Regents and antibodies

UC (TN7108, Topscience, Shanghai, China) and SC79 (T2274, Topscience, Shanghai, China) were dissolved in dimethyl sulfoxide (DMSO) (67-68-5, MP Biomedicals, Santa Ana, USA). The antibodies were listed as follows: rabbit anti‐GAPDH (10,494-1-AP, Proteintch, Wuhan, China), rabbit anti‐AKT (pan) (C67E7) (4691S, CST, Boston, USA), rabbit anti‐phospho-Akt (Ser473) (4060S, CST, Boston, USA), rabbit anti‐mTOR (7C10) (2983S, CST, Boston, USA), rabbit anti‐phospho-mTOR (Ser2448) (2971S, CST, Boston, USA), rabbit anti‐YBX1 (HY-P80936, MCE, State of New Jersey, USA), and the secondary antibody HRP-conjugated Affinipure Goat Anti-Rabbit IgG(H + L) (HY-P80936, Proteintech, Wuhan, China) for Western blot (WB).

Cell culture

All cell lines (CRC cell lines DLD1, HCT116 and RKO and the HEK293T cell lines) were purchased from the American type culture collection ATCC (Manassas, VA, USA). And all cells were cultured in RPMI-1640 (C22400500CP, Gibco, Waltham, MA, USA) and 10% fetal bovine serum (FBS) (F101-01, Vazyme, Nanjing, China) in an incubator with 5% CO2 at 37 °C.

Cell viability assay

A total of 4000 CRC cells were seeded in a 96-well plate and incubated overnight. CRC cells were subjected to treatment with dimethyl sulfoxide (DMSO) at a concentration of 0.1% as the control, along with varying concentrations of UC (12.5, 25, 50, 100, and 200 μM) for 24, 48, and 72 h. Cell viability was assessed using the Cell Counting Kit-8 (CCK-8) assay (C0005, Topscience, Shanghai, China) following the provided procedure. The absorbance at 450 nm was measured after a 2 h incubation at 37 °C using a microplate reader.

Colony formation assay

CRC cells were plated in six‐well plates and contained 2000 cells per well. After incubation overnight, these were treated with UC (15, 30 µM) for another 72 h. The medium was changed every 3 days. 14 days later, 4% paraformaldehyde (BL539A, Biosharp, Anhui, China) was used for fixing cells for 25 min and staining with 0.1% purple crystal (G1062, Solarbio, Beijing, China) for 25 min. Photographs were taken and the cell colonies were manually counted.

Apoptosis assays and cell cycle analysis

The FITC–Annexin V Apoptosis Detection Kit (A211-01, Vazyme, Nanjing, China) was used to detect apoptosis. CRC cells were harvested after incubation with 15 and 30 µM UC for 72 h and washed in cold PBS twice. Staining was performed using 5 µL of FITC–annexin V and 5 µL of propidium iodide. After 25 min of incubation at room temperature (25 °C) and protection from light, 400 µL of 1X binding buffer was added, then analyses were performed within 1 h using a flow cytometer (FCM) (BECKMAN COULTER, California, USA). The Cell Cycle Kit (F10797 Invitrogen, California, USA) was used to conduct the cell cycle analysis. CRC cells were harvested after incubation with 15 and 30 µM UC for 72 h and fixed in 70% ethanol overnight. Cells were stained with 500 µL FxCycle™ PI/RNase Staining Solution for 15–30 min at room temperature and protected from light. Then the FCM (BECKMAN COULTER, California, USA) was used to analyze PI-positive cells according to the given protocol.

Wound-healing assay

A sterile 200µl tip was used to create a straight wound for analysis. Cells were incubated with UC (15, 30 µM) in a six-well plate, and the straight wounds were photographed and measured under a microscope at 0, 24, and 48 h.

Transwell assay

1 × 105 (DLD1 and RKO) cells were cultured in the top chamber of the 24-well plate with serum-free FBS and incubated with UC (15, 30 µM). The bottom chamber was cultured in RPMI-1640 medium with 30% FBS [30]. 4% paraformaldehyde and 0.1% purple crystal were used to fix and stain the invading cells after incubation for 24 h, and then a microscope was used to count the cells.

Mouse xenograft model

To create the xenograft model, 5 × 106 DLD1 cells were injected into the flank of nude mice purchased from Beijing Huafukang Bio-Tech Co. Once the tumor volume reached 20 mm3, the mice were randomly divided into the control group and UC group (n = 5 per group). The mice received UC (intraperitoneal injection, 5 mg/kg, 3 times a week) or DMSO according to the indicated assay. Tumor volume was measured and calculated every 2 days, volume = length × width2/2. The tumors were harvested and weighed after 3 weeks. Paraformaldehyde was used to fix the tumor for H&E staining (HE). The Ethics Committee of Animal Experiments of Sichuan Provincial People’s Hospital approved all animal experiments.

H&E staining

Collected tissues were fixed and preserved by 4% paraformaldehyde at 4℃ for 72 h [31]. Then the tissue samples were sequentially fixed, dehydrated, embedded, and sectioned. Then, the sections were stained with the H&E staining kit (BL700A, Biosharp, Anhui, China) according to the manufacturer’s instructions.

RNA‐sequencing analysis

Trizol reagent (R411-01, Vazyme, Nanjing, China) was used to extract RNA in accordance with the protocol after treatment with DMSO and UC (15 µM) for 72 h. RNA was sequentially tested for quality, integrity, and quantity. Then the samples were submitted to sequencing (HaploX Biotech, Shenzhen, China). We screened the differential transcription factor genes among samples for Foldchange and Padj corresponding to the up- and downregulated transcription factor genes with the set threshold: | log2 (Foldchange) |> 1 and Padj < 0.05. The downregulated genes are in blue and upregulated genes in red.

Quantitative reverse transcription PCR (RT-qPCR)

The RNA extraction kit (RC112, Vazyme, Nanjing, China) and reverse transcription reagent (R302-01, Vazyme, Nanjing, China) were used to extract the RNA and reverse transcription of the aimed cDNA. The SYBR qPCR reagents (Q712, Vazyme, Nanjing, China) were used for processing qPCR experiment according to the manufacturer’s instructions. We used the 2−∆∆Ct method to calculate the relative expression levels of each gene. The primer sequences are presented in Table 1.

Table 1 Primer sequences for RT-qPCRLentiviral transduction assays

Lentiviral vectors with YBX1 and control sequences were designed by Geneppl (Geneppl technology Co. Nanjing, China) and transductions were conducted using the manufacturer’s protocol.

Western blot

Cells were lysed on ice for 15 min in RIPA cell lysis buffer (P70100, NCM, Suzhou, China) and phosSTOP phosphatase inhibitor (P002, NCM, Suzhou, China). The protein lysate was centrifuged and the upper clearance collected. Using the BCA Protein Assay Kit (PD101-250 T, Oriscience, Chengdu, China) to measure the concentration of the protein, the protein was separated on SDS–polyacrylamide gels and transferred to polyvinylidene difluoride membranes (10,600,023, Cytiva, Marlborough, MA, USA). After incubation overnight at 4 °C with the primary antibody and incubating with the secondary antibody for 1 h at room temperature, the protein bands were detected and visualized on Mini Chemiluminescent/Fluorescent Imaging and Analysis System (Sinsage, Beijing, China).

Statistical analysis

GraphPad Prism 9 was used to process the data. The Student’s t test was used for the two groups’ comparison. ANOVA was used for multiple comparisons between more than two groups. All experiments were presented three times independently, and the results were considered significant at p < 0.05 (*p < 0.05, **p < 0.01, ***p < 0.001).