Comparative infectivity of primary vascular endothelial cells and 293 cells as determined by RNA blot hybridization.

(A) At top, detection of T1.1 RNA by Northern blot hybridization of 15 μg total RNA from adult MVDECs (bMVDECs), HUVECs, and 293 cells infected at the same time and under identical conditions. Transcripts were analyzed on day 0 before infection and on days 4 and 8 after infection. BCBL-1 RNA was analyzed as a control. The position of the major T1.1 transcript is indicated on the right. The position of 18S RNA (2.37 kb) is indicated on the left. At bottom, the blot was reprobed to detect cellular GAPDH RNA. In bottom inset, T1.1 transcripts were detected by RT-PCR in 0.5 μg total RNA from 293L cells and fMVDECs that were infected with HHV-8 at the same time and under identical conditions. RNAs were analyzed on day 0 (D0) prior to infection and variably on days 1 through 7 (D1-D7) after infection as separately indicated for the various cells. BCBL-1 RNA was analyzed as a control (right lane). Primers are described in “Materials and methods.” Amplification products were detected by DNA blot hybridization with the use of probes as described in “Materials and methods.” The position of the 394-bp T1.1 amplification product is indicated on the right. (B) At top, detection of T1.1 RNA by Northern blot hybridization of total RNA from fMVDECs and mMVECs that were infected at the same time and under the identical conditions described in panel A. RNA loading was not equal (fMVDECs = 1.5 μg; mMVECs = 15 μg). However, comparison with GAPDH permits assessment of the relative amount of T1.1 RNA in the various cells. (C) At top, detection of T1.1 RNA by Northern blot hybridization of total RNA from 2 independent lots of fMVDECs (shown on the left and right with a lane between the 2 sets of experiments) that were infected after the same passage and under identical conditions with GAPDH purified HHV-8 (virus preparation 2 [VP2] and VP3) undiluted and diluted 1:5. RNA loading was not equal (left lot = 10 μg; right lot = 5 μg). BCBL-1 RNA was analyzed as a control. The position of the major T1.1 transcript is indicated on the right. The position of 18S RNA is indicated on the left. At bottom, the blot was reprobed to detect cellular GAPDH RNA.