Figure 6

DDX21 modulates AP-1 activity in MDA-MB-231 cells. (A) MDA-MB-231 cells were either infected with shSCR or shRNA-DDX21 lentiviruses. Whole cell lysates were subjected to western blot analysis with the indicated antibodies with tubulin as an internal control. Standard error of the mean is indicated with fold-change. ^*,P <0.05. (B) RT-PCR analysis was performed for cyclin D1 mRNA levels after normalization with GAPDH mRNA levels. Error bars were taken from three independent experiments. ^*,P <0.001. (C) RT-PCR analysis was performed for EGFR mRNA levels after normalization with GAPDH mRNA levels. Error bars were taken from three independent experiments. ^*,P <0.005. (D) MDA-MB-231 and SKBR3 cells were infected with AP-1 reporter lentiviruses to detect endogenous AP-1 activity. After verification of AP-1 luciferase activity, these cells were infected with either shSCR or shRNA-DDX21. Two days postinfection, equal numbers of cells were transfected with pGL-Renilla-luciferase plasmids. Equal numbers of cells were then analyzed for both firefly and Renilla luciferase activity. Data presented is firefly luciferase activity after normalization with Renilla luciferase and then further normalized to shSCR control. ^*,P <0.001 (n =3). (E) MDA-MB-231 cells infected with shSCR or shRNA-DDX21 lentiviruses were stained with antibodies recognizing NPM and visualized by indirect immunofluorescence. DAPI stain was used to mark nuclei. Images are representative of over 100 cells for each condition. DAPI, 4',6-diamidino-2-phenylindole; EGFR, epithelial growth factor receptor; GAPDH, glyceraldehyde 3-phosphate dehydrogenase; NPM, nucleophosmin; RT-PCR, reverse transcriptase-polymerase chain reaction.