Bioinformatics analysis

The Search Tool for Recurring Instances of Neighbouring Genes (STRING) database (https://cn.string-db.org/) was used to analyze SIRT5-related proteins. Besides, the GPSuc database (http://kurata14.bio.kyutech.ac.jp/GPSuc/index.php) was used to screen succinylation sites of c-myc.

Sample collection

This study was approved by BeiJing ChaoYang Hospital. The study included 26 patients diagnosed with chordoma. The complete clinical data of chordoma patients, including age, sex, tumor location, tumor size, whether they were primary tumor, and whether the tumor was totally resected were collected in Table 1. The tumor tissue specimens and corresponding adjacent ones (about 2 cm away from the tumor margin) were collected and stored in liquid nitrogen for use. All subjects consented to clinical examination and sampling. Written informed consent was obtained from all subjects.

Table 1 Association between SIRT5 expression and the clinicopathologic characteristics of patients with chordoma. SIRT, sirtuinCell culture

Chordoma cell lines (U-CH1 and U-CH2), human embryonic kidney (HEK)-293T cells, and human umbilical vein endothelial cells (HUVEC) were purchased from American type culture collection (Manassas, VA, USA). U-CH1 and U-CH2 cells were cultured in Iscove’s modified Dulbecco’s medium (IMDM)/Roswell Park Memorial Institute (RPMI) (4:1; Gibco; Thermo Fisher Scientific, Inc., Waltham, MA, USA) medium supplemented with 10% heat inactivated fetal bovine serum (FBS, Gibco), 100 U/mL penicillin and 100 µg/mL streptomycin [17]. HEK-293T and HUVEC cells were maintained in Dulbecco’s modified eagle medium (DMEM, Thermo Fisher) containing 10% FBS and 1% penicillin/streptomycin. All cells were incubated in a humidified incubator at 37 °C with 5% CO2.

Cell transfection

SIRT5 short hairpin (sh) RNA (sh-SIRT5), negative control shRNA (sh-NC), negative control pcDNA 3.1 vector, and pcDNA 3.1-c-myc overexpression vector were synthesized by GenesScript Biotechnology Co. Ltd. (Nanjing, China). The cells (5 × 105 cells/well) were inoculated in a 6-well plate (Corning, NY, USA) a few days before transfection. After the cell confluence reached about 60–80%, transfection was performed using Lipofectamine 3000 (Thermo Fisher) for 48 h. Next, reverse transcription-quantitative polymerase chain reaction (RT-qPCR) was performed to analyze the expression of SIRT5 and c-myc.

In addition, arginine (R) mutations were introduced at K369 (K369R), K385 (K385R) and K411 (K411R) sites of c-myc, respectively (Genscript). Then, c-myc-K369R, c-myc-K385R, and c-myc-K411R were transfected into HEK-293T cells for 24 h.

Animal study

Animal experiment protocols in this study were approved by the Animal Ethics Committee of BeiJing ChaoYang Hospital according to the Guide for the Care and Use of Laboratory Animals (National Research Council). Twelve male BALB/c nude mice (8 weeks old) with an average weight of 25 g purchased from Oricell Biosciences Co. Ltd. (Guangzhou, China) were housed under specific pathogen free conditions at 25℃ with a 12-h light/dark cycle. The mice were randomly divided into two groups: sh-NC and sh-SIRT5 groups (six mice per group). The mice were subcutaneously injected with U-CH1 cells stablely transfected with sh-NC and sh-SIRT5 plasmids (1 × 106 cells/100 µL). The volume of the tumors was measured every week, for four weeks. The tumor size was measured with a caliper and the volume was estimated using the formula: a × b2/ 2 (a, the longest diameter; b, the shortest diameter). The mice were euthanized using pentobarbital sodium (40 mg/kg, Sigma-Aldrich, St. Louis, MO,USA) after 28 days, and the tumors were harvested, weighed and subjected to immunohistochemistry (IHC).

RT-qPCR

Total RNA from tissues and cells was extracted by TRIzol reagent (Yuanye Biotechnology Co. Ltd., Shanghai, China). Then, RNA was reverse transcribed into cDNA using the HiScript IV RT SuperMix for qPCR (Vazyme Biotechnology Co. Ltd., Nanjing, China), and the qPCR amplification experiment was performed using the ChamQ Universal SYBR qPCR Master Mix (Vazyme) with the reaction conditions: 95 °C for 30 s, 40 cycles of 95 °C for 10 s, 60 °C for 30 s, and a melt curve stage. Primers used in this study are synthesized by Tsingke Biotechnology Co. Ltd. (Beijing, China) and listed as follows: sirtuin (SIRT)5, forward, 5′-GCCATAGCCGAGTGTGAGAC-3′ and reverse, 5′-CAACTCCACAAGAGGTACATCG-3′; c-myc, forward, 5′-TCAAGAGGCGAACACACAAC-3′ and reverse, 5′-TAACTACCTTGGGGGCCTTT-3′; glyceraldehyde-3-phosphate dehydrogenase (GAPDH), 5′-TGTGGGCATCAATGGATTTGG-3′ and reverse, 5′-ACACCATGTATTCCGGGTCAAT-3′. The gene expression was calculated by the 2−ΔΔCT method.

Western blot

The Total Protein Extraction Kit (Solarbio Science & Technology Co., Ltd, Beijing, China) was used to extract the protein from tissues and cells according to the provided instructions. Then, the homogenate was homogenized in an ice bath, shaken (2 h), and centrifuged (12,000 rpm, 20 min) at 4℃. Then, the supernatant was taken and stored at -80℃ for use. Bicinchoninic acid (BCA) method (Solarbio) was used to detect the protein concentration. After that, 50 µg of protein was separated by 10% SDS-PAGE (Sigma) and transferred to the PVDF membrane (Solarbio). The membrane was blocked in 5% skim milk for 1 h in order to remove nonspecific adsorption, and incubated with the primary antibodies (Sigma) overnight at 4 °C. Then, the secondary antibody (Sigma) was incubated with the membrane for 1 h at room temperature after washing the membrane three times with Tris-buffered saline Tween (TBST, Solarbio). Finally, protein signal detection was performed using an enhanced chemiluminescence solution (Sigma). The specific proteins were visualized using the Odyssey™ Infrared Imaging System (Gene Company Limited, Hong Kong, China). GAPDH expression was used as an internal control to show equal loading of the protein samples. The used antibodies were listed as follows: SIRT5 (Abcam, Cambridge, MA, USA; ab259967; 1/1000), c-myc (Abcam; ab32072; 1/1000), succinyl lysine (PTM Biolabs, Hangzhou, China; PTM-401; 1/1000), glyceraldehyde 3-phosphate dehydrogenase (GAPDH) (Abcam; ab181602; 1/10,000), and goat anti-rabbit IgG (Abcam; ab205718; 1/5000).

Cell counting kit-8 (CCK-8) assay

Cell viability was detected by the CCK-8 kit (Vazyme) according to the instructions. Three replicate wells were set up. Firstly, the cells were seeded into a 96-well plate (Corning) at the density of 1 × 103 cells/well, then maintained in the incubator for 24 h. After that, 10 µL of CCK-8 solution was added to each well and incubated with cells for 2 h. Finally, a microplate reader (Thermo Fisher) was used to assess the absorbance at 450 nm.

Colony formation assay

For colony formation detection, the cells were planted in 6-well plates and cultured at 37 °C for two weeks. Then, the cells were fixed with 4% paraformaldehyde (PFA, Sigma) and stained with 0.5% crystal violet (Solarbio). Finally, the stained cells were counted under a microscope (Olympus, Tokyo, Japan).

Transwell migration and invasion assays

The Transwell migration and invasion assays were performed as previously described [18]. For the migration assays, the cells (5 × 104/100 µL) were seeded onto the 24-well Transwells (Corning) uncoated with Matrigel. For the invasion assays, the cells (5 × 104/100 µL) were seeded onto the 24-well Transwells pre-coated with 100 µL of Matrigel. The U-CH1 and U-CH2 cells were resuspended in serum-free medium and added to the upper chamber. The bottom chamber was prepared using 800 µL of RPMI-1640 supplemented with 10% FBS as a chemoattractant. After 48 h of incubation, the cells on the lower surface were washed with phosphate buffer solution (PBS, Vazyme), fixed with 4% PFA for 20 min, and stained in a dye solution containing 0.5% crystal violet for visualization. Finally, the cells were counted and photographed under a microscope. All experiments were performed in triplicate.

Co-immunoprecipitation (Co-IP) assay

Co-IP assay was performed to detect the interaction relationship between SIRT5 and c-myc in HEK-293T cells. After washing by pro-cooled PBS twice, the cells were lysed in Radio Immunoprecipitation Assay Lysis buffer (RIPA) buffer (Vazyme) containing protease inhibitors on ice for 30 min. Then, the supernatant was collected after centrifugating (12,000 g, 10 min), and 10 µL of it was taken as the input group. After that, SIRT5, c-myc, or lgG antibody (2 µg) was added into the remaining supernatant and incubated with protein G Plus-Agarose Immunoprecipitation reagent (Abcam) at 4 °C overnight. IgG was used as a negative control. Next, the beads were washed with lysis buffer four times. Immunoprecipitates were eluted by boiling with 1% (wt: vol) SDS sample buffer (Vazyme) and boiled at 100 °C for 5 min. The protein-protein complexes were subsequently subjected to Western blot. The labeled protein membranes were observed and quantified using the Tanon 5200 system (Shanghai, China). The used antibodies were listed as follows: SIRT5 (Abcam; ab259967; 1/30), GAPDH (Abcam; ab181602; 1/60), c-myc (Thermo Fisher; PA5-85185; 1/500), and goat anti-rabbit IgG (Thermo Fisher; 31,460; 1/1000).

Ubiquitination assay

IP assay combined with Western blot were used to access the succinylation level of c-myc (PTM Biotechnology Co., Ltd, Hangzhou, China) in HEK-293T cells. Briefly, the HEK-293T cell lysates were obtained and immunoprecipitated with c-myc antibody c-myc (Thermo Fisher; PA5-85185; 1/500) and protein A/G agarose, followed by Western blot against ubiquitin.

IHC

Paraffin-embedded tumor tissues were cut into sections (about 5 μm), dewaxed, rehydrated, and treated with 3% H2O2, respectively. Then, the sections were treated with 10 mM citrate buffer (Yuanye) and boiled in microwave oven for 3 min to expose the site of the antigen. After cooling to room temperature, the citrate buffer was discarded and the sections were washed by PBS twice. Next, normal goat serum was used to block the sections at 37℃ for 30 min. After that, the sections were incubated with primary antibodies against SIRT5 and c-myc overnight at 4℃, and were then treated with goat anti-rabbit IgG at 37℃ for 30 min. After adding DAB solution (Vazyme), the sections were counterstained using hematoxylin (Vazyme) and blued in 1% ammonia water. Finally, the sections were dehydrated, sealed, and observed under a light microscope (Leica Microsystems Trading LTD., Shanghai, China). The used antibodies were listed as follows: SIRT5 (Abcam; ab259967; 1/100), c-myc (Abcam; ab185656; 1/500), GAPDH (Abcam; ab181602; 1/2000), and goat anti-rabbit IgG (Abcam; ab205718; 1/5000).

Protein stability assessment

The cells were treated with cycloheximide (CHX, 100 µg/mL, Yuanye), and the protein level of c-myc at different time points (0, 8, 16, and 24 h) was analyzed by Western blot.

Statistical analysis

The SPSS 21.0 software was used to analyze data. Data are expressed as mean ± standard deviation (SD). Student’s t-test was used for comparison between the two groups. One-way analysis of variance (ANOVA) was used for comparison among groups. Statistical analyses were performed using GraphPad Prism software (v8.0.1, GraphPad Software Inc., San Diego, CA, USA). p < 0.05 indicates that the difference is statistically significant.