Chemicals and reagents
The simpleChIP® plus enzymatic chromatin IP Kit was from Cell Signal Technology (Danvers, MA, USA). All-in-One First-Strand cDNA Synthesis Kit and All-in-One qPCR Mix were from GeneCopoeia (Rockville, MD, USA). Antibodies used for immunoblotting and IP assays were as follows: the NF-KB p65 primary antibody for chromatin IP was purchased from CST; primary antibodies against VEGF were from Abclonal (Cat: A17877, Wuhan, China);
Cell lines, cell cultures and transfection
The gastric cancer cell lines of poor differentiation, BGC823 and AGS-1, human umbilical vein endothelial cells (HUVECs) were obtained from Cell Bank of the Chinese Academy of Sciences (Chinese Academy of Sciences, Shanghai, China) and cultured in Dulbecco’s Modified Eagle’s Medium (DMEM, Gibco) supplemented with 10% (v/v) fetal bovine serum (FBS) at 37 ̊C in a humidified atmosphere of 5% CO2. The plasmids were transfected into cells with Hilymax according to manufacturer’s protocol.
Generation of stable cell line
The lentivirus control shRNA (shCTL: sc-108080) and REC8 shRNA (shREC8: sc-106878-V) were purchased from Santacruz Biotechnology, INC. Puromycin was purchased from sigma and used to select for stably infected cells.
RNA extraction and quantitative real-time PCR
Total RNA was extracted from cultured cells with trizol reagent (Invitrogen, Carlsbad, CA, USA) according to the manufacturer’s protocol. All cDNA samples were prepared using an All-in-one First-stand cDNA synthesis kit (GeneCopoeia, MD, USA). Quantitative real-time PCR (RT-qPCR) analyses were performed with an all-in-one qPCR mix (GeneCopoeia) according to manufacturer’s instructions using an ABI StepOne-Plus™ qPCR system. The primer for VEGF: Forward: 5′-TGCAGATTATGCGGATCAAACC-3′; Reverse: 5′-TGCATTCACATTTGTTGTGCTGTAG-3′; REC8: Forward: 5′-CATCCCACCAGAAGAACGG-3′; Reverse: 5′-GCACCAAAGGCATCTCCAT-3′; β-actin: Forward 5′-ATCGTGCGTGACATTAAGGAGAAG-3′; reverse:5′-AGGAAGGAAGGCTGGAAGAGTG-3’.
Endothelial cell tube formation assay
As described in Xu et al. study , 96-well plates were coated with matrigel basement membrane matrix (BD Biosciences) and then allowed to polymerize at 37 °C for at least 30 min. HUVECs were treated with conditional medium for 6–8 h, tubes formation of HUVECs can be visualized and the number of nodes (defined as when at least three cells formed a single point) per image was quantified as described .
As described in the study , cell lysates were lysed with 2 × loading sample buffer and analyzed by immunoblotting to detect proteins. Briefly, the protein was transferred to a 0.22 μm nitrocellulose transfer membrane. The membrane was blocked with 5% (w/v) milk in PBS/ 0.05% (v/v) Tween-20 and then incubated with the indicated antibody overnight. This was followed by incubation with a horseradish peroxidase secondary antibody (Jackson ImmunoResearch) for 1 h at room temperature. Proteins were detected using enhanced chemiluminescence substrates (Perkin Elmer). The antibodies listed as followed: REC8(abgent, AP13570c,1:2000 for WB; proteintech 11913-1-AP, 1:200 for IP), VEGF(abclonal, A12303,1:2000 for WB; MAB293, 1:50 for neutralize), NF-κB p65 (abclonal, A11202, 1:2000 for WB), Phospho-NF-κB p65(Ser536) (Ser536) (abclonal, AP0124, 1:2000 for WB). α-tubulin (abclonal, AC012); β-actin (abclonal, AC004).
Enzyme-linked immunosorbent assays (ELISA)
Quantitative measurement of VEGF secreted into conditional medium was determined using ELISA according to the manufacturer’s protocol (elabscience, E-EL-H0111c).
Immunohistochemistry (IHC) of human tissue microarrays
A human gastric cancer tissue microarray was purchased from Alenabio. Gastric cancer samples were immunostained against indicated antibodies as previously described . Briefly, the slides were dewaxed and rehydrated in distilled water, sections were immersed in citrate buffer(C6H5Na3O7·2H2O) and then microwaved for 20 min for antigen retrieval. Endogenous peroxidase activity was blocked with 0.5% (v/v) H2O2. The slides were then transferred into a humidified chamber, incubated with 5% (v/v) horse serum for 30 min and then incubated with primary antibodies overnight at 4 °C. After primary antibody CD31(Sangon Biothch, D260721,1:200 for IHC), REC8 (Proteintech 10793-1-AP, 1:200 for IHC) incubation, the slides were immersed in peroxidase-labeled secondary antibody for 30 min at room temperature. To detect the antibody-conjugated antigen reaction, the sections were incubated in 3-amino-9-ethylcarbazole substrate-chromogen for 30 min and counterstained with hematoxylin. The evaluation of the staining was performed as described in our pervious study . Quantitative analysis of the immunostained images of human biopsies was performed by positive cell number counting and computerized optical density (OD) measurements with Image Pro Plus 6.0 software (Media Cybernetics, MD, USA).
Chromatin immunoprecipitation (ChIP) assays
ChIP assays were performed in BGC823 cells by using a SimpleChIP® Plus Enzymatic Chromatin IP Kit as pervious work . Briefly, 1.0 × 107 cells were cross-linked with 1% (w/v) formaldehyde for 10 min and then quenched in 0.125 M glycine for 5 min. Cells were lysed and digested to collect the chromatin. IP was carried out by using the indicated antibody overnight. The precipitated DNAs were analyzed and quantified by using real-time PCR analysis. Primer sequences for VEGF (NM_001171622) listed as followed: forward 5′-CGTGTGGAAGGGCTGAGG-3′, reverse 5′-CCGCTACCAGCCGACTTTT-3′,
All statistical analyses and statistical graphing were done using GraphPad Prism 8 software. All the t test was used to determine the significance of differences in the qPCR assay. One-way ANOVA was performed on data from endothelial cell tube formation assay. For correlation analysis, p value of less than 0.05 was considered statistically significant.