Skip to main content
. 2019 May 13;20(6):e47283. doi: 10.15252/embr.201847283

Figure EV2. Protein phosphatase PP1α knockdown rescues the lamin aggregation phenotype in ELYS knockdown cells.

Figure EV2

  1. To confirm that lamin puncta were not an artifact of fixation, we performed live cell imaging. MCF‐10AT1k.cl2 cells were co‐transfected with control or ELYS siRNA and with plasmids expressing mCherry‐lamin B2 and H2B‐GFP. Representative images of live cells are shown. Also see Movies EV1, EV2 and EV3. Cytoplasmic lamin puncta were apparent in ELYS knockdown cells but not in control cells. In addition, time‐lapse imaging showed that cytoplasmic lamin aggregates exhibited dynamic movements, often appeared immediately after mitosis regardless of whether or not the mother cell contained lamin puncta, and coalesce or dissolve during interphase. In some extreme cases, cells contained abundant cytoplasmic lamin foci but no NE‐localized mCherry‐lamin B2.
  2. Because lamins are known to be regulated by phosphorylation 32, 125, 126, 127, we asked whether the appearance of cytoplasmic lamin aggregates might depend on the lamin phosphorylation state. To test this hypothesis, we knocked down two putative lamin protein phosphatases, PPP1CA (protein phosphatase 1 catalytic subunit alpha) and PPP2R4 (protein phosphatase 2A activator regulatory subunit 4) 65. MCF‐10AT1k.cl2 cells were transfected with control siRNA, ELYS siRNA, ELYS siRNA plus PPP1CA siRNA, or ELYS siRNA plus PPP2R4 siRNA and stained with antibodies against lamin B1 and B2. Representative images are shown.
  3. Cells with and without cytoplasmic lamin B aggregates were counted and the percentage of cells with aggregates was calculated. For each experiment, 28–120 cells were examined per condition. Two biological replicates, data from one representative experiment shown. While over 40% of ELYS knockdown cells exhibited cytoplasmic lamins, less than 10% of cells co‐transfected with siRNA against ELYS and PPP1CA showed cytoplasmic lamins. Knockdown of PPP2R4 had no impact on the appearance of cytoplasmic lamin accumulations.
  4. For each experiment, nuclear cross‐sectional areas were quantified for 70–184 nuclei per condition (117 nuclei on average) and averaged. Two biological replicates, data from one representative experiment shown. Dissolving cytoplasmic lamin aggregates through PPP1CA knockdown did not result in a significant increase in nuclear size in ELYS knockdown cells. Thus, cytoplasmic lamin aggregates formed in ELYS knockdown cells are not sufficient to cause reduced nuclear size.
Data information: Two‐tailed Student's t‐tests assuming equal variances: ***P < 0.005; NS, not significant. Scale bars are 10 μm. Error bars represent SD.