Fig 2: The HDACi SAHA Modulates miR-149 Expression in ERCC1-High NSCLC Cells(A) miRNA profiles were examined in SAHA-treated H1299 cells. (B) Differentially expressed miRNAs (>8-fold) in the SAHA-treated cells relative to the control. (C) H1299 and A549/DDP cells were treated with different concentrations of SAHA, and the expression of miR-149 was detected with qRT-PCR. (D) Sequence analysis of the promoter region revealed two conserved E2F1-binding sites at the core promoter region of miR-149. (E) Total protein extracts of H1299 cells in SAHA-treated cells or control cells were subjected to IP using E2F1 antibody or control IgG, followed by immunoblotting (IB) with acetyl-lysine antibody (left panels). Reciprocal IP was done using acetyl-lysine antibody or control IgG, followed by IB with the E2F1 antibody (right panels). (F) The binding of E2F1 on the miR-149 promoter region was measure by ChIP analysis. (G) H1299 cells were transfected with human cytomegalovirus promoter (pCMV)-E2F1 expression plasmid, together with the miR-149 promoter luciferase reporter construct or the E2F1-binding site mutant (mt) promoter luciferase reporter construct. Luciferase activity was measured after 48 h. (H) H1299 cells were treated with SAHA for 48 h, and the binding of E2F1 on the miR-149 promoter region was measure by ChIP analysis. (I) H1299 cells were transfected with E2F1 siRNA and then treated with SAHA; miR-149 promoter luciferase activity was measured after 48 h. (J) H1299 and A549/DDP cells were transfected with E2F1 siRNA and then treated with SAHA; the mRNA levels of miR-149 were measured by qRT-PCR. ∗∗p < 0.01.

Fig 3: Real-time PCR analysis of EMT-like (A-D) and radioresistance - related (E-F) gene expression changes in SCC-25 cells in control and CM-treated conditions. SCC-25 cells were treated for 72 hours with CM, followed by total RNA isolation, reverse transcription, and real-time PCR. Relative gene expression was calculated with the −δδCt method for E-cadherin (A), desmoplakin (B), vimentin (C), MMP-9 (D), ERCC1 (E), and survivin (F) in control and CM-treated conditions, where β-actin was used as housekeeping gene.

Fig 4: ERCC1 Enhances Cisplatin Resistance in NSCLC Cells(A) H1299 and A549/DDP cells were transfected with control shRNA (shControl) or ERCC1 shRNA (shERCC1), and the expression of ERCC1 was analyzed by western blotting. (B) H1299 and A549/DDP cells were transfected with ERCC1 shRNA and then treated with cisplatin at the indicated concentration for 48 h; cell viability was measured by an MTS assay. (C) A549 and H460 cells were transfected with ERCC1-expressing vector (LV-ERCC1), and the expression of ERCC1 was analyzed by western blotting. (D) A549 and H460 cells were transfected with LV-ERCC1and then treated with cisplatin at the indicated concentration for 48 h; cell viability was measured by an MTS assay. (E) A549 cells were transfected with LV-ERCC1 and then treated with 5 μM cisplatin for 48 h; cells were fixed and labeled with anti-γ-H2AX antibodies. The γ-H2AX foci were analyzed by immunofluorescence microscopy. (F) A549/DDP cells were transfected with ERCC1 shRNA and then treated with 20 μM cisplatin for 48 h; cells were fixed and labeled with anti-γ-H2AX antibodies. The γ-H2AX foci were analyzed by immunofluorescence microscopy. (G and H) A549/DDP cells transfected with control shRNA or ERCC1 shRNA were injected to the right shoulder of nude mice. Tumor-bearing mice were treated with either PBS or cisplatin (3 mg/kg body weight per day) for 4 weeks. (G) At the end of treatment, tumors were excised. (H) Tumor sizes were measured at every 3 days. ∗p < 0.05, ∗∗p < 0.01.

Fig 5: The HDACi SAHA Sensitizes ERCC1-High NSCLC Cells to Cisplatin via Regulating miR-149(A) H1299 and A549/DDP cells were transfected with anti-NC or anti-miR-149 and then treated with 2 µM SAHA for 48 h; the expression of ERCC1 was measured by western blotting. (B) H1299 and A549/DDP cells were transfected with anti-NC or anti-miR-149 and then treated with 2 µM SAHA for 48 h; cell viability was measured by an MTS assay. (C and D) H1299 and A549/DDP cells were transfected with anti-NC or anti-miR-149 and then treated with 2 µM SAHA for 48 h; cells were stained with annexin V-FITC and propidium iodide, and the cell apoptosis was analyzed by flow cytometry (C); Portions of the apoptotic cells are presented (D). (E) H1299 cells were transfected with anti-NC or anti-miR-149 and then treated with 2 µM SAHA for 48 h; cells were fixed and labeled with anti-?-H2AX antibodies. The ?-H2AX foci were analyzed by immunofluorescence microscopy. **p < 0.01.