Research Paper Volume 13, Issue 14 pp 18223—18237

Figure 5. Effects of combination treatment with RAPA and SAHA on IR after down-regulate the level of autophagy in NSCLC cells. A549 cells were infected by a lentivirus delivered Atg5 shRNA for 24h. (A, B) The relative protein and mRNA expression levels of Atg5 were determined by western blot and RT-qPCR analysis. (C) Quantitative data were calculating the number of LC3 dots per A549 cell by using an immunofluorescence confocal microscope. (D) Survival fractions of A459 cells were assessed by the colony formation assay. (E) γ-H2AX protein was determined by western blot analysis. A549 cells were treated with RAPA (100nmol/L) and SAHA (2.5μmol/L) for 24h and were subsequently exposed to IR (4Gy) for 1h and 24h after transfected with or without Atg5 shRNA for 24h. (F) Colony formation assay in A549 cells resulting from IR after treatment with different concentrations of 3-MA or without 3-MA. A549 cells were pretreated with 3-MA for 1h before RAPA and SAHA treatment.