Figure 4. Effects of combination treatment with RAPA and SAHA on autophagy and acetylation in NSCLC cells. (A–C) RAPA, SAHA, and IR induce cellular autophagy. A549 cells were treated with RAPA (100nmol/L) or/and SAHA (2.5μmol/L) for 24h or were exposed to IR (4Gy) and were tested after 4h. (A) The ultrastructures of autophagosomes in A549 cells were observed under a transmission electron microscope (TEM). Black arrows and rectangles indicate intracellular autophagosomes. (B) The distribution of LC3 dots in A549 cells was observed by using an immunofluorescence confocal microscope. Quantitative data were calculating the number of LC3 dots per cell. *p<0.05, compared with control, #p<0.05, compared with combined treatment. (C) The level of autophagy-related protein (LC3 I/II, p62, Atg5) was determined by western blot analysis. (D) SAHA induces acetylation of A549 cells. A549 cells were treated with SAHA (2.5μmol/L) and/or RAPA (100nmol/L) for 24h. The level of acetylation of histone H3 was determined by western blot analysis.