Effects of knockdown of ZEB1 or overexpression of miR-200c on EMT markers and erlotinib sensitivity in HCC4006ER cells.

<p>A, HCC4006ER cells were plated at 3x10⁵ per well in 6-well plate and transfected with ZEB1 or control siRNA at 5 nM as a final concentration. Cells were harvested for analysis at 96 hours post-transfection. Cell lysates were subjected to protein expression analysis with antibodies to ZEB1, E-cadherin, vimentin, and β-actin. B, HCC4006ER cells were plated at 3x10³ cells/ well in black wall 96-well plate, transfected with ZEB1 or control siRNA at 5 nM as a final concentration, and treated for 72 hours at 48 hours after siRNA transfection with increasing concentrations of erlotinib. HCC4006 cells were treated for 72 hours with increasing concentrations of erlotinib for 72 hours to plot a reference curve. Data generated by cell viability assay (CellTiter-Glo) are expressed as a percentage of the value for erlotinib-untreated cells with control siRNA. Control siRNA or transfection reagent did not affect cell viability in HCC4006ER cells at this concentration. The error bars represent SEM of 3 independent experiments. C, HCC4006 and HCC4006ER cells were plated at 3x10⁵ per well in 6-well plate. HCC4006ER cells were transfected with ZEB1 or control siRNA at 0.5 or 5 nM as indicated. HCC4006 cells and siRNA-transfected HCC4006ER cells were incubated for 6 hours ± erlotinib (1 μM) at 48 hours after siRNA transfection. Cell lysates were subjected to protein expression analysis with antibodies to pEGFR, pAkt, pErk, and β-actin. D, Cell lysates of H1975, H1975 BIBW-R, and H1975 WZ-R cells were subjected to protein expression analysis with antibodies to ZEB1, E-cadherin, and β-actin. H358 cells, with and without 5 ng/mL TGF-β treatment for 48 hours or with ZEB1 overexpression, served as positive controls (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0147344#sec002" target="_blank">Materials and Methods</a> for cell line details). TGF-β stimulation or ZEB1 overexpression reduced E-cadherin expression in H358 cells, as expected. E, Tumor samples from NSCLC patients who were treated with EGFR-TKI and developed resistance accompanied by an EMT phenotype (cases 2, 4, and 10 in our previous report (10); case 2 had T790M and PTEN loss in addition to EMT, whereas cases 4 and 10 did not have other known resistant mechanism after EGFR-TKI treatment). ZEB1 immunostaining analysis was done before and after EGFR-TKI treatment. Representative ZEB1 immunostaining images from cases 4 and 10 (before and after EGFR-TKI treatment) are shown in the upper panel. Percentage of ZEB1-positive nuclei per field was demonstrated for each case (before and after EGFR-TKI treatment) in the bottom panel. Determinations were done in triplicate. Bars, SEM. *<i>P</i> < 0.00001 for before versus after EGFR-TKI treatment (Student <i>t</i> test). F, Expression levels of miR-200c and RNU6B in HCC4006 and in HCC4006ER cells were measured by the quantitative real-time PCR with the corresponding TaqMan MicroRNA assay. Values are expressed as % of RNU6B. Determinations were done in triplicate with two machine replicates. *<i>P</i> < 0.005 for HCC4006 versus HCC4006ER (Student <i>t</i> test).