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. 2010 Feb 12;5(2):e9188. doi: 10.1371/journal.pone.0009188

Figure 4. mRNA and intracellular protein expression and identification of MMP, CXCL and CCL loci as SASP components.

Figure 4

A) IL-6 mRNA and intracellular protein in PRE and SEN mouse and human cells. IL-6 mRNA was quantified by RT-PCR (TaqMan). Intracellular protein was detected by immunofluorescence (green). Nuclei were stained with DAPI (blue). B) Intracellular IGFBP-6 protein was detected by immunofluorescence (green) of DAPI-stained (blue) cultures. C) mRNA levels of other senescence-associated genes (COX-2, TIMP-1, PAI-1 and VEGF) in mouse and human cells. mRNA was quantified by RT-PCR using TaqMan. D) Matrix metalloproteinases (MMP) are mouse and human SASP factors. Shown is the organization of the mouse (left) and human (right) MMP gene clusters. mRNA was isolated from mouse and human cells cultured as indicated: PRE cells in 3% (x) or 20% (gray dot) O2 (black line); SEN(XRA) cultured in 3% O2 (red line); SEN(OXI) mouse cells made senescent by passage in 20% O2 (green line); SEN(REP) human cells made senescent by passage in 20% O2 (green line). Abundance of the indicated MMP mRNAs was quantified by RT-PCR. PRE and SEN(XRA) mouse cells were immunostained for MMP-3 (bottom left). Conditioned media (CM) were assayed for MMP3 activity (zymography, bottom right) and protein level (western blotting, bottom right). E) Expression of Cxcl and Ccl gene clusters in PRE, SEN(XRA) and SEN(OXI) mouse cells. The genes are listed vertically in 5′ (top) →3′ order. Antibody arrays results are shown to the right. Bottom panels show immunostaining for intracellular Ccl2 and Ccl12.