Clinical samples

A total of 110 paired specimens, comprising GC tissues and matched non-cancerous adjacent tissues, were collected by the Department of General Surgery at the Affiliated Hospital of Nantong University, China, from 2010 to 2011. The clinical data of GC patients are detailed in Table 1. Follow-up was conducted until August 2015, with a median duration of 38.4 months (range: 1.5 to 66.4 months). Histological diagnosis of GC was independently verified by two pathologists. All patients were treatment-naive with respect to radiotherapy, chemotherapy, or immunotherapy prior to their surgical resection.

Table 1 Relationships between SCYL1 expression and clinicopathological characteristics of GC patients (*P < 0.05)Cell culture and in vitro functional experiments

Human gastric mucosal epithelial cells (GES-1) and a panel of GC cell lines (MKN-45, BGC-823, SGC-7901, AGS, HGC-27) were obtained from GeneChem (Shanghai, China). Cell cultivation was performed in RPMI-1640 medium enriched with 10% fetal bovine serum and antibiotics (100 U/mL of penicillin and streptomycin), procured from Clark (Shanghai, China) and Life Technologies (Shanghai, China), respectively. Wound healing assay and transwell assay were performed as previously described (Zang et al. 2022).

siRNA-mediated gene silencing

Specific siRNAs targeting SCYL1: si-SCYL1#1 (F:5′-GGCUACACCAGAUCGUGAATT-3′, R: 5′-UUCACGAUCUGGUGUAGCCTT-3′), si-SCYL1#2 (F:5′-CCGUGUCCAUCUUCGUCUATT-3′, R: 5′-UAGACGAAGAUGGACACGGTT-3′), si-SCYL1#3 (F:5′-GAGUAUCAGCAGAAGAUCATT-3′, R: 5′-UGAUCUUCUGCUGAUACUCTT-3′) and a non-targeting control were synthesized by Tsingke Biotech (Beijing, China). GC cells were transfected using jetPRIME (Suzhou, China), following the manufacturer's protocol (Liu et al. 2021).

Extraction of RNA and quantitative real-time PCR (qRT-PCR)

Total RNA was isolated from cells using standard protocols, and qRT-PCR was performed to quantify SCYL1: (F: 5′-TGACAGATGGGACGACGAAGA-3′, R: 5′-ATTTGGAGGATTTGTGGTCGG-3′) expression with specific primers, with GAPDH: (F: 5′-GGAAGCTTGTCATCAATGGAAATC-3′, R: 5′-TGATGACCCTTTTGGCTCCC-3′) serving as an internal control.

Western blot

Proteins were extracted from cell lines and subjected to Western blot analysis using antibodies specific to GAPDH (60,004-1-Ig), ULK1 (29,005-1-AP), p-ULK1 (80,218-1-RR), mTOR 66888-1-Ig) and p-mTOR (67,778-1-Ig) were purchased from Proteintech (Wuhan, China); SCYL1(A6735) from Abconal (Wuhan, China).

Autophagic flux determination

Autophagic flux was evaluated in SGC7901 cells transduced with GFP-mRFP-LC3 lentivirus (GeneChem, Shanghai, China). The specific operation is the same as before (Zang et al. 2022). Cells were treated with autophagy inhibitor 3-MA, fixed, and imaged to quantify autophagosome and autolysosome formation using fluorescence microscopy and the autophagosomes (yellow spots) and autolysomes (red spots) were counted using Image J software.

In vivo peritoneal metastasis assay in nude mice

Male athymic nude mice, four weeks old, were procured from the Animal Center of the Medical College of Nantong University and kept under controlled temperature and humidity conditions. All animal experiments were approved by the Animal Ethics Committee of Nantong University and conducted in accordance with the ARRIVE guidelines, the U.K. Animals (Scientific Procedures) Act, 1986, EU Directive 2010/63/EU for animal experiments, and the National Institutes of Health guide for the care and use of laboratory animals. Nude mice were utilized for the establishment of a peritoneal dissemination model, with injections of either siRNA-treated or control SGC7901 GC cells. Following a six-week period, mice were sacrificed, and metastatic nodules were quantified and subjected to immunohistochemistry (IHC) evaluation.

Tissue microarray (TMA) construction and IHC analysis

Tissue microarrays were assembled using a precision arraying instrument (Dako, Carpinteria, CA). The specific experimental procedure is as described earlier (Zang et al. 2021). All primary antibodies for IHC were shown below: SCYL1 (1:100), p-mTOR (1:200), LC3B (1:200). The IHC results were evaluated by two independent pathologists. Based on the expression level of SCYL1 in GC tissues compared to adjacent non-cancerous tissues, the samples were categorized into two distinct groups: those with high expression and those with low expression. The related scoring criteria refer to previous reports (Chen et al. 2022).

Microarray data acquisition and bioinformatics

The dependency of GC on SCYL family genes was interrogated using the DepMap portal (https://depmap.org/portal/), with expression analysis conducted on datasets from TCGA and GEO (GSE79973, GSE66229, GSE30727, GSE27342, GSE19826, GSE179252 and GSE13911). Survival analysis was performed using the Kaplan–Meier Plotter (www.kmplot.com), and GSEA (Gene Set Enrichment Analysis) generated angenerate an ordered list of all related genes based on their correlation with SCYL1 expression (Subramanian et al. 2005; Hu et al. 2024). The “c2.cp.all.v2022.1.Hs.symbols.gmt” and “h.all.v7.5.1.symbols.gmt” were selected as reference gene sets. The data for single-cell sequencing is from the GEO database GSE163558, and cell annotation was performed using the "Seurat" R package (Jiang et al. 2022; Hao et al. 2021).

Statistical analysis

Quantitative data were presented as mean ± SD. Statistical analysis was performed using SPSS version 22, with survival outcomes analyzed via Kaplan–Meier and Cox regression methods (Liu et al. 2023). The significance of differences between experimental conditions was determined using the t-test, with a p-value threshold of < 0.05 for significance.