Rotor gene a

MDA-MB-231 cells were seeded in 6-well plates and transfected with 25 nM of TAZ-siRNA for 24 h. Total RNA purification was performed by Direct-zol RNA MiniPrep, and total RNA (1 μg) was used to synthesize cDNA with the Takara cDNA synthesis kit (6110A, Takara, Otsu, Shiga, Japan). The relative mRNA expression of TAZ, RhoA, RhoC, ROCK1, ROCK2, Cdc42, Rac1, and Myosin Light Chain 2 (MLC2) was analyzed using a QuantiTect SYBR Green PCR Kit (204143, QIAGEN, Hilden, Germany). β-actin mRNA was used as an internal control. qRT-PCR was run on Rotor-Gene-A (QIAGEN, Hilden, Germany) and threshold was set by 0.02 to determine the threshold cycle (Ct) value. The relative mRNA expression was calculated, as we described previously (31 (link)). Each sample was tested in duplicate, and the primer sequences used for qRT-PCR are depicted in Table 1.

mpkCCDc14 cells were transfected with control-siRNA or SNX27-siRNA (50 nM) using a reverse transfection method and grown on semi-permeable filters of 6-well transwell plates for 9 days [38 (link)]. Total RNA purification was performed by Direct-zol^TM RNA MiniPrep (Zymo Research, Irvine, CA), according to the manufacturer’s instruction, and cDNAs were synthesized using 1 ug of total RNA by the Prime Script cDNA Synthesis kit (Takara Shuzo Co., Otsu, Shiga, Japan). The relative expression of the AQP2 and SNX27 mRNA was determined by real-time quantitative PCR (RT-qPCR), using a QuantiTect SYBR Green PCR Kit (QIAGEN), respectively. The PCR reaction was performed at 95 °C for 10 min followed by 30 cycles at 95 °C for 15 s, 58 °C for 30 s, and 72 °C for 30 s. β-actin was used as an internal control, and the threshold was set by 0.02 to determine the threshold cycle (Ct) value. The fold enrichment was calculated as 2^{−[ΔCt(AQP2 or SNX27)−ΔCt(β-actin)]}. RT-qPCR of mRNA was carried out using Rotor-Gene-A (QIAGEN), and each sample was tested in duplicate. Primers of mRNA were designed by Origene and purchased from Cosmogenetech (Seoul, Korea).

Total RNA was prepared by Direct-zol^TM RNA MiniPrep (R2050, Zymo Research, Irvine, CA, USA) in accordance with the manufacturer’s protocol. cDNAs were synthesized using 500 ng of total RNA by PrimeScript 1st strand cDNA Synthesis Kit (6110A, Takara, Otsu, Shiga, Japan), followed the manufacturer’s instruction. The RT-qPCR was performed using a QuantiTect SYBR Green PCR Kit (204143, QIAGEN, Hilden, Germany) to examine the relative expression of the AQP2 mRNA. β-actin mRNA was used as an internal control and each sample was tested in duplicate. RT-qPCR was run on Rotor-Gene-A (QIAGEN, Hilden, Germany) and threshold was set by 0.02 to determine the threshold cycle (Ct) value. The relative mRNA expression was calculated, as we described previously [21 (link)]. The primer sequences used for RT-qPCR were listed in Figure S1A.