Fig. 3.

Downregulation of Par3 suppressed invasion and proliferation of JHOC5 cells. a Effect of siRNA against Par3. JHOC5 cells were transfected with Stealth RNAi against PAR3 (A: HSS125534, B: HSS183488, C: HSS183489) or control siRNA for 48 h. Total RNA was reverse transcribed and PARD3 mRNA levels (Par3) were measured by a quantitative reverse transcription polymerase chain reaction. Expression was normalized to the expression of glyceraldehyde 3-phosphate dehydrogenase (GAPDH). Data are the mean (±SEM) of three independent experiments (A-1). siPar3-B was chosen for further analysis. Cells were transfected with siControl or siPar3, then 48 h after transfection, Par3 and α-Tubulin expression was analyzed by western blotting. The experiments were repeated at least 3 times (A-2). b Invasion assay. JHOC5 cells were transfected with the Par3 siRNA (siPar3) or control siRNA (siControl). Transfected cells were seeded in a Matrigel-coated Boyden chamber 48 h after transfection, and were allowed to invade for 24 h. Matrigel membranes were observed with an optical microscope. Scale bar indicates 100 μm (B-1). Numbers of cells invaded through matrigels were counted. Data are the mean (±SEM) of five different microscopic fields. The data is the representative of three independent experiments (B-2). c) Wound healing assay. JHOC5 cells were transfected with the Par3 siRNA (siPar3) or control siRNA (siControl), seeded onto 6-well culture plates, and grown as a monolayer for 48–60 h until 100% confluent. A scratch assay was then performed. Images of the same area of the wound were taken after 8 h to measure the width of the wound using fluorescence microscope. The data is the representative of three independent experiments. d Cell proliferation assay. To analyze the effect of Par3 knockdown on cell proliferation, 5000 cells were seeded onto 96-well plates 48 h after siRNA transfection. Cell Counting Kit-8 (Sigma Aldrich) was used to examine proliferation at 24, 48 and 72 h. Data are the mean (±SEM) of three wells. The data shown is representative of three independent experiments