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Figure 3: LC3B knockdown induces apoptosis in DLD-1 cells. (a) Expression of LC3B was knocked down in DLD-1 cells. Total protein was extracted from untransfected (WT), control siRNA-transfected, or LC3B siRNA-transfected cells. Forty-eight hours later, whole-cell lysates were subjected to western blot analysis for LC3B. Incubation with an anti-β-actin antibody was used as a protein-loading control. (b) Real-time PCR measurement of LC3B mRNA expression in DLD-1 cells. The relative LC3B levels normalized to 18S rRNA are expressed as the mean ± SEM ( ), ** . (c) Time-dependent cell growth inhibition was measured using Cell Counting Kit-8 at 6, 12, 24, 48, and 72 h after LC3B siRNA transfection. Data are expressed as mean ± SEM ( ; ** ). (d) Vacuolated cells were analyzed and counted as described in the experimental procedures. At least 3 fields of cells per sample were counted and tabulated. Data are expressed as mean ± SEM ( ; ** ). (e) siRNA transfected DLD-1 cells were stained with Hoechst 33342 and analyzed by fluorescence microscopy. Apoptotic nuclei stained more brightly than nuclei in untransfected cells or siRNA control-transfected cells. At least 5 fields of cells per sample were counted and tabulated. Values are expressed as the mean ± SEM ( ), ** based on Student’s -test. (f) LC3B siRNA transfected DLD-1 cells were collected in RIPA buffer, and 50 μg of protein was loaded for SDS-PAGE. Protein was analyzed by western blot using an anti-caspase-3 antibody to assess apoptosis.