PSF Knockdown Enhances Apoptosis via Downregulation of LC3B in Human Colon Cancer Cells
Figure 2
PSF knockdown decreases LC3B expression and inhibits the proliferation of DLD-1 cells. (a) Expression of PSF was knocked down in DLD-1 and HT-29 cells. Top panel: total protein was extracted from untransfected (WT), control siRNA-transfected, or PSF siRNA-transfected cells. Forty-eight hours later, whole-cell lysates were subjected to western blot analysis for PSF. Incubation with an anti-β-actin antibody was used as a protein-loading control. Bottom panel: Real-Time PCR measurement of PSF mRNA expression in DLD-1 and HT-29 cells. The relative PSF levels normalized to 18S rRNA are expressed as the mean ± SEM (), **. (b) Decreased LC3B expression after PSF knockdown in DLD-1 and HT-29 cells. Top panel: protein was analyzed by SDS-PAGE at the indicated times after PSF siRNA transfection, subjected to western blotting, and visualized with an enhanced chemiluminescence reagent. Each lane was loaded with 20 μg of whole-cell lysate. β-actin was used as an internal loading control and was detected using mouse anti-β-actin antibody. Bottom panel: (c) time-dependent cell growth inhibition was measured using Cell Counting Kit-8 at 6, 12, 24, 48, and 72 h after siRNA transfection. An equal number of cells (1 × 105 cells/well) were seeded in 6-well plates and then incubated overnight at 37°C in an incubator with 5% CO2. Cell Counting Kit-8 was added to the medium and incubated for 2 h in the incubator. The amount of orange formazan dye generated was calculated by measuring the absorbance at 450 nm in a microplate reader. Data are expressed as mean ± SEM (; **). (d) Top panel: protein was extracted from untransfected (WT), control pcDNA3.1-transfected, or pcDNA3.1-LC3B-transfected cells. Bottom panel: PSF knocked down DLD-1 cells were transfected with LC3B plasmid and incubated for 24 h. Cell proliferation was measured using Cell Counting Kit-8.
