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. Author manuscript; available in PMC: 2013 Sep 1.

Published in final edited form as: FEBS J. 2012 Jul 16;279(17):3048–3070. doi: 10.1111/j.1742-4658.2012.08686.x

Fig. 8 — (A) LECs overexpressing Senp-1 displayed elevated expression of LEDGF mRNA than cells overexpressing Sumo1. Cells were transfected either with vector plasmid (open bar), pEGFP-Sumo1 (gray bar) or pFlag-Senp-1 (black bar). Total RNA extracted from transfectant was submitted to real-time PCR using primers specific to LEDGF. Values were normalized with β–actin and presented (* p<0.05; **p<0.001).

(B) Senp-1-dependent transactivation of deletion mutants of 5′-proximal regulatory region of LEDGF gene promoter. (a) Schematic representation of partial constructs of LEDGF gene promoter linked to CAT vector [61]. Various deletion mutants of LEDGF gene promoter linked to reporter plasmid CAT were engineered as described in the text [61]. Extracts isolated from cells cotransfected with deletion mutants, plasmid constructs and pFlag-Senp-1 or pFlag-Senp-1 mutant as indicated were tested for CAT activity. The transactivation activities of truncated constructs in the presence of pFlag-Senp-1 mutant (B, black bar) or pFlag-Senp-1 (B, gray bar) are shown. Transfection efficiencies were normalized using pSEAP basic vector. The data represent the means ± SD from three independent experiments (* p<0.05; **p<0.001).

(C) Senp-1 dramatically enhanced LEDGF transcription in concentration-dependent fashion. hLECs were co-transfected with LEDGF-CAT plasmid (−170/+35; based on results obtained from Experiment 8B) with increasing concentration of pFlag-Senp-1 (gray bars; 0.05, 0.15, 0.5, 2 and 2μg) or its mutant plasmids (black bar). After 72h of transfection, extracted cell lysates from these transfectants were analyzed for CAT activity. CAT activity is shown as a histogram (pFlag-Senp-1 mut, black bar; pFlag-Senp-1, gray bar) (**p<0.001).

(D) LEDGF transcription was downregulated in cells overexpressing Sumo-1 in comparison to Senp-1. hLECs were co-transfected with LEDGF-promoter linked to CAT vector (−170/+35) along with either pEGFP-Sumo1 or pFlag-Senp-1. Cell lysates isolated after 72h of transfection were examined. The effects of pEGFP-Sumo-1 (black bar) or pFlag-Senp-1 (light gray bar) on CAT activity are shown. Empty CAT-vector served as a control (open bar). The transfection efficiencies were normalized using pSEAP basic vector. The data represent the means ± SD from three independent experiments (**p<0.001).

Fig. 8 — (A) LECs overexpressing Senp-1 displayed elevated expression of LEDGF mRNA than cells overexpressing Sumo1. Cells were transfected either with vector plasmid (open bar), pEGFP-Sumo1 (gray bar) or pFlag-Senp-1 (black bar). Total RNA extracted from transfectant was submitted to real-time PCR using primers specific to LEDGF. Values were normalized with β–actin and presented (* p<0.05; **p<0.001).

(B) Senp-1-dependent transactivation of deletion mutants of 5′-proximal regulatory region of LEDGF gene promoter. (a) Schematic representation of partial constructs of LEDGF gene promoter linked to CAT vector [61]. Various deletion mutants of LEDGF gene promoter linked to reporter plasmid CAT were engineered as described in the text [61]. Extracts isolated from cells cotransfected with deletion mutants, plasmid constructs and pFlag-Senp-1 or pFlag-Senp-1 mutant as indicated were tested for CAT activity. The transactivation activities of truncated constructs in the presence of pFlag-Senp-1 mutant (B, black bar) or pFlag-Senp-1 (B, gray bar) are shown. Transfection efficiencies were normalized using pSEAP basic vector. The data represent the means ± SD from three independent experiments (* p<0.05; **p<0.001).

(C) Senp-1 dramatically enhanced LEDGF transcription in concentration-dependent fashion. hLECs were co-transfected with LEDGF-CAT plasmid (−170/+35; based on results obtained from Experiment 8B) with increasing concentration of pFlag-Senp-1 (gray bars; 0.05, 0.15, 0.5, 2 and 2μg) or its mutant plasmids (black bar). After 72h of transfection, extracted cell lysates from these transfectants were analyzed for CAT activity. CAT activity is shown as a histogram (pFlag-Senp-1 mut, black bar; pFlag-Senp-1, gray bar) (**p<0.001).

(D) LEDGF transcription was downregulated in cells overexpressing Sumo-1 in comparison to Senp-1. hLECs were co-transfected with LEDGF-promoter linked to CAT vector (−170/+35) along with either pEGFP-Sumo1 or pFlag-Senp-1. Cell lysates isolated after 72h of transfection were examined. The effects of pEGFP-Sumo-1 (black bar) or pFlag-Senp-1 (light gray bar) on CAT activity are shown. Empty CAT-vector served as a control (open bar). The transfection efficiencies were normalized using pSEAP basic vector. The data represent the means ± SD from three independent experiments (**p<0.001).