Figure 4 : PCI-24781 treatment up-regulates NOTCH pathway genes and suppresses MYCN, as confirmed by RT-PCR.

(a) SMS-KCNR cells were treated in duplicate with 4 nM bortezomib, 125 nM PCI-24781, or the combination for 24 h. RNA
was isolated and hybridized to Affymetrix U133 2+ arrays. Rows represent genes; columns represent samples. P 1 and P 2
represent replicate PCI-24781 treatments; B 1 and B 2 represent Bortezomib treatments; P+B 1 and P+B 2 represent PCI-24781
in combination with Bortezomib. The color represents elevated (red) or diminished (green) expression relative to the mean
value for the gene and standardized. Rows were ordered to emphasize distinct expression patterns. (b) Real-time PCR for Notch
genes confirmed up regulation of expression seen in microarrays in SMS KCNR cells. (A) Notch expression after treatment
with PCI-24781 alone and in combination with bortezomib was statistically significantly increased (P<0.05) in each case except
for Notch 3 in SMS-KCNR cells (P=n.s.). Notch 4 was not expressed in any of the cell lines tested. (B) PCI-24781 treatment
significantly up regulated expression of the HES1 in both cell lines (P<0.05). JAG1 and JAG2 were both significantly up
regulated by PCI-24781 in SMS-KCNR cells (P<0.01) but JAG2 was not significantly changed in SKNBE(2)C cells. (C) MCYN
expression was significantly decreased by treatment with PCI-24781 along and in combination with bortezomib in both cell
lines (P<0.01), while treatment with bortezomib alone did not significantly change MYCN expression (P=n.s.).

Saulnier Sholler et al.Journal of Cancer Therapeutics and Research  2013 2:21DOI : 10.7243/2049-7962-2-21