Figure 1. Expression pattern of LEDGF/p75 in LECs from human eye lenses of different ages was associated with Sp1 expression.

A and B, mRNA expression levels of LEDGF/p75 (black bars) and Sp1 (gray bars) were analyzed by real time PCR. Total RNA was isolated from LECs separated from lenses of human subjects of different age groups and reverse transcribed cDNA was subjected to real time PCR analysis with specific primers as detailed in Materials and Methods. Age group 1 (n = 4, 16–26 years); Age group 2 (n = 3, 34–42 years); Age group 3 (n = 7, 52–75 years). n; denotes number of subjects. The data represent the mean ± S.D. from three independent experiments (** p<0.001). C, Western analysis of LEDGF/p75 and Sp1 protein using their corresponding specific antibodies. hLECs isolated from eye lenses of 24- and 64-year-old human subjects were cultured as described in Materials and Methods. Cellular proteins from confluent cells were extracted, and equivalent amounts were loaded onto SDS-PAGE, transferred to a PVDF membrane and processed for immunoblotting. Western analysis showed the expression levels of LEDGF/p75 (upper panel) and Sp1 (middle panel). Lower panel, membrane probed with β-actin antibody as loading/internal control. The same membrane was probed and reprobed with antibodies following stripping and restriping to obtain relative expression of Sp1, LEDGF/p75 or β-actin. Each band of blot was quantified using densitometer shown at the right. Images are representatives from three independent experiments. D and E, Sp1 upregulated expression of LEDGF/p75 protein and mRNA in hLECs in dose dependent fashion. hLECs were transfected with either pCMV-vector or increasing amounts of pCMV-Sp1 (2, 4 and 8 µg) as indicated and described in Materials and Methods section. Total Protein and RNA were extracted after 48 h of transfection and were used for Western analysis (D) and real time PCR (E) respectively, using specific probes. D, left, Western analysis data showing the expression levels of LEDGF/p75 (upper panel) in cells transfected with plasmid encoding Sp1 at different concentrations (middle panel). Lower panel, membrane probed with β-actin antibody. The same membrane was probed and reprobed with antibodies following stripping and restriping to obtain relative expression of Sp1, LEDGF/p75 or β-actin. Right, Histogram displaying relative protein band density indicated as values ± S.D. of three independent experiments. E, Histogram showing the values (mean ± S.D.) of Sp1 concentration-dependent expression of LEDGF/p75 mRNA (black bars vs gray bars) obtained from three independent experiments (**p<0.001). F, A Sp1 inhibitor, artemisinin, reduced expression of LEDGF/p75 in LECs in dose-dependent manner. Cultured cells were treated with either increasing concentrations of artemisinin (50, 150 and 300 µM) or with vehicle control. Cell lysates were resolved onto SDS-PAGE and analyzed by Western blot for the effects of artemisinin on expression of LEDGF/p75 and Sp1 protein. Relative band density in pixels is shown below the Western blot images (*p<0.01, **p<.001). β-actin was used as internal control. G and H, Representative immunoblots showing depletion of Sp1 using Sp1 Knockdown assay. Sp1-specific shRNA constructs were transiently (G) and stably (H) transfected as described in Materials and Methods section. Protein lysate was prepared and Western analysis was carried out. The same membrane was probed and reprobed with antibodies following stripping and restriping to obtain relative expression of Sp1 or LEDGF/p75 or β-actin. Relative band density in pixels is shown below the Western blot images (**p<.001).