Figure 8. Characterization of the WT and R202A mutant hSLIP1 proteins in vivo.

(A) The subcellular localization of endogenous hSLIP1, and exogenous wild type Flag-tagged hSLIP1 and Flag-tagged R202A hSLIP1 mutant in Hela cells was visualized in 0.5 micron optical sections using a CCD microscope and deconvolution system as described in supplementary methods. Hela cells were transiently transfected with flag-tagged wild type or mutant hSLIP1 as indicated. The cells were grown on coverslips, permeabilized, and pulse-labeled with EdU to stain for S-phase cells and labeled with DAPI for nuclear imaging. The hSLIP1 is shown in red, the DAPI blue, and the EdU is labeled in green. The first panel shows two cells in late S-phase with endogenous hSLIP1 present predominantly on the endoplasmic reticulum. Diffuse staining indicates that some hSLIP1 is also present in the nucleus and in the cytoplasm. The second panel shows cells transiently transfected with wild type hSLIP1. The cell is in mid-S phase and shows mostly nuclear and some cytoplasmic staining. The third panel shows the localization of R202A hSLIP1. The DAPI is in blue, the α-Flag hSLIP1 in red. The individual staining patterns are also shown in Supplementary Figure 6. In (B), the protein expression levels of transiently transfected Flag-tagged wild type hSLIP1 and Flag-tagged R202A hSLIP1 used for the CCD-microscopy and qPCR experiments is shown. The cells were harvested 24 hrs after transfection and probed by western blotting using an α-Flag antibody. (C) Real-time qPCR of histone genes in Hela cells was performed to compare mRNA levels in cells transiently transfected with either wild type hSLIP1 or the R202A hSLIP1. The average fold change determined from the Ct values using the comparative Ct method is depicted with the standard errors derived from three independent data sets. Only one band was observed for all genes analyzed and the primers were specific for the target genes being tested. The data was normalized to GAPDH. (D). hSLIP1 overexpression results in increased accumulation of cells in G₁ and reduced accumulation in S phase. Cell cycle profiles of Hela cells transiently transfected with either wild type or R202A hSLIP1. Cells were pulse labeled with propidium iodide and the DNA content was analyzed by flow cytometry and the cell cycle distribution was determined using the ModFit software.