Of note, using a siRNA approach, we found that NCX1 was the isoform specifically involved.
As control we used an siRNA targeting firely luciferase (CATCACGTACGCGGAATAC).
We used an siRNA and an overexpression plasmid to respectively downregulate or upregulate PDCD4 protein levels.
Cells were seeded at 2 × 10 cells per well onto 24-well plates and were transfected at 30 40% confluence approximately 16 24 h later, using a mixture of siRNAs from Dharmacon and Santa Cruz (200 n M) with the Genesilencer siRNA transfection reagent (Genlantis, San Diego, CA, USA), according to the manufacturer's instructions.
Considering that the expression level of TNRC6C is relatively low in HeLa cells compared to TNRC6A and TNRC6B (Yao et al., 2011), only TNRC6A and TNRC6B were downregulated using a mixture of siRNAs (TNi) targeting each to serve as a positive control.
BIRC3 and cREL expression was silenced using a mixture of three siRNA commercially available as Trilencer-27 siRNA knockdown duplexes kit (Origene, Rockville, MD, USA).
We found that inhibiting Nampt expression using a mixture of 4 siRNA oligo (siNampt) resulted in a 1.1-fold increase of GFP-positive cells in comparison to mock-depleted cells (siControl), and knockdown of Nampt expression by individual siRNAs derived from the mixture yielded similar results.
Inhibition of specific gene expressions was achieved by RNA interference using a mixture of four siRNAs (ON-TARGET plus SMARTpool; Dharmacon, Lafayette, CO, USA).
HCT116, HT-29 colon cancer cells (5 × 10 cells/well) were plated in 24-well plates and transiently transfected with 0.4 μg of the empty vector or the 100 nM of negative siRNA, DR4 or DR5 siRNA per well, using a mixture of plasmid and the WelFect-EX PLUS reagent in OPTI-MEM, according to manufacturer's specification (WelGENE, Seoul, Korea).
Immunoblotting for Beclin-1 and α-tubulin (loading control) was provided in cell extracts silenced using a random siRNA (Randsi) or Beclin siRNA (Beclinsi).
