Fig. 1
Auxin treatment allows for B-type lamin degradation without affecting Lamin A localization. A Schematic illustration showing the AID system. Auxin treatment promotes the interaction between OsTIR1 and the degron tag (mAID), which is fused to the target protein. This results in rapid degradation of the target protein(s) upon proteasomal mediated poly-ubiquitination. B Schematic illustration of creating the cell lines. Each gene of interest was targeted for degradation by co-transfecting progenitor cells with the donor template plasmid with Cas9 and sgRNAs targeting the STOP codon of the sequence. C Immunostaining of AID-tagged lamin proteins in relation to LMNA. Green: LMNB1/B2-AID, LMNB1-AID, and LMNB2-AID. Red: LMNA. Blue: DAPI staining. The maximum intensity projections of nuclear Z stacks are shown. Scale bars = 10 μm. Data are representative of two independent biological replicates (N = 2). D Flow cytometric analysis to determine the optimal auxin concentration ([IAA]) for maximal degradation of LMNB1 and LMNB2 in fixed HCT116LMN(B1&B2)−AID cells. At least 20,000 events were recorded during the experiment. E Western blot analysis shows drastically reduced AID-tagged B-type lamins within 24 h of auxin (IAA) treatment. Doxycycline (DOX) was added 24 h prior to IAA to induce OsTIR1 expression. Tubulin was used as a loading control. Data are representative of three independent biological replicates (N = 3). F Flow cytometric analysis to determine the optimal auxin treatment time for maximal degradation of LMNB1 and LMNB2 in fixed HCT116LMN(B1&B2)−AID, HCT116LMN(B1)−AID, and HCT116LMN(B2)−AID cells. At least 20,000 events were recorded during the experiment