Figures & data
Figure 1. Time course effect of HIV-1 on renal proximal tubular epithelial cell injury. Equal numbers of HIV-1-, vector alone,- or mock-transduced tubular cells were incubated in media for variable time periods (12, 24, 36, 48, 60, and 72 hrs). Subsequently, cellular injury was assessed by Trypan blue exclusion technique. Results (means ± SD) are from four series of experiments each carried out in triplicate. *p < 0.001 compared with respective mock- and vector- transduced cells.
Figure 2. Tubular cell expression of HIV-1 promotes apoptosis. Equal numbers of tubular cells were transduced with either HIV-1 or vector alone. After 24, 48, and 72 hours of transduction, cells were stained with annexin-V and then studied for apoptosis by flow cytometry. Vector and HIV-1 expressing HK-2 cells were gated for GFP positive cells. (A) Annexin V-stained cells; marker M1 indicates apoptosed cells. (B) Cumulative data showing percentage of apoptosed cells. Results (means ± SD) are from three sets of experiments each carried out in triplicate. *p < 0.001 compared with respective vectors; **p < 0.05 compared with respective vectors.
Figure 3. Effect of HIV-1 expression on cell death. Equal number of cells (2.5 × 104 cells/well) were seeded in a 96-well plate and incubated overnight at 37°C. Cells were transduced with either HIV-1 or vector alone. Cell death ELISA was carried out at 48 and 72 h post-transduction in quadruplicate wells. The results (mean ± SD) are from three sets of experiments plotted for each group of cells. *p < 0.001 compared with respective vector controls.
Figure 4. Effect of HIV-1 expression on tubular cell apoptosis. Equal number of HK-2 cells were grown on poly-L lysine coated glass coverslips. After incubation of 48 and 72 hours, cells were assayed for apoptosis by TUNEL assay. The upper panel shows representative micrographs of vector-transduced cells at (A) 48 and (B) 72 hours of incubation. The lower panel shows representative micrographs of HIV-1 transduced cells at (C) 48 and (D) 72 hours of incubation (×200). Apoptosis cells with dark brown nuclei are indicated by arrows. Cumulative data of apoptosed cells in vector- and HIV-1- transduced cells are shown in the bar diagram. Results (means ± SD) are from four sets of experiments each carried out in triplicate. *p < 0.001 compared with respective vector; **p < 0.001 compared with respective vector and HIV (48 h).
Figure 5. Tubular cell HIV-1 expression induces cell cycle arrest in G2/M phase. Equal numbers of cells were transduced with either HIV or vector alone. After 24, 48, and 72 hours of transduction, cells were stained with propidium iodide and examined for cell cycle stage by flow cytometry. Vector and HIV-1 expressing HK-2 cells were gated for GFP positive cells.(A) Histograms showing cell cycle stage of GFP positive cells. (B) Cumulative data showing percentage of cells in G2/M phase. Results (means ± SD) are from three sets of experiments each carried out in triplicate. *p < 0.001 compared with respective vector- and HIV-1-transduced cells at 24 and 48h.
Figure 6. Effect of HIV-1 expression on tubular cell cycle/checkpoint signaling pathways. Equal numbers of HK-2 cells were transduced with either HIV-1 or vector alone, followed by incubation for 72 hours. Subsequently, cells were harvested, proteins were extracted, and Western blots were prepared and probed for phospho-P53 (Ser 15), phospho-cdc-2 (Tyr 15), and phospho-chk-2 (Thr 68). Three sets of experiments were carried out. (A) The upper panel shows a representative gel of tubular cell expression of phospho-P53 (Ser 15) under HIV-1 and vector transduction states. The middle panel shows tubular cell content of actin under similar conditions. The lower panel shows cumulative data on densitometric analysis of the ratio of phospho-P53 and actin. *p < 0.01 compared with vector. (B) The upper panel shows a representative gel of tubular cell expression of phospho-Chk2 (Thr 68) under HIV-1 and vector transduction states. The middle panel shows tubular cell content of actin under similar conditions. The lower panel shows cumulative data on densitometric analysis of the ratio of phospho-Chk2 and actin. *p < 0.05 compared with vector. (C) The upper panel shows a representative gel of tubular cell expression of phospho-cdc-2 (Tyr 15) under HIV-1 and vector transduction states. The middle panel shows tubular cell content of actin under similar conditions. The lower panel shows cumulative data on densitometric analysis of the ratio of phospho-cdc-2 and actin. *p < 0.05 compared with vector.
