Skip to main content
. 2018 Feb 28;19(5):e45144. doi: 10.15252/embr.201745144

Figure 1. CIP2A localizes at the transition zone and regulates ciliogenesis.

Figure 1

  1. Immunofluorescence images of endogenous CIP2A and primary cilia in RPE1 cells. Cells were fixed with methanol and stained with antibodies specific for CIP2A (red) and ARL13b or γ‐tubulin, CEP164, CEP290, and TMEM67 (green). DNA was stained with DAPI (blue). Shown are the maximum projections from z stacks; scale bar = 5 μm.
  2. Cells were serum‐starved (SS) for the indicated time, and serum was added for 1 or 2 h. The expression of CIP2A was analyzed by immunoblot and β‐actin served as the loading control.
  3. RPE1 cells were serum‐starved (SS) for 24 h, and serum was added for the indicated times. For endogenous co‐immunoprecipitation analysis, cell lysates were immunoprecipitated (IP) with anti‐CIP2A at each time point and IP proteins were analyzed by immunoblot.
  4. Immunofluorescence images of RPE1 cells transfected with GFP or GFP‐CIP2A following serum starvation for 48 h. Primary cilia were stained with antibodies specific for ARL13b (red). DNA was stained with DAPI (blue). Shown are the maximum projections from z stacks of representative GFP‐ or GFP‐CIP2A‐transfected cells. Scale bar = 10 μm. The percentage of GFP‐positive cells with primary cilia was determined (n > 50 cells/condition). The average of three independent experiments is shown, with error bars representing s.d. **P < 0.01 compared with GFP‐transfected cells (one‐tailed Student's t‐test).
  5. Cilium length of each cell was measured using ImageJ software. Mean of each cilium length from 13 cells for GFP‐transfected condition and 7 cells for GFP‐CIP2A transfected condition is presented with error bars representing s.d. as a graph made using GraphPad Prism software. Data points are derived from a single experiment. *P < 0.05 compared to GFP‐transfected cells (one‐way ANOVA).
  6. RPE1 cells stably expressing Smo‐EGFP (RPE1‐Smo‐EGFP) transiently transfected with control, CIP2A #1, CIP2A #2, or CIP2A #3 siRNA, were serum‐starved for 48 h, fixed, and stained with antibodies against γ‐tubulin (red) and DAPI (blue) for immunofluorescence analysis. Shown are the maximum projections from z stacks of representative cells for each condition. Scale bar = 10 μm. The siRNAs against CIP2A were validated by immunoblot.
  7. Smo‐EGFP fluorescence was used to measure cilium length. The average of measured cilium length is presented with error bars representing s.d. as a graph made using GraphPad Prism software (n > 86 cells per condition). Data points are derived from a single representative experiment. *P < 0.05, **P < 0.01, ***P < 0.0001 compared with siControl (one‐way ANOVA).
  8. RPE1 cells transfected with control, CIP2A #2, CIP2A #3, or NEK2 siRNA were cultured in each indicated condition. For growing conditions, cells were cultured in complete media; for serum starvation, cells were cultured in serum‐starved media for 48 h. Serum was added for 24 h after serum starvation. Cells were fixed and stained with antibodies against ARL13b. The percentage of cells with primary cilia was determined for each condition (n > 20 cells/condition). The average of three independent experiments is shown, with error bars representing s.d. *P < 0.05, **P < 0.01, ***P < 0.0001 compared to siControl cells of each condition (one‐way ANOVA). The knockdown of siRNAs against CIP2A and NEK2 was validated by immunoblot.

Source data are available online for this figure.