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. 2022 Oct 6;221(12):e202112001. doi: 10.1083/jcb.202112001

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© 2022 Filippone et al.

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Figure 2. — Symmetric partitioning of Numb phosphomutants. (A) Numb mutants. The relevant Ser residues (S7, S276, and S295) and the mutations (red) are indicated. (B) MaSLCs (PKH^pos cells, green) were transduced (all constructs fused to Flag-tag), stained with an anti-Numb Ab (red), and analyzed. Blue, DAPI. Bar, 10 μm. (C) Mammospheres (MS) transduced with the indicated constructs (DsRed fusion proteins) were dissociated and single cells were analyzed by time-lapse video microscopy. Epifluorescence (red). Bar, 10 μm. (D) Quantitation of the experiment in C, showing the frequency of symmetric (Sym) vs. asymmetric (Asym) partitioning. Significance was calculated vs. Numb-FL. (E) MS were transduced with Numb-DsRed (all panels) and then silenced as indicated (KD), or transduced with PKCζ (PKCζ-OE), or treated with BIS (3 μM). Cells were then analyzed as in C and D. P was calculated vs. matching control condition (Ctr-KD, EV or Ctr). When shown: N, number of doublets analyzed. Data are reported ± SD. Statistical analysis was with the nonparametric Fisher’s exact test.