Real-time PCR was performed using the SYBR PrimeScript RT-PCR Kit II (Takara)

Real-time PCR was performed using the SYBR PrimeScript RT-PCR Kit II (Takara). reduced intracellular Ca2+ release via IP3Rs, altered cell morphology and significantly inhibited the migration of A549 cells. These phenomena were primarily dependent on IP3R2 because wound healing in A549 cells with IP3R2 rather than IP3R1 or IP3R3 siRNA was markedly inhibited. Moreover, the overexpression of ERP44 did not affect the migration of the human neuroblastoma cell line SH-SY5Y, which mainly expresses IP3R1. Based on the above observations, we conclude that ERP44 regulates A549 cell migration mainly via an IP3R2-dependent pathway. (Fig. ?(Fig.4D).4D). The physical centre of gravity in ERP44 overexpressed A549 cells was nearly maintained at its original location during the 1.5 h tracking time. Open in a separate window Figure 4 ERP44 inhibits cell migration by reducing intracellular Ca2+ release(A) Identification of ERP44 overexpression (ERP44-OE) system in A549 cells via western blot and immunofluorescence. Overexpressed ERP44 were co-located with ER marker Bip. (B) ERP44 overexpression inhibited 10 M ATP-induced calcium release via IP3Rs. (C) Wound healing was significantly inhibited by overexpressed ERP44. (D) Overexpression of ERP44 inhibited A549 cells random motility. A549 cells were recorded in real time after adenovirus infection. Circled cells are DsRed-positive cells. The right panel shows the movement tracking of A549 cells. As we noted above, 2-APB inhibited Ca2+ release and resulted in an inhibitory effect on A549 cell migration by affecting the cell cytoskeleton. Thus, we examined whether ERP44, similar to 2-APB, also inhibited cell migration by affecting the cell cytoskeleton. In the control, A549 cells stained with Phalloidin-FITC exhibited a clear structure consisting of F-actin microfilaments (Supplementary Fig. 2) and polarized cells presented a network arrangement of microfilaments at the CUDC-427 forefront of the cells. In addition, stress fibres were observed throughout the cells. However, the microfilaments were not clearly observed or only some circular microfilaments were observed around the edge of the cells in ERP44 overexpressed A549 cells, suggesting that ERP44, similar to 2-APB, inhibited A549 cell migration by affecting the cell cytoskeleton. ERP44 inhibition of A549 cell migration is mainly dependent on IP3R2 It has been reported that ERP44 inhibits intracellular Ca2+ release by binding to IP3R1 [15]. We confirmed that all three types of IP3R were expressed in A549 cells (Fig. ?(Fig.5A).5A). However, the subtype of IP3Rs that mediates the inhibitory effect of ERP44 on A549 cell migration remains unknown. To clarify this, we performed RNA interference studies. We synthesized siRNAs for and according to a previously reported method [4] and the real-time PCR results indicated the interference efficiency of single siRNA to be 50% after transfection for 72 h (Fig. ?(Fig.5A).5A). Wound-healing studies demonstrated that all types of IP3Rs exhibited a inhibition of wound healing of A549 cells compared to the control (Fig. 5B & E, p 0.001 vs. control). However, among these receptors, IP3R2 displayed a CUDC-427 remarkable inhibitory effect on A549 cell wound healing (Fig. 5B & E, p 0.001 vs. IP3R1 and IP3R3). To further confirm, we carried out wound-healing studies CUDC-427 with combined siRNA of 30% interference efficiency. As the Fig. 5D & F shown, wound healing in A549 cells with treatment involved siRNA was markedly inhibited while in A549 cells with and siRNA was mildly inhibited. These results suggested that IP3R2 plays a predominant role in mediating the inhibitory effect of ERP44 on A549 cell migration. Moreover, we performed scratch experiments in ERP44 stably transfected SH-SY5Y cells, which mainly express IP3R1 [20](Fig. ?](Fig.5G5G left-upper), indicated that the overexpression of ERP44 did not significantly inhibit cell migration, confirming that ERP44 inhibition of cell migration is independent of MAP2K1 IP3R1 (Fig. ?(Fig.55). Open in a separate window Figure 5 IP3R2 plays a dominant role in regulating A549 cell migration(A) RT-PCR analysis for the three subtypes of expression in A549 cells with control or single siRNA. (B) Wound healing in A549 cells with control or single siRNA. (C) The interference efficiency detection in A549 cells with control or combined siRNA. (D) Wound healing in A549 cells with control or combined siRNA. (E) Statistical analysis of single siRNA affecting wound healing in A549 cells. (F) Statistical analysis of combined siRNA affecting wound healing in A549 cells. (G) Overexpression of ERP44 did not affect wound healing in SH-SY5Y cells. RT-PCR assay shows that IP3R1 is specifically expressed in SH-SY5Y cells (left-upper). The.