Figure 3.

STAT3 inhibition suppresses human diffuse intrinsic pontine glioma cell migration and invasion by regulating EMT. SF8628 cells were treated with vehicle control (DMSO) or AG490. SF8628 cells were transfected with shCtrl or shSTAT3. (A) Western blot analysis of EMT markers. Protein samples were extracted from SF8628 control cells and STAT3-inhibited cells. Protein samples were tested for E-cadherin, N-cadherin, Vimentin, Twist, Snail, MMP-9 and β-actin (loading control) expression. Exposure time was 5 min for E-cadherin, 1 min for N-cadherin, Twist and Snail, 10 sec for Vimentin, 3 min for MMP-9 and 15 sec for β-actin. (B) Organization of the actin cytoskeleton was determined by immunofluorescence staining. Alexa Fluor 633-conjugated phalloidin was used to visualize F-actin (red), and DAPI staining (blue) was used for visualization of cell nuclei. Original magnification, ×400. Scale bar, 2 µm. (C) Cells were seeded in the upper chamber of Transwell inserts, and the cell migration ability was assessed 48 h after cell plating. Magnification, ×40. Scale bar, 100 µm. (D) Cells were seeded in the upper chamber of Transwell inserts coated with Matrigel, and the cell invasion ability was measured 48 h after cell plating. Magnification, ×40. Scale bar, 100 µm. The results were calculated as percentages relative to control cells. Representative images of invasive cells are shown next to each bar graph. Data are presented as the mean ± SD. *P<0.05 using a two-tailed unpaired Student's t-test. STAT3, signal transducer and activator of transcription 3; shCtrl, control short hairpin RNA; shSTAT3, STAT3 short hairpin RNA; EMT, epithelial-mesenchymal transition; MMP-9, matrix metallopeptidase-9; DAPI, 4′6′-diamidio-2-phenoylindole.