Figure 3.
Figure 3. Arpp19 deletion decreases MEF viability without affecting S phase progression. (A) Arpp19Lox/Lox MEFs were transduced with GFP- (GFP) or GFP-Cre–expressing (CRE) adenoviruses, and GFP-positive cells were sorted by FACS. ARPP19 expression was evaluated by immunoprecipitation (IP) using the anti-NterARPP19 antibody at day 3 after viral transduction. Band intensity was quantified from six independent experiments. (B) Clonogenic assays using critical dilutions of GFP or Cre MEFs. Arpp19Δ/Δ MEFs were transiently transfected with HA-ARPP19 or the HA-ARPP19 S62A mutant. Cell colonies were counted and plotted as percentages relative to GFP cells. Values are from three different experiments. Expression of HA-ARPP19 and HA-ARPP19 S62A was confirmed by Western blotting. Band intensities measured from three different experiments. (C) HeLa and U2OS cells were transfected with scramble (CT) or with two different siRNAs against ENSA (siE1 and siE2) and processed for Western blotting. (D) Gwl, Ensa, or Arpp19 floxed MEFs were transduced with the indicated adenoviruses and used for treslin analysis by Western blotting (n = 3, 6, and 3, respectively). (E) Arpp19 floxed MEFs were transduced with GFP-Cre or GFP-expressing adenoviruses, kept in G0 for 72 h, and then forced to reenter the cell cycle and blocked at the S-phase boundary by aphidicolin. After aphidicolin washout, MEFs were harvested and processed for FACS analysis. (F) MEFs in E were pulsed with EdU for 30 min and, at the indicated time points after aphidicolin release, were analyzed by immunostaining using anti-EdU antibodies and ImageJ software for quantification of the percentage of EdU-positive cells over time. (G) EdU-pulsed HeLa cells were fixed and counterstained with anti-ENSA or anti-ARPP19 (green) and anti-EdU (red) antibodies (1–2 mm confocal sections). Insets represent zooms of the boxed areas using ImageJ. Bar, 10 µm. Bar magnifications, 1 µm. All band intensities were quantified by densitometry using ImageJ and normalized to loading control. All data are means ± SD from the indicated number of experiments (n).

Arpp19 deletion decreases MEF viability without affecting S phase progression. (A) Arpp19 Lox/Lox MEFs were transduced with GFP- (GFP) or GFP-Cre–expressing (CRE) adenoviruses, and GFP-positive cells were sorted by FACS. ARPP19 expression was evaluated by immunoprecipitation (IP) using the anti-NterARPP19 antibody at day 3 after viral transduction. Band intensity was quantified from six independent experiments. (B) Clonogenic assays using critical dilutions of GFP or Cre MEFs. Arpp19Δ/Δ MEFs were transiently transfected with HA-ARPP19 or the HA-ARPP19 S62A mutant. Cell colonies were counted and plotted as percentages relative to GFP cells. Values are from three different experiments. Expression of HA-ARPP19 and HA-ARPP19 S62A was confirmed by Western blotting. Band intensities measured from three different experiments. (C) HeLa and U2OS cells were transfected with scramble (CT) or with two different siRNAs against ENSA (siE1 and siE2) and processed for Western blotting. (D) Gwl, Ensa, or Arpp19 floxed MEFs were transduced with the indicated adenoviruses and used for treslin analysis by Western blotting (n = 3, 6, and 3, respectively). (E) Arpp19 floxed MEFs were transduced with GFP-Cre or GFP-expressing adenoviruses, kept in G0 for 72 h, and then forced to reenter the cell cycle and blocked at the S-phase boundary by aphidicolin. After aphidicolin washout, MEFs were harvested and processed for FACS analysis. (F) MEFs in E were pulsed with EdU for 30 min and, at the indicated time points after aphidicolin release, were analyzed by immunostaining using anti-EdU antibodies and ImageJ software for quantification of the percentage of EdU-positive cells over time. (G) EdU-pulsed HeLa cells were fixed and counterstained with anti-ENSA or anti-ARPP19 (green) and anti-EdU (red) antibodies (1–2 mm confocal sections). Insets represent zooms of the boxed areas using ImageJ. Bar, 10 µm. Bar magnifications, 1 µm. All band intensities were quantified by densitometry using ImageJ and normalized to loading control. All data are means ± SD from the indicated number of experiments (n).

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