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. 2015 Mar 30;208(7):961–974. doi: 10.1083/jcb.201410095

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© 2015 Howell et al.

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Figure 7. — WISp39 KD can be rescued by expression of Cofilin(S3A) and WT Coronin 1B. (A and B) Images of control and WISp39 KD cells. U2OS cells were transfected with control (A) or WISp39 (B) siRNA and with GFP plasmid to visualize transfected cells. 20× DIC images were taken at 48 h after transfection over a period of 16 h. A control cell (A) retains normal apolar/unipolar morphology throughout the experiment, unlike a WISp39 KD cell (B), which becomes elongated and multipolar. (C) Images of failure of Cofilin(S3A) to rescue WISp39 KD. U2OS cells were cotransfected with human WISp39 siRNA and GFP-Cofilin(S3A) for 48 h, and one cell was imaged as in A. WISp39 KD cell transfected with GFP-Cofilin(S3A) shows abnormal cell polarity similar to that of WISp39 KD cells. (D) Images of rescue of WISp39 KD by Cofilin(S3A) and Coronin 1B. U2OS cells were cotransfected with human WISp39 siRNA and GFP-Cofilin(S3A) and HA–Coronin 1B WT for 48 h, and 20× DIC images were taken. WISp39 KD cell transfected with GFP-Cofilin(S3A) and HA–Coronin 1B (Coro 1B) retains normal apolar/unipolar morphology throughout the experiment. (E) Quantitation of WISp39 KD rescue by Cofilin(S3A) alone or Cofilin(S3A) and Coronin 1B WT. U2OS cells were cotransfected for 48 h. Cells were scored as either apolar/unipolar or bipolar/multipolar and presented as a percentage of total cells scored. Number of rescued cells scored: control siRNA + GFP (22); WISp39 KD + GFP (21); WISp39KD + GFP-Cofilin(S3A) (25); WISp39KD + GFP-Cofilin(S3A) + HA–Coronin 1B WT (25). Note that only fluorescent cells were scored. Data represent the means ± SD. Student’s t test; **, P ≤ 0.01; ns, not significant. CTRL, control; post trans., posttransfection. Bars, 50 µm.