Figure 4. Oncogenic Kras activation triggers TSP-1 upregulation in a p53-dependent manner.

(A) TSP-1 protein expression in lungs harvested from p53^+/+, p53^+/–, and p53^–/– mice. Isolated lung tissue was probed for TSP-1 expression by immunoblotting. Tsp-1^–/– lung tissue served as negative control. TSP-1 expression relative to β-actin (loading control) was quantified by densitometry. (B) TSP-1 expression in the absence of p53 after Kras^G12D activation in primary lung fibroblasts. Kras^G12D×Tsp-1^+/+, Kras^G12D×p53^fl/fl, and Kras^G12D×Tsp-1^–/– lung fibroblasts were isolated, infected with Ad-Cre to activate oncogenic Kras and delete p53, harvested at the indicated time points, and probed for TSP-1 expression by immunoblotting. (C) Western blot of p53 and p21^cip1 expression in Kras^G12D×Tsp-1^+/+ and Kras^G12D×Tsp-1^–/– primary lung fibroblasts isolated at the indicated times after Kras^G12D activation. β-actin served as loading control. (D) Immunohistochemistry for p21^cip1 in Kras^G12D×Tsp-1^+/+ and Kras^G12D×Tsp-1^–/– lung sections isolated 21 weeks after Kras^G12D activation. Arrows indicate p21^cip1-positive cells. (E) pp53^Ser15 after oncogenic Hras activation. Tsp-1^+/+ and Tsp-1^–/– lung fibroblasts were infected with Hras^V12 retrovirus, harvested at the indicated days, and probed for pp53^Ser15, p53, p21^cip1, and β-actin by immunoblotting. Uninfected lung fibroblasts served as negative controls. pp53^Ser15 expression relative to total p53 was quantified by densitometry. (F) Nutlin-3 treatment restored Hras^V12-induced senescence in Tsp-1^–/– lung fibroblasts. Lung fibroblasts were infected with Hras^V12 or mock retrovirus, treated with 5 μM nutlin-3 or DMSO for 9 days, then stained for SA–β-gal to detect senescent cells. Scale bars: 50 μm (D); 200 μm (F).