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. 2016 Dec 27;8(4):6589–6607. doi: 10.18632/oncotarget.14244

Figure 7. Complementation of FL splice variant in GAS5 knocked-down cells stabilizes HDM2.

Figure 7

A. qRT-PCR of HDM2 (MDM2) expression in IMR-32 cells after transfection with: Negative Control siRNA & Vector only (VO), GAS5 siRNA + VO, GAS5 siRNA complemented with FL-expressing plasmid and GAS5 siRNA complemented with C2-expressing plasmid. Samples were normalized to the Ct value of GAPDH, with relative expression calculated by normalizing to the ΔΔCt of the Negative Control siRNA & Vector only sample. Data were expressed as a means of +/- SD using three biological replicates. B. Western blot analysis of p53 and HDM2 (MDM2) in IMR-32 cells after transfection with: Negative Control siRNA & Vector only (VO), GAS5 siRNA + VO, GAS5 siRNA complemented with FL-expressing plasmid, and GAS5 siRNA complemented with C2-expressing plasmid. GAPDH was used as a load control. * and ** p < 0.05, Student's t-test. C. Schematic illustration of the effects of GAS5 knockdown in neuroblastoma cells. Briefly, loss of GAS5 induces ser1457 phosphorylation of BRCA1, an ATM-specific phosphorylation site. Simultaneously, loss of GAS5 also induces multiple p53 phosphorylation events, leading to increased stability and a decrease in cellular HDM2 levels (likely due to auto- ubiquitination). Phospho-activated p53 and BRCA1 then induce increased transcription of GADD45A, leading to the induction cell cycle arrest.