Forced over-expression of <i>ALK, PHOX2B</i> and <i>PHOX2A</i> in HeLa and NB cell lines.

<p>A) Transcription levels of the <i>ALK</i>, <i>PHOX2B</i> and <i>PHOX2A</i> genes were evaluated in HeLa cells 48 hours post-transfection with the corresponding gene-specific cDNA expressing vectors by real-time RT-qPCR. Besides the dramatic increase of gene transcripts by each respective transfectants, <i>ALK</i> expression results enhanced by the <i>PHOX2</i> genes over-expression. Values are the mean ± s.d. of N = 3 independent experiments performed in duplicate. B) Upper (I) and lower (II) lanes from immunofluorescence analysis report examples of HeLa cells transfected with the PHOX2B-Myc expression construct. From left to right images show DAPI stained cell nuclei (blue), staining for the PHOX2B-Myc protein (green), and staining for the ALK protein (red). The most distal image is the merge of the three nearby figures. C) Western blot evaluating protein amounts of ALK and PHOX2B in HeLa cells at 72 hours post-transfection with gene-specific cDNA constructs. A consistent transcript increment of each gene is observed but ALK starts to be expressed also following the forced expression of PHOX2B-Myc protein. Gel was loaded with 100 µg of total protein extracts except for evaluation of ALK in <i>ALK</i>-transfected cells and PHOX2B in <i>PHOX2B</i>-transfected cells, for which 1∶10 (10 µg) of protein extracts was loaded to avoid over-saturation of autoradiograph films. D) Western blot evaluating protein amounts of ALK in ACN cells (left) at 72 hours post-transfection with the PHOX2B-Myc construct, in a clone of IMR-32 cells stably expressing the same PHOX2B-Myc fusion protein (right). A marked increment in ALK expression is detected following PHOX2B-Myc expression in transient –transfected ACN cells compared to both native and mock-transfected cells and in a clone of IMR-32 cells, stably expressing PHOX2B-Myc, compared to native cells. An anti-cMyc antibody was specifically used to distinguish PHOX2B-Myc fusion protein from endogenous PHOX2B of NB cells (lower blots). E) Two stable IMR-32 clones, one negative (104) and one positive (49) for PHOX2B-Myc expression were analyzed for ALK expression (upper panels); expression of the fusion PHOX2B-Myc protein was assessed by Western Blot using anti –cMyc (middle panels) and the anti-actin antibodies (bottom panels).