Journal: Cancer biology & therapy

Article Title: RIalpha influences cellular proliferation in cancer cells by transporting RFC40 into the nucleus.

doi: 10.4161/cbt.4.4.1621

Figure Lengend Snippet: Figure 1. RIα and RFC40 are localized in the nucleus as well as the cytoplasm of asynchronous MCF7 cells. MCF7 cells (1.2 x 105) were incubated with polyclonal anti-RIα antibody (A) and polyclonal anti-RFC40 antibody (B), respectively, followed by staining with FITC-labeled anti-rabbit secondary antibody. Cells were then visualized by immunofluorescence microscopy (Nikon Microphot FXA, objective Fluor 60X). (C) Nuclear and cytosolic extracts were prepared from asynchronous MCF7 cells as described in materials and methods. Blots in the top panels were immunoblotted with monoclonal anti-RIα antibody (left) and polyclonal anti-RFC40 antibody (right) respectively. Blots in the bottom panels were immunoblotted with monoclonal anti-histone H1 antibody (left) and polyclonal anti-lactate dehydrogenase antibody (right) as positive controls for nuclear and cytosolic extracts, respectively. Input lanes in all four panels contained 37 µg protein.

Article Snippet: Goat polyclonal anti-lactate dehydrogenase antibody was obtained from Novus Biologicals, Inc. (CO, USA).

Techniques: Incubation, Staining, Labeling, Immunofluorescence, Microscopy

Journal: Cancer biology & therapy

Article Title: RIalpha influences cellular proliferation in cancer cells by transporting RFC40 into the nucleus.

doi: 10.4161/cbt.4.4.1621

Figure Lengend Snippet: Figure 3. RIα and RFC40 are colocalized in the nucleus and the cytoplasm of asynchronous MCF7 cells. (A) Nuclear (left panel) and cytosolic (right panel) extracts were prepared from asynchronous MCF7 cells as described in materials and methods and immunoprecipitated with preimmune serum (Pre) and polyclonal anti-RFC40 antibody (IP). Western blot analyses were carried out using monoclonal anti-RIα antibody. (B) MCF7 cells (2 x 104) were incubated with mouse monoclonal anti-RIα antibody (red) and rabbit polyclonal anti-RFC40 antibody (green), followed by staining with Cy5- labeled anti-mouse secondary antibody (red) and FITC-labeled anti-rabbit antibody (green). DNA was counterstained with DAPI (blue) and the DNA distribution of cells was then measured by LSC (CompuCyte; 40X). The DNA histogram (inset) shows the distribution of MCF7 cells in the G1, S and G2/M phases, respectively. The localization of RIα (red) and RFC40 (green) was examined in cells corresponding to their positions in different cell cycle phases as established by the DNA histogram. (B and C) show two repre- sentative staining patterns obtained for cells in G1; (D) cells in S phase; (E) cells in G2/M. The right hand panels represent the merged images of RIα (red) and RFC40 (green) in G1, S and G2/M phases.

Article Snippet: Goat polyclonal anti-lactate dehydrogenase antibody was obtained from Novus Biologicals, Inc. (CO, USA).

Techniques: Immunoprecipitation, Western Blot, Incubation, Staining, Labeling

Journal: Cancer biology & therapy

Article Title: RIalpha influences cellular proliferation in cancer cells by transporting RFC40 into the nucleus.

doi: 10.4161/cbt.4.4.1621

Figure Lengend Snippet: Figure 2. The levels of RIα and RFC40 shift significantly from the cytoplasm to the nucleus of MCF7 cells as they progress from G1 to S phase. MCF7 cells (1.2 x 105) were incubated with polyclonal anti-RIα antibody and polyclonal anti-RFC40 antibody, respectively, followed by staining with FITC-labeled anti-rabbit secondary antibody. DNA was counterstained with PI/RNase A and visualized by laser scanning cytometry (CompuCyte; 20X). The instrument allows thresholds to be set separating nuclear from cyto- plasmic fluorescence in individual cells. The graphs represents the statistical analyses of the mean cytoplasmic and nuclear RIα (n = 15; A) and RFC40 (n = 16; B) fluorescence in G1 and S phase, respectively. Arrows indicate the expected 50% increase in RIα and RFC40 protein levels as the cells progress from G1 to S phase, if the increase were due solely to the increase in cell volume. The cytoplasmic and nuclear RIα and RFC40 fluorescence are reported as mean ± SEM. Statistical analysis was performed using the student t-test and P<0.05 was considered significant. The DNA histograms (inset) show the distribution of MCF7 cells in the G1, S and G2/M phases respectively.

Article Snippet: Goat polyclonal anti-lactate dehydrogenase antibody was obtained from Novus Biologicals, Inc. (CO, USA).

Techniques: Incubation, Staining, Labeling, Cytometry, Fluorescence

Journal: Cancer biology & therapy

Article Title: RIalpha influences cellular proliferation in cancer cells by transporting RFC40 into the nucleus.

doi: 10.4161/cbt.4.4.1621

Figure Lengend Snippet: Figure 4. RIα has a nonconventional nuclear localization sequence at residues 91–98. 293 cells (1 x 106) were transiently transfected with 1–2.5 µg of GFP vector alone (A; top panel), GFP-RIα-wt (RFC40+/NLS+, A; bottom panel), GFP-RIα-dmt1 (RFC40-/NLS+, (B) GFP-RIα-dmt2 (RFC40-/NLS-, C) and GFP-RIα-SDM (RFC40+/NLSM, D) for 48 hrs and subcellular localization of these constructs were examined by immunofluorescence microscopy (Nikon Microphot FXA, objective fluor 40X). Figure 5. RIα transports RFC40 into the nucleus. 293 cells (1 x 106) were transiently transfected with 1–2.5 µg of GFP vector alone (A, left panel), GFP-RIα-wt (RFC40+/NLS+, B; left panel), GFP- RIα-dmt1 (RFC40-/NLS+, C; left panel), GFP-RIα-dmt2 (RFC40-/NLS-, D; left panel) and GFP-RIα-SDM (RFC40+/NLSM, E; left panel) for 48 hrs. Cells were stained for endogenous RFC40 using polyclonal anti-RFC40 antibody and Rhodamine Red X conju- gated anti-rabbit secondary antibody (A–E; center panels) and visualized by immunofluorescence microscopy (Nikon Microphot FXA, objective fluor 60X). The combined images on the right were separated by filters into green (GFP constructs of RIα) and red (RFC40) images.

Article Snippet: Goat polyclonal anti-lactate dehydrogenase antibody was obtained from Novus Biologicals, Inc. (CO, USA).

Techniques: Sequencing, Transfection, Plasmid Preparation, Construct, Immunofluorescence, Microscopy, Staining

Journal: Cancer biology & therapy

Article Title: RIalpha influences cellular proliferation in cancer cells by transporting RFC40 into the nucleus.

doi: 10.4161/cbt.4.4.1621

Figure Lengend Snippet: Figure 7. Downregulation of endogenous RIα reduces nuclear localization of RFC40 and influ- ences the cell cycle under physiological conditions. MCF7 cells (2 x 104) were grown on coverslips in 6-well plates for 24 hr. After 24 hrs incubation, cells were treated with 0, 25, 50 and 100 µM of 8-Cl-cAMP, for 48 hrs. Cells were fixed with 1% formaldehyde for 15 min at 4˚C and incubated overnight at -20˚C in 70% ethanol for permeabilization. Cells were then incubated with 1:100 dilution of polyclonal anti-RFC40 antibody for 2 hr at room temperature, followed by staining with FITC-labeled anti-rabbit secondary antibody for 1hr at room temperature. DNA was counter- stained with PI/RNase A and visualized by laser scanning cytometry. The instrument allows thresholds to be set separating nuclear from cytoplasmic fluorescence in individual cells. Graphs represent (A) mean nuclear and cytoplasmic RFC40 fluorescence, (B) nuclear to cytoplasmic ratio of RFC40 fluorescence (n = 4; p < 0.05), (C) the percentage of S phase cells and (D) the percentage of G1 phase cells respectively, when treated with 0, 25, 50 and 100 µM of 8-Cl-cAMP (n = 4). The nuclear to cytoplasmic ratio of RFC40 levels, the percentages of S phase and G1 phase cells are reported as mean ± SEM. Statistical analysis for each graph was performed using the student t-test and P < 0.05 was considered significant.

Article Snippet: Goat polyclonal anti-lactate dehydrogenase antibody was obtained from Novus Biologicals, Inc. (CO, USA).

Techniques: Incubation, Staining, Labeling, Cytometry, Fluorescence