Fig 2: Western blot analysis of survivin and ERCC1 in SCC-25 and Detroit 562 cells in control and CM - treated conditionsSCC-25 and Detroit 562 cells were treated with albumin - containing (control) or with CM medium (stimulation, CM) and subjected to western blot analysis of ß-actin (red), survivin (green) or ERCC1 (green) in a two channel near infrared fluorescence detection system. ß-actin loading control (42 kD), a single band was detected at 700 nm and is presented in red, survivin or ERCC1 were detected at 800 nm and are presented in green. (a) Survivin was detected as a single band in both SCC-25 (lanes 1-4) and Detroit 562 cells (lanes 5-8). ERCC1 was detected as multiple bands with two more intensive ones at 38 and 30 kD in both cell lines. More bands might be present in certain cell lines, which was also described by the data sheet of the antibody provider. Lanes 1-2: SCC-25 control, lanes 3-4: SCC-25 stimulation with CM, lanes 5-6: Detroit 562 control, lanes 7-8: Detroit 562 stimulation with CM; MW: molecular weight standard. (b-c) Column diagrams representing mean and standard error of measurement of normalized (to loading control, ß-actin) survivin (b) and ERCC1 (c) band volumes from 8 gel lanes for each SCC-25 and Detroit 562 control and CM stimulated protein samples. The mean band volume of SCC-25 control was set as “1”. *: p < 0.05.

Fig 3: Activation and expression levels of Akt, Erk1/2, BRCA1, PTEN, ERCC1, EGFR and DNA-PKcs on the three non-small-cell lung cancer cells after different treatments. (A) Effects of Akt, Erk1/2, BRCA1, PTEN, and ERCC1 on cisplatin combined with gefitinib. (B) Expression levels and activation of EGFR and DNA-PKcs in the three NSCLC cells after the combination of gefitinib and cisplatin. p-, phosphorylated; BRCA1, BRCA1 DNA repair associated; ERCC1, ERCC excision repair 1, endonuclease non-catalytic subunit; DNA-PK, DNA-dependent protein kinase; EGFR, epidermal growth factor receptor.

Fig 4: 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 -ddCt method for E-cadherin (A), desmoplakin (B), vimentin (C), MMP-9 (D), ERCC1 (E), and survivin (F) in control and CM-treated conditions, where ß-actin was used as housekeeping gene.

Fig 5: The HDACi SAHA Sensitizes ERCC1-High NSCLC Cells to Cisplatin In Vitro(A) H1299 and A549/DDP cells were co-treated with cisplatin and SAHA at the indicated concentration for 48 h, and cell viability was measured by an MTS assay. (B) A549 cells transfected with LV-ERCC1 were co-treated with cisplatin and SAHA at the indicated concentration for 48 h, and cell viability was measured by an MTS assay. (C) H1299 and A549/DDP cells were co-treated with 10 µM cisplatin and 2 µM SAHA for 48 h, cells were stained with annexin V-fluorescein isothiocyanate (FITC) and propidium iodide, and cell apoptosis was analyzed by flow cytometry. (D) H1299 and A549/DDP cells were co-treated with 10 µM cisplatin and 2 µM SAHA for 48 h, and cells were fixed and labeled with anti-?-H2AX antibodies. The ?-H2AX foci were analyzed by immunofluorescence microscopy. *p < 0.05, **p < 0.01.