Cell culture

Human gastric cancer cell lines, NCI-N87, HGC27, and AGS, were purchased from ATCC and were cultured in Dulbecco’s modified Eagle’s medium (DMEM) (Gibco) supplemented with 10% fetal bovine serum (FBS) (EallBio, # 03.U16001). All cells were maintained at 37 °C in a saturated humidity atmosphere containing 95% air and 5% CO2

Immunoblotting

Immunoblotting analyses involved lysing cells or tissue samples in RIPA lysis buffer (Invitrogen, #89900). The protein concentration of each sample was assessed using the bicinchoninic acid (BCA) kit (Yeasen, #20201ES86) according to the manufacturer’s instructions. Equal amounts of protein extracts were separated by electrophoresis on appropriate Tris-Glycine gels (Yeasen, #36252ES10) and subsequently transferred to polyvinylidene difluoride (PVDF) membranes (Millipore, #IPVH00010). The membrane was probed with primary antibodies listed below, followed by secondary antibodies conjugated to horse radish peroxidase (HRP). The immunoblot bands were analyzed using Image Lab software.

Cell counting kit-8 (CCK-8) assay

Cells were dispensed into 96-well plates after indicated treatments and incubated with the medium containing 10% Cell Counting Kit-8 reagent (ApexBio, #K1018) for 2 h at 37 °C. The OD450 nm value was measured using a microplate reader.

Pro-senescence drug screening

Cells were seeded onto 96-well plates at a density of 3000 cells per well followed by sequentially treating with individual inhibitors from the Epigenetics Compound Library (MCE, #HY-L005, 10 nM) and dimethyl sulfoxide (DMSO) or SSK1 (MCE, #HY-138936, 1 μM) for 96 h. Cell viability was determined using a CCK-8 assay, and the data were visualized by GraphPad Prism 8.0.

Senescence-associated β-galactosidase (SA-β-gal) analysis

Senescence-associated expression of β-gal activity was determined by a Senescence Detection kit (Solarbio, #G1580-100T) according to the manufacturer’s instructions. Briefly, cells were seeded onto 6-well plates at a density of 1 × 105 and treated with DMSO or gradient doses of QC6352 (20 nM) for 96 h. Subsequently, cells were fixed by adding 1 mL β-Gal Fixative buffer at room temperature for 15 min followed by staining with Dye Working Solution at 37 °C overnight.

Immunofluorescence

Cells were seeded on a 6-well plate with coverslips, followed by treatment with QC6352 (20 nM) for 96 h. The culture medium was removed, and coverslips were carefully washed three times with phosphate-buffered saline (PBS). The cells were fixed by incubating in 4% paraformaldehyde for 5 min at room temperature, washing twice with PBS and twice with washing buffer. Cells were then permeabilized with 0.5% Triton X-100 for 5 min, blocked in PBS containing 1% bovine serum albumin (BSA), and subsequently incubated with primary antibodies against H3K9me3 (CST, #9649S) at 4 °C overnight. Cells were washed three times with PBS and then incubated with Alexa Fluor 488 goat anti-rabbit (Life Technologies) at room temperature for 1 h in the dark. The nuclei were extensively washed with ice-cold PBS and then counterstained with 4’,6-diamidino-2-phenylindole (DAPI) for 10 min. Specimens were mounted in 70% glycerol and sealed with nail polish. Fluorescent images were taken using an Olympus fluorescence microscope.

Cell cycle analysis

Cells treated with QC6352 were collected and washed with PBS, fixed with 70% ethanol, and resuspended in PBS containing propidium iodide and RNase A (200 µg/mL). Cell cycle analyses were performed using a NovoCyte flow cytometer and FlowJo software v10 (Treestar, Ashland, USA).

Enzyme linked immunosorbent assay (ELISA)

Cell-cultured medium was centrifuged, and the supernatant was subjected to ELISA detection according to the manufacturer’s instructions of the Human IL-6 ELISA Kit (BOSTER, #EK0410) and IL-8 Elisa Kit (BOSTER, #EK0413).

For tumor tissues, 0.1 g of tissue blocks were first transferred into a glass homogenizer containing 1 mL of pre-cooled PBS and 10 μL 100mM phenylmethylsulfonyl fluoride (PMSF), followed by thorough grinding and ultrasonication on ice. The prepared homogenate was centrifuged at 5000 × g for 5 min, and the supernatant was collected for ELISA assays.

7-amino-actinomycin D (7-AAD) staining

Gastric cancer cells (2 × 105 cells/well) were seeded in 6-well plates, treated with DMSO or QC6352 (20 nM), followed by DMSO or SSK (1 μM) treatment. Subsequently, cells were collected and washed with PBS. After centrifugation, the cell pellets were resuspended and then incubated with 50 μL 1 × Assay Buffer containing 5 μL 7-AAD Solution at room temperature for 15 min, followed by mixing with 450 μL 1×Assay Buffer working solution. After that, the death cell rates (positive 7-AAD cells) were assessed by NovoCyte flow cytometer.

Animal studies

Male nude mice (BALB/C, 15–20 g, 4–6 weeks old) were obtained from SPF (Beijing) Biotechnology and maintained under pathogen-free conditions. A total of 3 × 106 NCI-N87 cells resuspended in a 1:1 solution of PBS and Matrigel Matrix (Corning) were subcutaneously injected into the dorsal flanks of mice.

The mice were numbered and randomized into vehicle, 10 mg/kg QC6352, and / or 1 mg/kg SSK1 treatment groups when tumor volume reached approximately 13.5 mm3. Here, QC6352 and SSK1 were resolved and suspended in 10% DMSO (MCE, #HY-Y0320) and 90% corn oil (MCE, #HY-Y1888).

Mice weight and tumor volume was recorded daily by caliper measurements using the following formula: π (width × length) / 6 (mm3). Mice were sacrificed when the tumor size in the vehicle-treated group exceeded 1000 mm3 (21 days after treatment). The tumors were dissected, weighed, and photographed.

Bioinformatic analysis

RNA-sequencing expression (level 3) profiles and corresponding clinical information for gastric cancer were downloaded from The Cancer Genome Atlas (TCGA) dataset (https://portal.gdc.com). The two-gene correlation map is realized by R software package ggstatsplot, and the multi-gene correlation pheatmap is displayed by R software package. Spearman’s correlation analysis was used to describe the correlation between quantitative variables without a normal distribution. P values less than 0.05 were considered statistically significant.

The ATF6, SP1, and KDM4C expression levels in normal and gastric cancer tissues were determined by the online tool GEPIA2 (http://gepia2.cancer-pku.cn/).

Kaplan–Meier survival curves were generated using the Kaplan–Meier Plotter website for gastric cancer (Version 2020, http://kmplot.com) and statistical significance was determined by the log-rank test.

The correlation between candidate genes and age was obtained from UALCAN (http://ualcan.path.uab.edu)

Colony formation assay

Cells were cultured in 6-well plates at a density of 3000 cells per well, followed by treatment with the indicated compounds for 14 days and staining with crystal violet (Beyotime, #C0121). The colonies were analyzed by ImageJ software.

Transwell assay

In vitro migration assays used an 8 µm pore size Boyden chamber (Corning Costar). Cells (200 µL, 1 × 105) in serum-free DMEM were plated in the upper chamber, and 500 µL 10% FBS was added to DMEM in the lower chamber as a chemoattractant. After 12 h, cells on the upper side of the filter were removed and cells that remained adherent to the underside of membranes were fixed in methanol, followed by staining with 0.1% crystal violet. The number of migrated cells was counted using a microscope. Five contiguous fields of each sample were examined using a 20 × objective to obtain a representative number of cells that migrated across the membrane.

Study approval

Prior to obtaining patient samples, requisite approval from the Medical Ethics Committee of the affiliated hospital of Jiangnan University and written informed consent from patients were obtained. The mouse experiments were approved by Institutional Animal Care and Use Committee of the affiliated hospital of Jiangnan University.

Statistical analyses

All statistical analyses were performed using GraphPad Prism 8.0 (GraphPad Software, USA). Differences between two groups were analyzed using an unpaired Student’s t test, while differences among multiple groups were analyzed by one-way analysis of variance (ANOVA). Results were considered statistically significant when p < 0.05.