Figure 4

Expression of MCP-1 in Li Fraumeni fibroblasts and after p53 knockdown in A172 cells. (A) RT-PCR of MCP-1 and GAPDH in Li Fraumeni cells (MDAH041) in passage 8 (p:8, p53 mut/wt) and passage 160 (p:160, p53 mut/mut), respectively. Cells were treated with 250 U/ml of TNF-α for 6 h (+). Untreated control cells: (-). (B) Western blot analysis of p53 in MDAH041 cells (p:8) and (p:160) treated with TNF-α (250 U/ml) (+) for 6 h. (-): untreated control. Cytosolic extracts (50 μg per lane) were separated in a 12% SDS-PAGE. Actin confirms equal loading. (C) A172 cells were transiently transfected with pSUPER-p53 or with the empty pSUPER vector. After 24 h, cells were stimulated with TNF-α for additional 5 h. Cells were harvested and Western blot analysis was performed. Filters were probed with anti-p53 (DO-1) (see also panel B). (D) 4 μg of total RNA were separated in a 1% agarose gel and transferred to a Gene screen Plus membrane. The filter was subsequently hybridized with a MCP-1 cDNA probe. Hybridization of the same filter with a cDNA probe coding for the housekeeping gene β-actin confirmed equal loading. The positions of the 28S and 18S ribosomal RNA are indicated.