Cell lines and clinical samples

CRC cell lines, including SW48, HT29, LoVo, HCT116, DLD1, SW1116, SW480, SW620, CT26, MC38, and Caco2, were acquired from the American Type Culture Collection (ATCC). The KRAS G12D mutation was introduced into wild-type SW48 and HT29 cells using CRISPR/Cas9 technology. Briefly, the sgRNA targeting KRAS codon 12 was cloned into pSpCas9(BB)-2 A-Puro (PX459, Addgene, #62988) and transfected using Lipofectamine 3000 (Invitrogen, #L3000015) with 2 µg plasmid DNA per 10⁶ cells. Stable clones expressing KRAS G12D were selected using puromycin, and the successful introduction of the mutation was confirmed by Sanger sequencing. Elevated KRAS protein expression was further validated by Western blot analysis. Cells were maintained in DMEM (Dulbecco’s Modified Eagle Medium, Gibco, #11965092) supplemented with 10% FBS (fetal bovine serum, Gibco, #10099141) and 1% penicillin/streptomycin (Gibco, #15140122) at 37 °C in a 5% CO₂ humidified incubator. All the cell lines were authenticated via short tandem repeat (STR) analysis, and they were confirmed to be free from mycoplasma contamination. A total of 120 paired samples comprising human CRC tissues and adjacent noncancerous tissues (NCTs) were obtained from Nanfang Hospital. Written consent was obtained from all the subjects, with approval from the relevant institutional review boards. This study adhered to the ethical standards set forth in the Declaration of Helsinki and the CIOMS International Ethical Guidelines for Biomedical Research Involving Human Subjects.

Reagents and antibodies

The commercial antibodies used in this study and their manufacturers were as follows: anti-beta actin (Cat No. 66009-1-Ig, Proteintech), anti-beta-tubulin (Cat No. 66240-1-Ig, Proteintech), anti-DBT (Cat No. 12451-1-AP, Proteintech), anti-OXCT1 (Cat No. 12175-1-AP, Proteintech), anti-BCKDK (Cat No. 15718-1-AP, Proteintech), anti-ECHS1 (A3845, ABclonal; 11305-1-AP, Proteintech), anti-KRAS (ab191595, Abcam), anti-phospho-AKT (Ser473, #4060, CST), anti-phospho-S6K (Thr389,#9205,CST), anti-S6K (#9202,CST), Akt Antibody (#9272,CST), SIRT3 (AF5135, Affinity), anti-Ki67 (#9449,CST), anti-PCNA (#13110,CST), anti-e4E-BP1 (9452,CST), phospho-4E-BP1 (Thr37/46, #2855), DYKDDDDDDK tag polyclonal antibodies (14793,CST), Mouse Anti-rabbit IgG (Conformation Specific, 5127, CST), MRTX1133 and bevacizumab were purchased from Selleck, NAM (72345, Sigma), MA132(M8699, Sigma), Cycloheximide(CHX, 239763, Sigma). Primary antibodies were used at the following dilutions for Western blot: anti-ECHS1 (A3845, ABclonal, 1:1000), anti-KRAS (ab191595, Abcam, 1:500), and anti-beta actin (66009-1-Ig, Proteintech, 1:5000).

Mice and diet

Male athymic nude mice (BALB/c-nu/nu, 6 weeks old) purchased from the Animal Center of Guangdong Province were used to construct subcutaneous xenograft models. HCT116, HT29, CT26, and MC38 cell lines (2 × 10⁶ cells per mouse) were resuspended in 100 µL of PBS and subcutaneously injected into the right flank of each mouse using a 1 mL syringe with a 26-gauge needle. Tumor growth was monitored starting 6–7 days post-injection, when tumors became visible to the naked eye. Tumor volume was measured every 3 days using the formula: Volume = length × width × width / 2. The starting time for measurement varied depending on the cell line, with measurements taken accordingly. Tumor measurements began when tumors reached approximately 50–100 mm³ (typically 6–7 days post-injection), recorded every 3 days using digital calipers. MRTX1133 (10 mg/kg, i.p., daily) and bevacizumab (5 mg/kg, i.p., twice weekly) [43] were administered with CD or BRD for 3 weeks. Diet compositions were detailed in Supplementary Tables S2 and S3. Mouse weight and adverse effects were monitored, with data detailed in Supplementary Tables S4, S5 and S6. All animal care and experiments were approved by the Institutional Animal Care and Use Committee (IACUC) of Nanfang Hospital. All animal studies complied with relevant ethical regulations for animal testing and research. The mice were fed either a control diet (CD) or the BCAA-restricted diet (BRD). Diets were purchased from the Dyets Company.

Quantification of BCAA concentrations

BCAA concentrations in the culture medium were quantified via a BCAA kit (Sigma, MAK003) following the manufacturer’s instructions. Twenty-four hours postcultivation on a 10 cm plate, the medium was collected, and BCAAs were measured in a 100 µl reaction mixture. The absorbance at 450 nm was recorded to determine the BCAA concentration. The cells were subsequently washed, detached, and counted for normalization.

CCK8 and colony formation assays

For cell proliferation assays, cells (1,000 cells per well) were seeded in 96-well plates, and their viability was assessed daily for 6–7 consecutive days using the CCK8 assay. Specifically, 10 µL of CCK8 reagent (Dojindo Laboratories, Japan, #CK04) was added per well, incubated for 2 h at 37 °C, and absorbance was measured at 450 nm with a SpectraMax i3x (Molecular Devices). For the colony formation assays, cells (500 cells per well) were seeded in 6-well plates and cultured in DMEM supplemented with 10% FBS at 37 °C in a 5% CO₂ humidified incubator for 8–12 days. At the end of the experiment, colonies were washed with PBS, fixed in methanol, and stained with 0.1% crystal violet. Colonies exceeding 50 cells per well were counted in triplicate.

EdU assay

Cell proliferation was evaluated using a 5-ethynyl-2’-deoxyuridine (EdU) assay kit (RiboBio, Guangzhou, China, #C10310-1). Cells were seeded at a density of 3,000 cells per well in 96-well plates and cultured in DMEM supplemented with 10% FBS and 1% penicillin/streptomycin at 37 °C in a 5% CO₂ humidified incubator. After 72 h of incubation, cells were treated with 50 µM EdU (prepared by diluting the stock solution in culture medium according to the manufacturer’s instructions) and incubated for 2 h at 37 °C. Following this, cells were fixed with 4% formaldehyde (Sigma, #F8775) in PBS for 30 min at room temperature, then permeabilized with 0.1% Triton X-100 (Sigma, #X100) in PBS for 20 min. The EdU detection solution (100 µL per well, containing Alexa Fluor 488 azide) was added to the culture medium and incubated for 30 min at room temperature in the dark, per the kit protocol. Nuclear staining was performed by adding 100 µL of Hoechst 33,342 (1:1000 dilution in PBS, RiboBio) and incubating for 10 min at room temperature. After washing three times with PBS (5 min each), fluorescence was visualized using a fluorescence microscope (Nikon Eclipse Ti2) with excitation/emission wavelengths of 495/519 nm for EdU (green) and 350/461 nm for Hoechst (blue). Images were captured and analyzed for EdU-positive cells in triplicate experiments.

Immunoprecipitation and Western blotting

For immunoprecipitation (IP), cells were harvested, washed twice with ice-cold PBS, and lysed in a lysis buffer containing 50 mM Tris-HCl (pH 7.5, Sigma, #T3253), 150 mM NaCl, 0.5% Nonidet P-40 (NP-40, Sigma, #74385), and 1× protease inhibitor cocktail (Sigma-Aldrich, #P8340, diluted 1:100 from stock), unless otherwise specified. Approximately 1 × 10⁷ cells were lysed in 1 mL of buffer on ice for 30 min with intermittent vortexing. Lysates were centrifuged at 12,000 rpm for 15 min at 4 °C using a refrigerated centrifuge, and the supernatant was collected. For FLAG-tagged protein IP, 500 µg of protein (quantified by BCA assay, Thermo Fisher, #23225) was incubated with 20 µL of anti-FLAG M2 agarose beads (Sigma-Aldrich, #A2220) in a 1.5 mL microcentrifuge tube for 3 h at 4 °C with gentle rotation (10 rpm) on a rotary mixer. Alternatively, for specific antibody IP, lysates were incubated with primary antibodies (e.g., anti-ECHS1, ABclonal, A3845, 2 µg) and 20 µL of Protein G agarose beads (Thermo Fisher, #20398). Beads were washed three times with 1 mL of lysis buffer (5 min per wash at 4 °C), pelleted by centrifugation at 3,000 rpm for 1 min, and resuspended in 40 µL of 2× SDS loading buffer (125 mM Tris-HCl pH 6.8, 4% SDS, 20% glycerol, 10% β-mercaptoethanol, 0.02% bromophenol blue). Samples were boiled at 95 °C for 5 min using a heat block and cooled on ice.

For co-immunoprecipitation (co-IP) experiments, cells were lysed in a milder buffer containing 50 mM Tris-HCl (pH 7.5), 150 mM NaCl, 0.1% NP-40, and 1× protease inhibitor cocktail, prepared fresh and chilled to 4 °C. Approximately 2 × 10⁷ cells were lysed in 1 mL of buffer, and after centrifugation as above, the supernatant (1 mg protein) was incubated with anti-ECHS1 antibody (ABclonal, A3845, 2 µg) and 30 µL Protein G agarose beads for 4–5 h at 4 °C with rotation. Beads were washed three times with 1 mL of co-IP lysis buffer and processed as described for standard IP.

For Western blotting, extracted proteins or IP eluates were separated by SDS-PAGE using 10-12% polyacrylamide gels at 120 V for 90 min. Proteins were transferred to a PVDF membrane using a wet transfer system (Bio-Rad, #1703930) at 100 V for 1 h in transfer buffer (25 mM Tris, 192 mM glycine, 20% methanol). Membranes were blocked with 5% skim milk (Sigma, #70166) in TBST (Tris-buffered saline with 0.1% Tween-20, 20 mM Tris-HCl pH 7.5, 150 mM NaCl, 0.1% Tween-20) for 1 h at room temperature with gentle shaking. Primary antibodies were diluted in blocking buffer and incubated with membranes overnight at 4 °C with rocking. Membranes were washed three times with TBST (10 min each), then incubated with HRP-conjugated secondary antibodies in TBST with 5% milk for 1 h at room temperature. After three additional TBST washes (10 min each), protein bands were visualized using an enhanced chemiluminescence (ECL) kit (Vazyme, E412-02) and imaged with a ChemiDoc Imaging System (Bio-Rad). All experiments were performed in triplicate.

Immunohistochemistry (IHC) staining and scoring

For immunohistochemical staining of human CRC tissues, paraffin-embedded sections were first baked at 65 °C for 1–2 h. The tissues were then deparaffinized by placing them in three jars of xylene for 10 min each, followed by rehydration through a graded alcohol series (100% ethanol 1, 100% ethanol 2, 95% ethanol, 90% ethanol, 80% ethanol, and 70% ethanol), with each step lasting 1 min. After rehydration, the sections were washed three times in deionized water (ddH2O) for 1 min each. Antigen retrieval was performed using citric acid or EGTA, and endogenous peroxidase activity was blocked by incubating the sections with a peroxidase blocking agent for 10–15 min, followed by three washes with PBS. To block non-specific binding, the sections were incubated with goat serum working solution at room temperature for 1 h, and then washed once with PBS. Human CRC tissues were stained with antibodies against ECHS1(1:400). Paraffin-embedded tissues were sectioned at 4 μm and incubated with the appropriate antibodies at 4 °C overnight. The subsequent steps followed the protocols provided by the GTVision III Detection System/Mo&Rb manufacturer (Gene Tech, China). Additionally, paraffin-embedded xenograft tissue sections were stained with anti-Ki67(1:800), anti-ECHS1(1:400), and anti-PCNA (1:800) antibodies. The staining results were evaluated by two independent authors who were blinded to the patients’ clinicopathological data. Score 0: no staining or incomplete and faint/barely perceptible cytoplasmic staining in ≤ 10% of tumor cells; Score 1: incomplete and faint/barely perceptible cytoplasmic staining in > 10% of tumor cells; Score 2: weak-moderate complete cytoplasmic staining in > 10% of tumor cells OR intense cytoplasmic staining in ≤ 10% of tumor cells; Score 3: complete and intense membrane staining in > 10% of tumor cells.

Expression and purification of Recombinant human ECHS1

The mutants pRH281-ECHS1 K101R, K118R, K204R, and K204Q were constructed via a site-directed mutagenesis kit (C215-01, Vazyme, China). Protein expression was achieved in Escherichia coli BL21 (DE3) cells. A single colony was isolated, propagated in LB medium supplemented with ampicillin (100 mg/L), and incubated at 37 °C with shaking at 200 rpm. Protein induction was triggered at 18 °C using 400 µmol/L 3-indoleacrylic acid upon reaching an optical density of 0.4–0.6 at 600 nm (OD600). Following a 10-hour induction period, the cells were collected and disrupted. The lysates were centrifuged at 12,000 rpm for 30 min, and the supernatants were subsequently incubated with His-tag affinity beads for 5 h at 4 °C. The proteins were eluted with a 6× His peptide at 4 °C for 2 h and concentrated via ultrafiltration.

Immunofluorescence (IF) analyses

For cell fluorescence, cells were pre-seeded on 24-well plates with coverslips (biosharp, BS-14-RC, 14 mm) at a density of 100,000 target cells per well. After appropriate treatments, the cells were fixed according to established protocols and incubated with primary antibodies at a 1:100 dilution. Then, they were treated with fluorescent dye-conjugated secondary antibodies and stained with DAPI.

For tissue fluorescence, tissue preparation (repair and blocking) followed the same protocol as immunohistochemistry (IHC). Primary antibodies were added at a 1:100 dilution and incubated overnight at 4 °C. The next day, the samples were warmed to room temperature for 30 min, washed three times with TBST, and incubated with corresponding secondary antibodies at a 1:400 dilution for 1 h at room temperature. After three washes with TBST, fluorescent antibodies were applied and incubated for 30 min at room temperature. After another three washes with TBST, DAPI working solution was added and incubated for 5 min at room temperature. The tissues were then washed three times with TBST, and an anti-fluorescence quencher was applied before the slides were sealed with a coverslip.

For multicolor cell fluorescence, the same procedure was used with cell coverslips. After the first round of staining, microwave antigen retrieval was performed, and the same staining steps were repeated. Finally, DAPI was added and the slides were sealed with a coverslip.

Identification of ECHS1 acetylation sites

HEK293T cells were transfected with FLAG-tagged ECHS1 and incubated for 48 h. Prior to harvesting, the cells were treated with 5 mM nicotinamide (NAM, 72345, Sigma, USA) for 6 h. Total cell lysates were then incubated with Flag (M2) magnetic beads for more than 4 h, followed by three washes with ice-cold NP40 buffer. Proteins were eluted from the beads by incubation with a 3× Flag peptide mixture (P9801, Beyotime) at 4 °C for 2 h. The eluted proteins were separated via SDS‒PAGE and stained with Coomassie Brilliant Blue. Protein bands at 21–34 kDa were excised and analyzed by mass spectrometry via PTM Biolabs (Shenzhen, China).

Mass spectrometry analyses

Total lysates from HEK293T cells transfected with ECHS1-Flag were incubated with anti-FLAG M2 magnetic beads for over 4 h at 4 °C. The protein-bead complexes were washed three times with ice-cold NP40 buffer. Proteins were subsequently eluted from the beads by incubation with a 3× FLAG peptide solution (P9801, Beyotime, China) at 4 °C for 2 h. The eluted proteins were then subjected to mass spectrometry analysis via PTM Biolabs (Shenzhen, China).

Measurement of ECHS1 activity

ECHS1 enzyme activity was measured via previously established methods. The assay involved adding ECHS1 to a reaction mixture containing 130 µl of 50 mM Tris-HCl (pH 8.0) and 0.25 mM crotonyl-CoA. The reaction progress was monitored by quantifying the decrease in absorbance at 263 nm.

SiRNA transfection and RNA interference

CBP expression is reduced by RNA interference. Synthetic siRNA oligonucleotides were sourced from Shanghai Gene Pharma Co., Ltd., employing the following sequences: siCBP-1, 5′-GGAAGCAGCUGUGUACCAUTTdTdT-3′; siCBP-2, 5′-GCAUGAAUGCUAACUUUAAdTdT-3′; and siCBP-3, 5′-CCUACAGAUAUCAAGAAUAdTdT-3. Transfection of each siRNA was carried out via Lipofectamine RNAiMAX (Invitrogen, Thermo Fisher Scientific) according to the manufacturer’s protocol. The knockdown efficacy was confirmed by western blot analysis.

Ubiquitinylation assay

For the ubiquitinylation analysis, the protease inhibitor MG132(100µmol, Sigma) was administered 4 h before the cells were harvested. The cells were then harvested and lysed in 1% SDS buffer (Tris-HCl pH 7.5, 0.5 mM EDTA, and 1 mM DTT), followed by boiling for 10 min post lysis. Immunoprecipitation of the lysed proteins was performed by adding antibodies to the lysate, which was diluted tenfold with 10 mM Tris-HCl buffer.

Statistical analyses

The experiments were independently performed three times, yielding similar results. Statistical analyses were conducted via an unpaired two-tailed Student’s t test in GraphPad Prism 8.0. The data are presented as the mean ± standard deviation (S.D.). Statistically significant differences are denoted by the p values: *p < 0.05, **p < 0.01, ***p < 0.001, and ****p < 0.0001.