Figure 1
Figure 1. HHV-8–induced acute infection and PAX2 expression in HMECs. (A) Subconfluent HMEC monolayers were infected with HHV-8 and analyzed at different productively infected times for virus transcription and release. RT-PCR amplification was performed for the indicated HHV-8 genes using RNA extracted from infected cells. β-actin levels were determined in the same samples as control. Positive controls of amplification of the genes analyzed (C+) are shown. Virus release was evaluated in the culture supernatant by PCR amplification of HHV-8 ORF50 gene. β-actin gene was also amplified to verify the absence of cellular contaminating DNA. (B) Representative micrographs of the same microscopic field observed by phase contrast and by immunofluorescence showing that approximately 20% of the cells stained for the viral HHV-8 antigen ORF K8.1A. Micrographs were obtained by Zeiss Axioskop (Jena, Germany) using 20× objective. (C) Representative confocal micrographs costaining of PAX2 (green) and the viral HHV-8 antigen ORF K8.1A (red) in control uninfected dermal HMECs and renal-HMECs (r-HMECs) and, in HHV-8–infected HMECs, r-HMECs, HMEC-AS, and r-HMEC-AS. Uninfected HMECs and r-HMECs did not express PAX2, but 3 days after infection they showed nuclear staining for PAX2. HHV-8 infection failed to induce PAX2 expression in HMEC-AS and r-HMEC-AS (Zeiss LSM 5 Pascal confocal laser scanning microscope equipped with a helium/neon 543 mm laser, an argon 450 to 530 mm laser, and an EC planar NEOFluar 40×/1.3 oil DIC objective lens; acquisition software, Zeiss LSMS, version 3.2). (D) Representative RT-PCR showing the expression of PAX2 mRNA in cells derived from renal carcinoma used as positive control (lane 2), in HMECs infected with HHV-8 after 8 hours (lane 3), 16 hours (lane 4), 24 hours (lane 5), and 3 days (lane 6) and in HMEC-AS infected with HHV-8 after 8 hours (lane 7), 16 hours (lane 8), 24 hours (lane 9), and 3 days (lane 10). (E) Representative Western blot analysis of PAX2 protein expression by HMECs and HMEC-AS after 8 hours, 1, 3, 7, and 14 days after HHV-8 infection showing a band corresponding to 46 kDa. As positive control, lysates of cells derived from renal carcinoma (RCC) were used. Three individual experiments were performed with similar results.

HHV-8–induced acute infection and PAX2 expression in HMECs. (A) Subconfluent HMEC monolayers were infected with HHV-8 and analyzed at different productively infected times for virus transcription and release. RT-PCR amplification was performed for the indicated HHV-8 genes using RNA extracted from infected cells. β-actin levels were determined in the same samples as control. Positive controls of amplification of the genes analyzed (C+) are shown. Virus release was evaluated in the culture supernatant by PCR amplification of HHV-8 ORF50 gene. β-actin gene was also amplified to verify the absence of cellular contaminating DNA. (B) Representative micrographs of the same microscopic field observed by phase contrast and by immunofluorescence showing that approximately 20% of the cells stained for the viral HHV-8 antigen ORF K8.1A. Micrographs were obtained by Zeiss Axioskop (Jena, Germany) using 20× objective. (C) Representative confocal micrographs costaining of PAX2 (green) and the viral HHV-8 antigen ORF K8.1A (red) in control uninfected dermal HMECs and renal-HMECs (r-HMECs) and, in HHV-8–infected HMECs, r-HMECs, HMEC-AS, and r-HMEC-AS. Uninfected HMECs and r-HMECs did not express PAX2, but 3 days after infection they showed nuclear staining for PAX2. HHV-8 infection failed to induce PAX2 expression in HMEC-AS and r-HMEC-AS (Zeiss LSM 5 Pascal confocal laser scanning microscope equipped with a helium/neon 543 mm laser, an argon 450 to 530 mm laser, and an EC planar NEOFluar 40×/1.3 oil DIC objective lens; acquisition software, Zeiss LSMS, version 3.2). (D) Representative RT-PCR showing the expression of PAX2 mRNA in cells derived from renal carcinoma used as positive control (lane 2), in HMECs infected with HHV-8 after 8 hours (lane 3), 16 hours (lane 4), 24 hours (lane 5), and 3 days (lane 6) and in HMEC-AS infected with HHV-8 after 8 hours (lane 7), 16 hours (lane 8), 24 hours (lane 9), and 3 days (lane 10). (E) Representative Western blot analysis of PAX2 protein expression by HMECs and HMEC-AS after 8 hours, 1, 3, 7, and 14 days after HHV-8 infection showing a band corresponding to 46 kDa. As positive control, lysates of cells derived from renal carcinoma (RCC) were used. Three individual experiments were performed with similar results.

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