Figure 1.

Regulation of 53BP1 protein levels by CTSL. (A) WT and Lmna^−/− MEFs were treated with cycloheximide (CHX) to inhibit protein synthesis and with either a proteasome inhibitor (MG-132) or a CTSL inhibitor (Z-FY-CHO). Global levels of 53BP1 were monitored by western blot. β-Tubulin was used as loading control. Note the stabilization of 53BP1 by both treatments. (B) Western blots showing higher levels of active CTSL and lower levels of 53BP1 in Lmna^−/− MEFs with respect to WT. Graph showing CTSL enzymatic activity in WT and Lmna^−/− MEFs. (C) Acute depletion of A-type lamins by lentiviral transduction with an shRNA followed by western blots to monitor the levels of A-type lamins, 53BP1, and CTSL. Graph showing CTSL enzymatic activity upon depletion of A-type lamins. Note how acute depletion of A-type lamins leads to increased levels of CTSL activity and decreased levels of 53BP1. (D) Levels of CTSL mRNA detected by northern blot. Average of three independent experiments is shown. (E) Western blots showing that acute depletion of CTSL by lentiviral transduction with an shRNA leads to stabilization of 53BP1 protein in Lmna^−/− MEFs. Graph shows the decrease in CTSL enzymatic activity. (F) WT and Lmna^−/− MEFs transduced with an shRNA control and Lmna^−/− MEFs depleted of CTSL were incubated with cycloheximide and either proteasome or CSTL inhibitors. The levels of 53BP1 were monitored by western blots. Graphs show the quantitation of two independent experiments. Note how depletion of CTSL prevents stabilization of 53BP1 by the CTSL inhibitor, but not by the proteasome inhibitor. Values in bar graphs are expressed as mean±s.e.m. In ‘bee swarm’ plots, horizontal bar indicates the average value. N represents the number of independent experiments. ^*P-value of statistical significance (P⩽0.05). RFU stands for relative fluorescence units and RU stands for relative units (normalization to control).