Knockdown and overexpression of FSTL1 caused altered cell cycle. increased cell apoptosis. Moreover, the changed migration and invasion ability in FSTL1 sufficient or deficient cells may be caused by alterations in MMP2, MMP3 and MMP9 expression. Altogether, our results revealed the crucial tumor-suppression function of FSTL1 in NSCLC progression, suggesting that FSTL1 might be an important factor in NSCLC progression. migration ability of NSCLC cells was assessed by scrape assay. Cells were seeded in 6-well plates and the monolayer was scratched with 10-l pipette tips. The wound areas were photographed 0 and 20 h after scratching and measured using a caliper. The wound-closure percentages were calculated using the following formula: [1-(current wound size/initial wound size)] 100. Cell invasion assay Cells were detached and re-suspended in a serum-free medium and seeded around the upper chamber of Matrigel-coated Transwell inserts with a pore size of 8 m. The culture medium made up of 10% FBS as a chemo-attractant was added to the lower chamber. After 24-h incubation, the cells around the upper surface of the insert were gently removed with a cotton swab. Invading cells (lower surface of the insert) were fixed with 4% paraformaldehyde (Sigma-Aldrich), stained with crystal violet, and counted under a microscope. Five random microscopic fields were examined for each insert. Flow cytometry analysis Cells were seeded into 6-well plates PROTAC ERRα ligand 2 at a density of 1106 cells/well for 24 h. Subsequently, the cells were collected and stained with the ANXA5 (Annexin V)-PE apoptosis detection kit (4A Biotech Co. Ltd., FXP018-100) according to the manufacturer’s instructions and analyzed by flow cytometry (FACSCalibur, BD Bioscience, San Jose, CA, USA). Statistical analyses Unless stated otherwise, data are presented as mean SD in the figures. A Student’s t-test was performed to compare the two groups of data. For more than two groups, we analyzed HMOX1 with one-way ANOVA followed by Tukey’s multiple comparison test. All statistical assessments were two-sided. Results FSTL1 is usually downregulated in NSCLC cells In order to explore the function of FSTL1 in NSCLC, we collected an array of lung cancer cells and lung normal epithelial cell line, BEAS-2B. Expression of FSTL1 was examined by qRT-PCR and western blot analysis. As shown in Fig. 1A, the mRNA levels of FSTL1 in NSCLC cells were PROTAC ERRα ligand 2 much lower than normal BEAS-2B cells. Consistently, the protein level of FSTL1 in BEAS-2B was higher than NSCLC cells (Fig. 1B). These results suggest that FSTL1 is usually downregulated in NSCLC cells. Open in a separate window Physique 1. Expression of FSTL1 in lung cancer cells and lung normal epithelial cell line. qRT-PCR (A) and western blot analysis (B) of FSTL1 mRNA expression level in human lung normal epithelial cell line and NSCLC cell lines. Overexpression of FSTL1 in H446 cell line. FSTL1 expression was analysis with qRT-PCR (C) and western blot analysis (D). Knockdown of FSTL1 in A549 cell line with 5 different shRNA sequences. FSTL1 expression was analyzed with qRT-PCR (E) and western blot analysis (F). Student’s t-test; N=3; error bars, SEM. ***P<0.001. We then constructed FSTL1 overexpression in H446 cells. Both RT-PCR and western blot analysis revealed the successful establishment of FSTL1 overexpression (Fig. 1C and D). Then FSTL1 expression was knocked down in A549 cells. The results of qRT-PCR and western blot analysis shown, FSTL1 was effectively suppressed by SH1 and SH4 (Fig. 1E and F). FSTL1 reduced NSCLC cell proliferation with altered cell cycle To analyze the function of FSTL1 in NSCLC cells, we PROTAC ERRα ligand 2 examined the cell proliferation ability using CCK8. The results showed that A549 cells with FSTL1 knockdown proliferated faster than control cells (Fig. 2A). On the contrary, H446 cells with FSTL1 overexpression proliferated slower than control cells (Fig. 2B). In order to further clarify the function of FSTL1 in NSCLC cells, we.