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. 2022 May 5;221(7):e202008116. doi: 10.1083/jcb.202008116

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© 2022 Huang and Winklbauer

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Figure 4. — Diffuse Pk1 and Dvl2 control cortical F-actin levels. (A) Single PCM cells stained with phalloidin Alexa Fluor 488, pseudo-colored using MPL-Inferno to show intensity spectrum. Pk1-MO 40 ng/bl; Dvl2-MO 24 ng/bl; TBB 10 μM; Pk1-OE 500 pg/bl. Scale bars, 10 μm. (B) Differently treated cells were stained simultaneously in a single dish to minimize unspecific intensity differences. (C–F) Cortical F-actin intensity in phalloidin-stained cells. Average intensity was calculated from pooled measurements of WT cells, and treated cells were all normalized to this average to give the percentage intensity. n, number of cells. (C) Relative staining intensity of cells with low (L, 13 ng/bl, n = 42), medium (M, 27 ng/bl, n = 49), and high (H, 40 ng/bl, n = 139) doses of Pk1-MO and rescue with Pk1-venus-RNA (500 pg/bl) in high Pk1-MO background (n = 109). WT, n = 406. ****, P ≤ 0.0001 in a two-tailed Student’s t test. Data distribution was assumed to be normal but was not formally tested. (D) Relative staining intensity of Dvl2 knockdown cells with low (L, 12 ng/bl, n = 47), medium (M, 24 ng/bl, n = 17), and high (H, 36 ng/bl, n = 48) doses of Dvl2-MO. ****, P ≤ 0.0001 in a two-tailed Student’s t test. Data distribution was assumed to be normal but was not formally tested. (E) Relative cortex intensity of Pk1 and Dvl2 double-knockdown cells with combinations of low (L), medium (M), and high (H) morpholino doses, and of cells treated with TBB (10 μM) in a WT, Pk1-MO, or Dvl2-MO background. H Pk1-MO and M Dvl2-MO from C and D are included for comparison. H Pk1-MO + L Dvl2-MO, n = 27; H Pk1-MO + M Dvl2-MO, n = 23; H Pk1-MO + H Dvl2-MO, n = 27; TBB, n = 100; H Pk1-MO + TBB, n = 33; M Dvl2-MO + TBB, n = 65. *, P ≤ 0.05; ***, P ≤ 0.001; ****, P ≤ 0.0001 in a two-tailed Student’s t test. Data distribution was assumed to be normal but was not formally tested. (F) Relative cortex intensity of Pk1 overexpressing (Pk1-OE 500 pg/bl, n = 43) and TBB (10 μM)-treated Pk1-OE cells (n = 27). WT, n = 60. ****, P ≤ 0.0001 in a two-tailed Student’s t test. Data distribution was assumed to be normal but was not formally tested.