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

Figure 3.

Figure 3.

WISp39 KD effect on motility is rescued by WT WISp39 and requires Hsp90. (A) Images of WISp39 KD rescue by WT WISp39. U2OS cells were cotransfected with human WISp39 siRNA and GFP-mouse WISp39 WT plasmid. 20× phase-contrast images were taken after 48 h of transfection. Top images show a cell transfected with rescue GFP plasmid (yellow arrows), whereas the neighboring cell is not. This cell (yellow arrows) retains normal apolar/unipolar morphology throughout the experiment unlike its neighbor (red arrows), which becomes elongated and multipolar. post trans., posttransfection. Bar, 10 µm. (B) Images of failure of WISp39 KD rescue by WISp39ΔTPR. U2OS cells were cotransfected with human WISp39 siRNA and mouse GFP-WISp39ΔTPR (which does not bind Hsp90) for 48 h and imaged as in A. The cell containing GFP-WISp39ΔTPR (top, red arrows) gradually loses its normal appearance and becomes elongated. Bar, 10 µm. (C) Quantitation of WISp39 KD rescue experiments. U2OS cells were cotransfected with human WISp39 siRNA and a plasmid expressing GFP-tagged mouse WISp39 WT or WISp39ΔTPR. In additional experiments, cells were transfected with WISp39 siRNA, HA-tagged mWISp39-WT or -ΔTPR, and GFP-H2B to mark transfected cells. Abnormal cell polarity of WISp39 KD cells is rescued by WT WISp39 but not by WISp39ΔTPR. Cells were scored as either apolar/unipolar or bipolar/multipolar and presented as a percentage of total cells scored from more than three independent experiments. Number of rescued cells scored: GFP-mouse WISp39 WT (30); GFP-mouse WISp39ΔTPR (75); HA-mouse WISp39 WT (172); HA-mouse WISp39ΔTPR (105). Note that only fluorescent cells were scored. (D) Migration assay of WISp39 KD rescue with WT WISp39 versus WISp39ΔTPR. Migration was initiated by wounding and the experiment was conducted as in Fig. 2 A. GFP-WISp39 WT rescues the WISp39 KD phenotype, whereas WISp39ΔTPR does not. The data shown are from a single representative experiment (n = 10), out of three independent experiments. (E and F) Quantitation of cell motility parameters in U2OS cells cotransfected with human WISp39 siRNA and either GFP-mouse WISp39 WT or GFP-mouse WISp39ΔTPR plasmid. Controls were transfected with control siRNA. Measurements of wound directionality and persistence were performed as described in Fig. 2. Number of control and WISp39 KD cells scored is 25 each from three independent experiments. Data shown in C, E, and F represent the means ± SD. ANOVA followed by Tukey’s test; ***, P ≤ 0.001. a.u., arbitrary unit; deg, degree.