ITSN2 knockdown ameliorates ING1a-induced senescence phenotypes.

(A) Biotin internalization assay in A431 cells expressing Ad-ING1a+Control siRNA or ITSN2 siRNA. Cells were serum-starved overnight and stimulated with EGF for the indicated times. Biotinylated cell surface EGFR was quantified using scanning densitometry. Results of three trials are shown in the graph. (B) A β-gal assay was carried out in cells expressing GFP and ING1a after transfection with control or ITSN2 siRNA. (C) Hs68 cells expressing Ad-GFP, Ad-ING1a, or Ad-ING1a together with control siRNA or ITSN2 siRNA were analysed for proliferation using a BrdU incorporation assay. The percentage of cells that incorporated BrdU is presented in the histogram (p<0.05). (D) Low passage (young) Hs68 cells were transfected with control siRNA (50 nM) or ITSN2 smartpool siRNA (50 nM). Twenty-four hours later cells were infected with Ad-GFP or Ad-ING1a-expressing viruses, and 48 h later the levels of ITSN2, p16, RB, and HSP70 mRNA were measured by quantitative real-time PCR using gene-specific primers. The mRNA levels were normalized to β-actin levels. (E) Senescent Hs68 cells (MPD80) were transfected with control siRNA or ITSN2 siRNA and checked for the relative levels of the indicated mRNAs using quantitative real-time PCR. The values were normalized to actin. (F) Relative mRNA levels of representative E2F target genes in ING1a-expressing cells transfected with control siRNA or ITSN2 siRNA. The values are plotted after normalizing to actin (p<0.06).