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. 2020 Jan 17;9:e53402. doi: 10.7554/eLife.53402

Figure 5. Kif2C mediates DNA double strand break mobility and foci dynamics.

(A) Examples of 10 min mobility traces of EGFP-53BP1 foci in WT and Kif2c knockout (KO) U2OS cells after etoposide (20 μM) treatment. Kif2C depletion did not impact 53BP1 expression (Figure 5—figure supplement 1A). (B) Mean-square displacement measurements of EGFP-53BP1 foci in WT and Kif2C KO U2OS cells, shown in black in red, respectively. (C) Mean-square displacement measurements of EGFP-53BP1 foci in WT U2OS cells treated with the vehicle control (DMSO), Taxol (5 μM), or DHTP (20 μM), as indicated. (D) Quantification of the distance travelled by EGFP-53BP1 foci over 10 min in the corresponding cells described in B-C. (E) Examples of disappearance (yellow arrowheads) and fusion (red circle) events of EGFP-53BP1 foci induced by etoposide in U2OS cells. (F, G) Number of EGFP-53BP1 foci in WT or Kif2C KO U2OS cells, treated with the vehicle control (DMSO), Taxol, or DHTP. These inhibitors were added either 5 min before (F) or 5 min after (G) etoposide treatment. (H–J) Numbers of fusion (H) and disappearance (I–J) events of EGFP-53BP1 foci in the corresponding cells in panel G are shown. A total of 15 randomly selected cells were analyzed over three independent experimental runs. For disappearance events, number of occurrence in the first 30 min under each treatment condition is shown in (I) and the time required for foci disappearance (min) over the entire hour of recording is shown in (J) (>150 events quantified per condition). The box represents 50% of the foci disappearance events and the line shows the median of the data set. All microscopy image acquisitions began five minutes after final compound treatment, either every 30 s for 10 min (A–D) or every 3 min for one hour (H–J). All data were collected from at least three independent experimental sets. Error bars, S.D.; ns: p>0.05; *p≤0.05; **p≤0.01; ***p≤0.001, by Student’s t-test.

Figure 5.

Figure 5—figure supplement 1. Kif2C depletion did not affect the expression of GFP-53BP1 (A).

Figure 5—figure supplement 1.

Kif2C depletion increased g-H2AX induced by etoposide (B).
Figure 5—figure supplement 2. Kif2C depletion did not influence the general nuclear dynamics.

Figure 5—figure supplement 2.

(A) Representative examples of 10 min mobility traces of EGFP-53BP1, mCherry-CENP-B and EGFP-PRPF6 in U2OS cells. The tracked traces were overlaid over the corresponding images (upper panel). The lower panel shows the same traces aligned with a common starting point (indicated by circles). Note that the randomness of 53BP1 traces and the coordinated movement of the CENP-B and PRPF6 foci, representing general nuclear dynamics. (B) Quantification of the distance travelled by EGFP-53BP1, mCherry-CENP-B and EGFP-PRPF6 foci over 10 min in WT-U2OS and KIF2C-KO-U2OS cells. 53BP1 mobility was tracked 5 min after etoposide treatment, while the mobility of nuclear CENP-B and PRPF6 was tracked either with or without the addition of etoposide. (C) Examples of the intra-nuclear foci localization of CENP-B and PRPF6.
Figure 5—figure supplement 3. Taxol, but not DHTP, reduced the general mobility of CENP-B and PRPF6.

Figure 5—figure supplement 3.

Quantification of the distance travelled by nuclear envelop-associated mCherry-CENP-B foci (A), intra-nuclear mCherry-CENP-B foci (B), or intra-nuclear PRPF6 foci (C) over 10 min in U2OS cells without or with etoposide treatment, in the presence or absence of DHTP (20 μM) or taxol (5 μM).