br H cells transfected with nM
H1975 cells transfected with 220 nM AAMP siRNAs for 24 h were reseeded in 96-well plates and then treated with icotinib [20 μM] for 0, 24, 48 and 72 h. Cells were fixed suing 10% trichloroacetic LY3009120 (TCA) for 2 h at 4 °C and then washed with sterile water 3 times. Samples were stained with SRB (sulforhodamine B) for 10 min followed by rinse with 1% acetic acid. Absorbance at 450 nm was detected after SRB was dissolved by Tris-base (10 mM, pH = 10). Cell survival rate is positively with 450nM absorbance.
Cells were transfected with 220 nM AAMP siRNAs or 1 μg AAMP plasmid for 24 h and reseeded in 6 cm dishes at a density of 125 cells/ ml. During culture process, culture medium was refreshed every 3 days. Cells were fixed with 10% trichloroacetic acid (TCA) for 2 h at 4 °C followed washing with sterile water 3 times. Cell number was calcu-lated after SRB staining without dissolve following above.
2.10. Soft agar clone formation assay
The soft agar clone formation assay was performed using 6-well plates. The bottom layer contained 1.5 mL of 0.7% agar in complete medium and the top layer included 1.5 mL of 0.35% agar in complete
medium containing 3000 cells. 200 μL fresh medium with 10% (v/v) fetal bovine serum (FBS, Gibco) was added into each hole every 3 days. Cells were cultured for 2–3 weeks. After culturing, the colonies were stained with nitroblue tetrazolium chloride solution (200 μL/well), and the number of clones were counted.
H1792 and Calu-1 cells were transfected with 220 nM AAMP siRNAs in 6-well plates for 24 h. The cells were suspended and seeded in 96-well plates at 6000 cells per 100 μl, and then cultured for 18 h. Cell proliferation was assessed using an EdU Cell Proliferation ELISA kit (Beyotime Biotechnology, Shanghai, China).
Female B-NSG 5-week-old mice were purchased from Biocytol (Beijing, China). These mice were divided into 3 groups: H1792/pGIPZ-luc, H1792/pGIPZ-shAAMP-1 and H1792 /pGIPZ-shAAMP-2. A total of 2 × 106 cells suspended in 100 μl of PBS were injected subcutaneously. The tumor volume was calculated according to the formula V = π × (length × width2)/6. All the animal experiments were carried out in accordance with the approval of the Animal Research Committee of Shandong University.
2.13. Apoptosis evaluation by flow cytometry
Cells were incubated with annexinV-FITC and PI using an Apoptosis Detection Kit (EnoGene, #BA11100) according to instructions. Flow cytometry was utilized to separate apoptosis cells from normal cells.
2.14. Wound-healing scratch assay
Cells were seeded in 6-well plate respectively and cultured over-night. Scratches were made using a p-200 pipette tip when cells were grown to monolayer confluency. Suspended cells were wiped oﬀ using PBS washing for 3 times and adherent cells were cultured with optimal medium supplemented with 1% FBS for 48 h–60 h. Images were cap-tured every 12 h. The migration rate was calculated according to images.
2.15. Statistical analyses
Image-Pro Plus was used for wound-healing scratch assay. GraphPad Prism 5.0 and Image J were used for statistical analyses. Values are expressed as the mean ± standard deviation. Student's t-test was used for analysis, and P < .05 was considered significant.
3.1. AAMP promoted cell proliferation and clonogenic ability in NSCLC in vitro
Although many reports have studied AAMP in some cancer cells, expression level of AAMP in NSCLC cells has gone undefined. Thus, we measured expression levels of AAMP in several non-small lung cancer cell lines by western blot (Fig. 1A). Results show AAMP was highly expressed in H1792, H157, H1299 and Calu-1; expression was sig-nificantly less in H460 and A549 cell lines. Therefore, H1792 and Calu-
1 cell lines were chosen for suppression analysis; H460 and A549 cell lines were selected for overexpression experiments.
We generated two siRNAs, AAMP siRNA #1 and #2, to investigate the function of AAMP in NSCLC cells. Data show cell number was sig-nificantly reduced compared with control after AAMP siRNA transfec-tion for 48 h in H460 and Calu-1 cell lines (Fig. S1A). In addition, cell number was significantly less after AAMP knockdown in both Calu-1 and H1792 cells (Fig. 1B and Fig. S1B). In contrast, cellular prolifera-tion rate increased after overexpression of AAMP (Fig. 1C). Con-sistently, EdU assay results show cell proliferation was significantly less after AAMP knockdown (Fig. 1D and S1C) and increased when AAMP was overexpressed (Fig. S1D).