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. 2015 Jun 4;6(6):e1779. doi: 10.1038/cddis.2015.151

Figure 4.

Figure 4

Microglia-derived IL-1β and rIL-1β induce p53 and p53 target gene expression in NPCs. NPCs were cultured in unconditioned stem cell media (Ctrl), conditioned media from microglia activated with LPS/γIFN in the presence or absence of yVAD-CMK (20 μM) or LPS/γIFN-aMCM supplemented with IL-1RA (50 ng/ml). (a) NPCs were harvested after 48 h and protein extracts were subjected to sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and immunoblotted for p53, Puma, p21 and actin as a loading control. Representative immunoblots and densitometric analysis of three independent experiments is shown (n=3; *P<0.05). (b) RNA was harvested after 24 h and mRNA levels of Puma, Noxa and p21 were determined by quantitative real-time-polymerase chain reaction (qRT-PCR). mRNA levels are reported as fold increase over NPCs incubated in unconditioned media (n=4, *P<0.05). (c) NPCs treated with recombinant IL-1β (50 ng/ml) were harvested after 48 h and subjected to SDS-PAGE and immunoblotted for p53 and actin as a loading control. A representative blot from three independent experiments is shown. (d) RNA was harvested from p53+/+ and p53−/− NPCs treated with rIL-1β for 24 h and mRNA levels of Puma and p21 were examined by qRT-PCR. mRNA levels are reported as fold increase over control NPCs treated with vehicle (n=3; *P<0.01)