000), anti-cleaved caspase 9 (catalog #7237, 1:1000), anti-cleaved PARP (catalog #5625, 1:1000), anti-cyclin D1 (catalog #2978, 1:1000), anti-cyclin D3 (catalog #2936, 1:1000), anti-cyclin A2 (catalog #4656, 1:1000), anti-cyclin E1 (catalog #20808, 1:1000) and anti-α-tublin (catalog #2148, 1:2000) were purchased from Cell Signaling Technology (Danvers, MA, USA); anti-ATM (catalog #70103, 1:1000), anti-ATR (catalog #70109, 1:1000), anti-TKT (catalog #101477, 1:1000) and anti-TALDO1 (catalog# 102076, 1:1000), which were purchased from GeneTex (California, TX, USA). On the other hand, anti-CHK1 (catalog #8048, 1:1000), anti-CHK2 (catalog #5278, 1:1000) anti-RPI (catalog #515328, 1:1000) and anti-RPE (catalog #393655, 1:1000) were purchased from Santa Cruz Biotechnology, (Santa Cruz, CA, USA) while anti-FLAG (catalog #F1804, 1:1000, Sigma-Aldrich); anti-HA (catalog #MMS-101P, 1:1000, Convance, Princeton, NJ, USA); anti-pan succinylation (catalog #PTM-401,1:1000), anti-pan glutarylation (catalog #PTM-1151, 1:2000) and anti-pan malonylation (catalog #PTM-901, 1:1000) were obtained from PTM Biolabs (HangZhou, China). The secondary antibodies included; the Anti-beta Actin antibody (catalog #KC-5A08, 1:2000, Kang Cheng, China), Peroxidase-conjugated anti-rabbit antibody and anti-mouse antibodies (1:5000, Kangcheng). Thereafter, the western blot bands were detected using an ECL western blotting substrate (Thermo Scientific, Waltham, MA, USA) and the analysis was repeated at least three times.
Assessment of the cell cycle and apoptosis. Progression of the cell cycle and apoptosis were analyzed through flow cytometry. Briefly, the HCT116 and LoVo cells were cultured under the specified conditions. In the cell cycle assay, cells were stained with 50 µg/mL of propidium iodide containing 20 µg/mL RNase (DNase-free) after which they were analyzed using flow cytometry according to the manufacturer’s instructions. The G1, S (DNA synthesis phase), G2 and M (mitosis) phases were then identified based on the content of DNA and the study obtained the percentage of cells in the distinct phases. All the experiments were performed at least three times. Analysis was also conducted using an FITC Annexin V fluorescein isothiocyanate (FITC)/propidium iodide (PI) double stain assay (BD Pharmingen, San Diego, CA, USA). The stained cells were then analyzed through flow cytometry.
Half of the medium was replaced with a fresh medium containing 20 µM of EdU for 2 h followed by fixation in 3.7% formaldehyde in PBS then a 0.5% TritonX-100 permeabilization step.
500 µL of the Click-iT reaction cocktail was used per coverslip, according to the manufacturer’s protocol (Click-iT EdU Iamge kits, Life Technologies, catalog no. C10338). Thereafter, the hoechst 33342 dye was bound to DNA and the average fluorescence intensity was quantified using the Zeiss digital image processing software, ZEN® (blue edition). In the EdU Flow Cytometry Assay, EdU was detected using Pacific Blue™ azide at an excitation of 405 nm in a violet emission filter (Click-iT EdU Flow Cytometry Assay Kits, Life Technologies, catalog no. C10418). The percentage of EdU+ cells was then analyzed using the FlowJo software.
The Soft Agar Colony Formation Assay. The soft agar colony formation assay was conducted 48 h after transfection with the SIRT5 siRNA. The cells were counted and seeded in six-well plates at 104 cells in a layer of 0.7% agar/complete growth medium over a layer of 1.2% agar/complete growth medium in the wells of a 6-well plate. Then, a cell medium containing the indicated concentration of four nucleotides (A, U, C, G) was replenished every 3 days. Cultures were grown in a humidified incubator at 37℃. 21 days after seeding, the cells were incubated with 4% paraformaldehyde for 10 min and 0.5% crystal violet was used to stain the colonies. Thereafter, megascopic colonies were quantified under a light microscope. Colonies, whose diameter was larger than 50 µm were counted and analyzed.
Measurements of enzyme activity. The cells were homogenized with ice-cold 0.1 M Tris-HCl buffer (pH 7.6) and centrifuged at 12000 rpm for 15 min after which the supernatant was collected. The protein concentration was then determined using the BCA Protein Assay Kit and recorded as C (g/L). Then, TKT activity was measured as previously described by Bayoumi and Rosalki, with minor modifications. Briefly, the supernatant (50 µL) was mixed with a 200 µL reaction mixture containing 14.4 mmol/L ribose-5-phosphate, 190 µM/L NADH, 380 µM/L TPP, > 250 U/L glycerol-3-phosphate dehydrogenase and > 6500 U/L triose phosphate isomerase. The Optical Density (OD) of TKT was then measured immediately at 340 nm, then once every 5 min for 1h. Moreover, TKT activity was deduced from the difference in absorbance measured at 15 and 45 min. The enzyme activity assay was repeated three times for each group. The TKT activities (%) of the treated group were then normalized to those of the control category (100%).
Immunofluorescence. The HCT116 or LoVo cells were plated in 4-well chamber slides and transfected with siRNA or plasmids as indicated. Forty-eight hours after transfection, cells were fixed with 4% formaldehyde, permeabilized with 0.2% Triton X-100 and blocked in 1% BSA in PBS for 1 h at room temperature. The cells were then stained using rabbit polyclonal anti-γH2A.X (1:400), rabbit polyclonal anti-TKT (1:100), rabbit polyclonal anti-TALDO1 (1:100), mouse monoclonal anti-RPI (1:100), mouse monoclonal anti-RPE (1:100), mouse monoclonal anti-FLAG (1:1200), rabbit polyclonal anti-SIRT5(1:100, Sigma) and mouse polyclonal anti-SIRT5(1:100, Santa Cruz) antibodies followed by secondary antibodies (donkey anti-mouse Dylight 488 and donkey anti-rabbit Dylight 594), respectively. Thereafter, fluorescence was analyzed using a confocal laser scanning microscope (Carl Zeiss, AG, Germany). After staining with DAPI (1:10,000), the media chamber was removed from the glass slide, which was treated using the Prolong Gold antifade reagent (P7481) and sealed with a coverslip. 24 h later, the average fluorescence intensity was quantified using the Zeiss digital image processing software, ZEN® (blue edition). Finally, co-localization analysis was performed using the “colocalization” module of the ImageJ program.
The Comet Assay. The HCT116 and LoVo cells were transfected with SIRT5 or negative control siRNA. DNA damage was then measured using the comet assay (single-cell gel electrophoresis) 48 h after transfection, where the comet assay kit (Cell Biolab, San Diego, CA, USA) was used according to the manufacturer’s instructions. Biefly, the cells (1 x 105 cells/mL ) were re-suspended in ice-cold PBS (without Mg2+ and Ca2+). Thereafter, the cell samples were mixed with Comet Agarose at a ratio of 1:10 (v/v), homogenized by pipetting then the mixture (75 µL/well) was transferred immediately onto the OxiSelect™ Comet Slide. The slide was maintained horizontally and carefully transferred from the alkaline solution to a horizontal electrophoresis chamber. Electrophoresis was then run in an alkaline buffer. Notably, the assay was performed under low/dim light conditions to avoid damage to the cell samples by ultraviolet light. Following this, the slides were viewed through epifluorescence microscopy using a FITC filter. In addition, measurement was done using a public domain PC-image analysis programme CASP software, version 1.2.2 (CASPLab, University of Wroclaw, Wroclaw, Poland). The comet assay was repeated at least three times and the tail moment was calculated.
Immunohistochemistry (IHC). Tumors dissected from nude mouse xenograft models of CRC were subjected to IHC to detect γH2AX and SIRT5. Sections (3 mm-thick) were incubated with the antibodies against γH2AX (1:400; CST) and SIRT5 (1:400, Sigma). HRP-conjugated secondary antibodies were used and subsequently mounted with Diaminobenzidine (DAB). Pathological evaluation was then conducted in a blinded manner. Moreover, apoptosis was detected using the Terminal Deoxynulceotidyl Transferase Nick-end-labeling (TUNEL) staining Kit (Keygen Biotech, Nanjing, China) according to the manufacturer’s instructions.
In vivomodels. For the rescue-function experiments, nude mice (nu/nu, male, 5 weeks old) were weighed and injected subcutaneously with HCT116 cells (2 × 106 cells). Thereafter, the mice were randomly divided into four groups, including the control vector or TKT WT with or without SIRT5 knockdown. Then, two adenoviruses targeting the SIRT5 and TKT genes were used every 3 days. Moreover, the diameters of the tumors were measured every 3 days using calipers. Additionally, the tumor volumes were calculated using the formula: (shortest diameter)2 × (longest diameter) × 0.5. The tumors were eventually dissected and analyzed.
All animal studies were conducted according to the guidelines published by the Animal Ethics Committee of Renji Hospital, Shanghai Jiao Tong University, School of Medicine.
Measurement of Metabolite Levels.
For stable isotope-tracing analysis, LoVo cells (2 × 106/sample) were grown to 80% confluence in complete media. The cells were briefly rinsed twice with PBS then the medium was replaced with DMEM (glucose-free) supplemented with 11.11 mM [1,2-13C2]-glucose, 2 mM label-free glutamine in 10% dialyzed FBS, 100 units mL− 1 Penicillin-Streptomycin and 3.7g/L sodium bicarbonate for 0.5h, 1h, 6h, 12h and 24h. Thereafter, the cell metabolites were extracted by adding pre-cold 80% (vol/vol) methanol and centrifuged at 15000g for 15 minutes, at 4 ℃. The supernatant was evaporated until it became dry. The residues were then reconstituted in 100 µL of 50% aqueous acetonitrile (1:1, v/v) prior to UHPLC-HRMS/MS analysis. Additionally, chromatographic separation was performed on a ThermoFisher Ultimate 3000 UHPLC system with a Waters BEH Amide column (2.1mm × 100 mm, 1.7 µm). The injection volume was 2 mL, the flow rate was 0.4 mL/min and the column temperature was 10℃. The mobile phases consisted of water (phase A) and acetonitrile/water (90:10, v/v) (phase B), both with 15mM ammonium acetate (pH = 9, modified by ammonium hydroxide). Moreover, a linear gradient elution was performed using the following program: 0 min, 95% B and held to 2 min; 5 min, 85% B; 7 min, 80% B; 11 min, 75%B; 12 min, 55% B and held to 13.5 min; 14 min, 95% B and held to 18 min. Thereafter, the eluents were analyzed separately using a ThermoFisher Q Exactive™ Hybrid Quadrupole-Orbitrap™ Mass Spectrometry (QE) in a Heated Electrospray Ionization Negative (HESI-) mode. For stable isotope-tracing analysis, the measured distribution of mass isotopomers was corrected for the natural abundance of isotopes using the IsoCor software.
Finally, the study used Liquid Chromatography Tandem Mass Spectrometry (LC-MS/MS) techniques, for targeted metabolomic analysis. A standard curve for the (d)NTP standard was prepared and used to determine the concentration of (d)NTPs in each unknown sample.
Statistical analysis. Data are shown as the means ± standard deviation (SD). Comparisons of data between two groups were performed using Student’s t-test (two-tailed). For multiple comparisons, an analysis of variance (ANOVA) test was used. The correlation between the expression of SIRT5 and TKT was analyzed using the χ2-test. P values < 0.05 was accepted as statistically significant.