Figure 2

Expressions of NF-κB target genes are elevated by lapatinib in both HER2-positive and –negative breast cancer cells. (A) Total RNA extracted from two pairs of parental and lapatinib-resistant cells (SkBr3 and SkBr3/Lap#6; BT474 and BT474/Lap#3) was subjected to reverse transcription followed by cDNA microarray analysis. Gene expressions of NF-κB target gene with more than two folds change were analyzed with Ingenuity Pathway Analysis (IPA). (B-E) Total RNA extracted from SkBr3, MDA-MB-231, and their lapatinib-selected clones was subjected to RT-qPCR analysis for mRNA levels of IL-1β (B), IL-6 (C), TRAF-1 (D) and COX-2 (E). (F-I) SkBr3/Lap#6 and 231/Lap#2 cells were infected with lentiviral shRNA against luciferase (shLuc) or p65 (shp65) for three days, and then total RNA was extracted and subjected to RT-qPCR analysis for mRNA levels of IL-1β (F), IL-6 (G), TRAF-1 (H) and COX-2 (I). (J) MDA-MB-231 and 231/Lap#6 cells were transiently transfected with the luciferase reporter gene of IL-6 promoter containing wild-type (WT) or mutation of NF-κB-binding sites (NF-κB^mut). The promoter activity of IL-6 was normalized with β-galactosidase activity. (K) MDA-MB-231 and 231/Lap cells were infected with lentivirus of shRNA against luciferase (shLuc) or p65 (shp65) for three days, and then whole protein lysates were prepared and subjected to Western blot analysis with antibodies against indicated molecules. shRNA, short hairpin RNA.