Figure 3.

NGAL-mediated decrease of Bim protein is responsible for apoptosis resistance. A: Bim expression is downregulated in NGAL overexpressing H3255 and H441 cells (NG) compared to vector control cells (V). Bim EL: Bim extra-long isoform; Bim L: Bim long isoform. B: H3255 and H441 cells were either untreated or transfected with negative control siRNA (Neg) or Bim siRNA (Bim). After 48 hours, cells were treated with erlotinib (8 μM for H441 and 0.05 μM for H3255) or vehicle control (DMSO) for 24 hours. Cell lysates were analyzed for Bim and PARP cleavage by Western blot. C: Expression of apoptosis effector proteins Bcl-xL, Bax, Mcl-1, Bad, Puma and Bcl-2 NGAL is not altered in NGAL overexpressing (NG) H3255 and H441 cells compared to vector control (V). D: NGAL knockdown by shRNA leads to an increase in Bim levels in H358 erlotinib-resistant cells. Neg - negative control shRNA; NG-NGAL shRNA; Ctrl - non-transduced cells. E: NGAL knockdown increases the apoptosis sensitivity of cells with acquired resistance to erlotinib. H358 erlotinib-resistant cells were transduced with NGAL shRNA (NG) or negative control shRNA (Neg) and treated with erlotinib (10 μM) for 72 hours. Cell lysates were analyzed for cleaved PARP protein by Western blot.