Figure 3

Effects of stable silence of Tβ10 on cell migration and monolayer wound healing in KKU-M214 cell line. Stable cell lines expressing small hairpin Tβ10 RNA (sh-Tβ10) or control vector (sh-vector) were generated in KKU-M214. (A) All clones of control and Tβ10 silencing cells were confirmed by real time RT-PCR. β-actin was used as an internal control. Arrows indicate selected clone for use in subsequent experiments; and insert pictures show immunocytochemistry results of selected clones. (B) Stable Tβ10 silence led to enhanced KKU-M214 cell migration in vitro in 24 and 48 h incubation by the modified Boyden chamber assay. Bar graphs represent fold change. n = 3; *P<0.05 versus the control. (C) Wound healing assay was carried out in M214-sh-vector and M214-sh-Tβ10 cells using DMEM medium supplemented with 10% FBS. Representative images were taken from the same field at 0, 24, 30 and 36 h. (D) M214-sh-vector-GFP and M214-sh-Tβ10-GFP cells were established, showing GFP signals. (E) The expression levels of Tβ10 in stable silence cells (M214-sh-Tβ10-GFP cells and M214-sh-vector-GFP cells) were determined by real time RT-PCR. n = 3, *P<0.05 versus the control. (F) Migration assay was carried out with M214-sh-vector-GFP and M214-sh-Tβ10-GFP cells at 48 h incubation by the modified Boyden chamber assay. n = 3, *P<0.05 versus the control. (G) Wound healing assay for M214-sh-vector-GFP and M214-sh-Tβ10-GFP cells. Representative images were taken from the same field at 0, 20, and 24 h. (H) M214-sh-vector-GFP and M214-sh-Tβ10-GFP cells were pre-treated with a Ras-GTPase inhibitor (FPT inhibitor III) for 2 h. Wound healing assay was performed. Representative images were taken from the same field at 0, 20, and 24 h.