2.1. Cell lines and cultures
All cell lines (HGC-27, MKN-45, SGC-7901, BGC-823, GES-1 and 293FT) were derived from American Type Culture Collection (ATCC, Beijing, China) and were cultured as described . All cell lines tested negative for mycoplasma.
2.2. Protein extraction, western blotting, and co-immunoprecipitation (co-IP) assays
RIPA buffer, supplemented with protease and phosphatase inhibitors, was utilized in cell lysis. Then sediments were removed by centrifugation at 12,000 rpm and 4℃ for 10 min, and the supernatants were separated for western blot experiments. Nuclear and Cytoplasmic Protein Extraction Kit (Beyotime, Shanghai, China) was utilized to analyze protein distribution. 30 µg of protein were used in equal amounts for western blot experiments. For co-IP assays, cell lysates with specific antibodies were incubated on a turn table at 4°C overnight. Then, Protein A/G Agarose (Santa Cruz, Dallas, Texas, USA) was added for antibody attachment and the immunoprecipitants were boiled for SDS-PAGE. Details about the antibodies used in this study are described in Supplementary Table 1.
2.3. immunohistochemical (IHC) staining
Embedded tumor tissue sections in paraffin were probed with MESP2 primary antibody (1:200, #bs-18796R, BIOSS, Beijing, China) or Ki67 primary antibody (1:200, #27309-1-AP, Proteintech, Wuhan, China), then tested as described [17, 18].
2.4. RNA extraction and qRT-PCR
Total RNA was extracted using TRIzoL as previously described . NanoDrop ND-2000 instrument (Yeasen, Shanghai, China) was used for RNA concentration measurement. GoScript™ Reverse Transcription System Kit (Promega, Beijing, China) was used for complementary DNA synthesis. qRT-PCR was used to examine cell mRNA levels. Details about the primers used in qRT-PCR are described in Supplementary Table 2.
2.5. shRNAs and Plasmids or lentiviruses for transfection or infection
shMESP2, shSKP2 and shGFP were purchased from Sangon and cloned into the pLKO.1 vector. Sequences of all the shRNAs and plasmids used are provided in Supplementary Table 3. Lentiviruses were used for transfection or infection as described . The 293-FT cell lines were co-transfected with the packaging plasmids pLP1, pLP2, and pLP/VSVG (Invitrogen) and the corresponding shRNA plasmids or overexpressing plasmids to produce the lentivirus. Lipofectamine 2,000 (Invitrogen) was used for all transfections according to the manufacturer’s instructions. After 48 h transfection, the virus supernatant was collected to infect HGC-27 and MKN-45 cells, with a final concentration of 4 µg/mL polybrene. 2 mg/mL puromycin was used to select stable knockdown cells.
2.6. Cell proliferation, migration, and invasion assays
As previously mentioned, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenylte-trazolium bromide (MTT) assays and bromodeoxyuridine (BrdU) staining were used to assess cell proliferation .
For cell cycle assay, cells were harvested and washed by 1×PBS for three times, and then samples were fixed in ice-cold 75% ethanol at 4°C for 24 h. After being washed and re-suspended in 1×PBS. Cells were incubated with PI and RNase A (Sigma Aldrich, USA) at 37°C for 45 min. The samples were analyzed using FACS C6 (BD, USA) with the software Flow jo (Three Star, Ashland, OR, USA).
Migration, invasion, and wound healing assays were used for assessing the migratory and invasive behaviors of cells as described .
2.7. Animal studies
All animal experiments were permitted by the Animal Care and Use Committee of Southwest University and carried out in accordance with the Animal Care and Use Guidelines (Ministry of Science and Technology, Beijing, China). Five-week-old female nude mice were purchased and were raised in SPF room for a week to adapt to the new environment.
Subcutaneous xenotransplantation. Human GC lines (HGC-27) cells (1 × 105 cells) were stably transfected with shGFP or shMESP2, grown in 100 µl medium (mixed with Matrigel at a 1:1 ratio), and injected hypodermically into the mice using a 1 mL Hamilton microliter syringe. After euthanizing the mice, the tumors were removed, photographed, and weighed.
In vivo metastasis assay. Five million HGC-27 cells that stably expressing shGFP or shMESP2 were injected into the lateral tail vein of 5-week-old female NOD/SCID mice. Six female NOD/SCID mice were used in each group. All mice were raised 25 days in SPF room and finally killed by cervical dislocation. All lung samples were collected.
2.8. Ubiquitination assay
The 293FT cells were co-transfected with relevant plasmid for 48 h. Then cells were dealt with MG132 (50 µg/mL) for 6 h. Western blot and co-IP assays were performed after cell lysis.
2.9. Expression microarray profiling
Total RNAs were extracted from shGFP- and shMESP2-transfected HGC-27 cells using TRIzoL reagent. All samples were analyzed by Sangon (Shanghai, China) according to mRNA expression microarray protocols.
2.10. GSEA and GO analysis
The gene expression datasets (GSE15459 and GSE34942) analyzed in this study were obtained from the GEO database (https://www.ncbi.nlm.nih.gov/geo/). GSE15459 and GSE34942 was based on platform GPL570 ([HG-U133_Plus_2] Affymetrix Human Genome U133 Plus 2.0 Array). All of the data were freely available online, and this study did not involve any experiment on humans or animals performed by any of the authors.
2.11. GSEA and GO analysis
GSEA analysis was performed by folding change data from differential expression analysis into GSEA software (Broad Institute). GO analysis was performed with marker genes from each subcluster by tools from DAVID.
2.12. Chromatin immunoprecipitation
GC cells were cultured in 10 cm culture dish followed by 1% formaldehyde treatment for 10 min. Cells were pelletized and a supersonic device was used for shearing DNA into fragments between 200 and 500 bps. After chromatin sonication and centrifugation, 2 µg of Flag antibody (#8146, CST, Shanghai, China) or normal IgG were added overnight. The precipitated DNA was analyzed via qRT-PCR and the primers used are listed in Supplementary Table 4.
2.13. Luciferase reporter assay
The SKP2 promoter and TCF-reporter plasmid (TOP Flash and FOP Flash) were subcloned into pGL-basical vector (Promega), and 293FT cells were co-transfected with luciferase reporter and pGL-TK reporter for 48 h. Then, luciferase activity was detected after cell lysis.
2.14. Statistical analysis
All experiments were carried out with three technical and biological replicates. All the results acquired in this study are presented as the means ± standard deviation (SD). Differences between means were determined via unpaired Student’s t-tests, and p < 0.05 was considered statistically significant.