Figure 5.

A. DUSP4 knockdown enhances growth of lung adenocarcinoma cell lines. In 3/3 cell lines with moderate to high DUSP4 expression, DUSP4 knockdown by two different siRNAs (D1, D2) resulted in growth enhancement (all p<0.05 except H1975) relative to control siRNA (C). In contrast, in 3/3 EGFR-mutant cell lines with low baseline DUSP4 expression, DUSP4 knockdown had no significant effect. For comparison, EGFR knockdown reduced the growth of 4/5 EGFR-mutant lines. EGFR knockdown did not suppress growth of the KRAS-mutant H358 line. Thymidine incorporation was measured 48-72 hours after transfection. DUSP4 expression was measured by Q-RT-PCR, normalized to TBP expression and expressed as the ratio of the cell line expression to that of HBECs. B. DUSP4 knockdown preferentially enhances growth of human bronchial epithelial cells (HBEC) in the presence of mutant EGFR. To confirm the findings shown in A. in an isogenic background, we examined the effect of DUSP4 knockdown in HBECs stably transfected with EGFR L858R. Reducing DUSP4 levels (using D1 siRNA as in A.) enhances growth compared to control siRNA (C) in the presence of EGFR L858R (p<0.05) but not in the parental line (thymidine incorporation measured 48-72 hours after transfection). DUSP4 knockdown was confirmed by RT-PCR as shown in the adjacent agarose gel image. C. DUSP4 cDNA transfection inhibits cell growth of lung adenocarcinoma cell lines. Thymidine incorporation was measured 24 hours after DUSP4 cDNA transfection. Transfection efficiencies, estimated by FACS analysis of GFP-positive cells after 24 hours of transfection, were 54-60% for H1650 cells, 20-44% for H3255 cells, and 46% for H358 cells. V: vector-transfected cells; D: DUSP4-GFP cDNA transfected cells. For all three figure parts, significant differences relative to control are shown by asterisks if p<0.05 (*) or p<0.01 (**) (t-test, 2 tails, equal variance analysis).