Pegfp c1 plasmid vector

pEGFP-C1 plasmid vector for eukaryotic expression was purchased from Clontech (Palo Alto, CA, USA). pGFP-Klf9 (pCMV-AC-GFP-Klf9) plasmid was purchased from OriGene (RG210147, OriGene Technologies, Inc., Rockville, MD, USA). pcDNA3-EGFP-C4-Nrf2 (pEGFP-Nrf2) plasmid were purchased from Addgene (Watertown, MA, USA). Control shRNA (sc-108060) and Nrf2 ShRNA (sc-37030-SH) plasmids were purchased from Santa Cruz Biotechnology. For experimentation, LECs were transfected with Control ShRNA or Nrf2 ShRNA using the Neon Transfection System (Invitrogen, Waltham, MA, USA).

To synthesize dsRNA for Tm14-3-3ζ, specific primers conjugated with T7 promoter sequences were designed using the Snapdragon dsRNA design software [40 (link),41 (link)]. The primer sequences are available in Table 1. Enhanced green fluorescent protein (EGFP) dsRNA was synthesized from pEGFP-C1 plasmid vector (Clontech Laboratories, CA, USA) and injected as the negative control. Template cDNAs for dsTm14-3-3ζ synthesis were amplified by using ExTaq polymerase (Takara, kusatsu, Japan) with specific primers as described in Table 1. PCR products containing the T7 promoter sequences were purified using the AccuPrep PCR purification kit (Bioneer). dsTm14-3-3ζ was synthesized with 1 µg of purified PCR products using the Ampliscribe T7-flash transcription kit (Epicentre Biotechnologies, Madison, Wisconsin, USA). Synthesized dsRNAs were confirmed by electrophoresis on 1% agarose gels. For gene specific silencing, 1 µg of dsTm14-3-3ζ were injected into T. molitor larvae using Picospritzer III micro-dispense system (Parker). dsEGFP was injected as a negative control using the same concentration. Transcript levels of Tm14-3-3ζ were analyzed using real-time PCR.