Fig. 1.

Lack of effect of SAT1 overexpression on mRNA levels of GFP and two endogenous genes. (A) Fold change in GFP and SAT1 mRNA levels compared with untransfected control shown by real-time PCR. Total RNA was isolated from HeLa cells using the RNeasy kit and reverse transcription was performed using ThermoScript reverse transcription-PCR kit (Invitrogen) in a 20-µL reaction mixture containing 500 ng purified RNA, according to the manufacturer’s instructions. PCR was performed in triplicates using the IQ SYBR Green Super mix (Bio-Rad) and the Bio-Rad Q-cycler machine, as follows: 50 °C for 2 min and 95 °C for 10 s followed by 40 cycles at 95 °C for 30 s, 60 °C for 1 min, and 72 °C for 20 s. The generation of specific PCR products was confirmed by melting curve analysis. Error bars refer to SD for three independent experiments done in triplicate. GFP and SAT1 mRNA levels were normalized using GAPDH as an internal control. (B) RNA FISH image showing GFP mRNA (Q570, yellow) and SAT1 mRNA (Q670, red) in high and low magnifications. HeLa cells cultured on glass coverslips were washed with PBS, fixed in 3.7% (vol/vol) formaldehyde for 10 min at room temperature, washed twice with PBS, and permeabilized at 4 °C in 70% (vol/vol) EtOH for 1 h. A total of 1 μL of respective probe stock (12.5 μM) in 100 μL of hybridization solution was added and cells were incubated in a dark chamber at 37 °C overnight. After washing with the wash buffer [2× SSC with 10% (vol/vol) formamide], DAPI nuclear stain in PBS (5 ng/mL) was added to counterstain the nuclei. (C) RNA FISH of endogenous GAPDH and eIF5A-1 mRNAs (Q570, yellow) and SAT1 mRNA (Q670, red) in cotransfected HeLa cells. Images were obtained using a Zeiss LSM510 META inverted confocal system. pGFP, pCEFL/GFP; pEV, pCMV7.1. 3xFLAG empty vector; and pSAT1, pCMV7.1.3xFLAG/SAT1.