Fig. 7.

Ectopic expression of a TNFα receptor sensitizes H19-7 cells in N2 to TNFα. A, H19-7 cells were cultured as indicated and harvested in 2× buffer at the indicated times. The samples were electrophoresed and blotted as described in Materials and Methods and then probed for TNFR1 (55 kDa).B, TNFR1 or TNFR2 expression vector was transiently expressed in H19-7 cells along with a GFP vector. TNFα (10 ng/ml) was added to triplicate cultures 1 d after the switch to N2 medium, and the survival of transfected cells was determined 1 d later. These data are representative of three experiments for TNFR1 (p = 0.001) and four experiments for TNFR2 (p = 0.001). C, Populations of H19-7 cells stably expressing ectopic TNFR1 or TNFR2 or neither were shifted to N2 medium at 39°C, and some cultures were treated with TNFα (10 ng/ml) 1 d later. Höechst 33342 was added to the cultures, and triplicate counts of apoptotic cells were done 1 d after the addition of TNFα. These data are from one of two experiments for TNFR1 (with TNFα added, p = 0.025) and one of three experiments for TNFR2 (with TNFα added,p = 0.01).