Pgfp 5 rs shrna vector

Pregnant mice were maintained under inhaled isoflurane anaesthesia for the duration of the procedure. The uterine horns were exposed. In some experiments, 1 to 2 μl of Vismodegib (5 mM solution in DMSO; Stratech) or DMSO alone was injected into the lateral ventricle of each embryo’s brain with a glass micropipette. In other experiments, plasmids were injected into the lateral ventricle of each embryo’s brain at 1 to 4 mg mL⁻¹, the embryo in the uterus was placed between tweezer-type electrodes (CUY650; Nepagene), and an electroporator (CUY21E; Nepagene) was used to deliver 6 pulses (30 V, 50 ms each, 950 ms apart). In both cases, the uterine horns were replaced, the abdominal wall was sutured, and animals recovered. Processing was as described above.
Plasmids used in this study were as follows: CAG-FoxG1-IRES-mCherry (kindly provided by Goishi Myioshi, Tokyo Women’s Medical University, Japan); Scrambled shRNA control in pGFP-V-RS shRNA Vector (TR30013, Origene); Smoothened shRNA in pGFP-V-RS shRNA Vector (TG510788, Origene).

To establish a stable LARS1-overexpressing SW480 cell line, cells were transfected with 4 μg of empty vector (pCMV6-AC-GFP; PS100010) or LARS1 expression vector (LARS1 human tagged ORF clone; RG221682) from Origene (Rockville, MD, USA) using Lipofectamine 2000 (Invitrogen, Carlsbad, CA, USA) following the manufacturer’s recommended procedures. After transfection, stable cell lines (LARS1-2- and -4-SW480 cells) were established after G418 selection (800 μg/mL) for 14 days. To obtain a stable LARS1-knockdown cell line, SW620 cells were transfected with 4 μg of nontargeting control (NC) shRNA (pGFP-V-RS shRNA vector; TR30007) or LARS1 shRNA plasmid (TG303577; Origene, Rockville, MD, USA) using Lipofectamine 2000 (Invitrogen, Carlsbad, CA, USA) following the manufacturer’s instructions. After transfection, cells were treated with G418 (800 μg/mL) for 14 days and six clones were isolated. Among them, LARS1 shRNA-4- and -5-SW620 cells were efficiently knocked-down and used in this study.

PGC-1α-HEK293, PGC-1α-1-SW480, PGC-1α shRNA-1-SW620, or NC shRNA-SW620 cells were transfected with NC shRNA (pGFP-V-RS shRNA vector; TR30007) or LARS1 shRNA expression vector (TG303577; Origene, Rockville, MD, USA) using lipofectamine 2000 according to the manufacturer’s instructions. In addition, PGC-1α shRNA-1-SW620 cells were transfected with 4 μg of LARS1 expression vector (LARS1 human tagged ORF clone; RG221682) or empty vector (pCMV6-AC-GFP; PS100010) using lipofectamine 2000 according to the manufacturer’s instructions. After transfection, cells were cultured in 10% FBS-supplemented DMEM for 48 h. These cells, pcDNA-HEK293, pcDNA-SW480, and NC shRNA SW620 cells were then used for cell counting followed by transwell migration and invasion assays.