Figure 4
From: Fanconi anemia proteins counteract the implementation of the oncogene-induced senescence program
FANCD2 depletion increased cathepsin L1 expression during OIS, a protease targeting FANCD2 downstream oncogene induction. (A,B) Representative Western blots showing cathepsin L (CTSL1) behavior in IMR90* (A) or WI38* (B) cells depleted or not depleted of FANCD2. The three forms of CTSL1 could be observed. ERK1, actin or vinculin were used as loading control. (C,D) Histograms representing the time-dependent relative levels of total and mature CTSL1 in oncogene-active IMR90* (C) or WI38* (D) cells expressing or not expressing FANCD2. Bars indicate the mean +/− SEM of at least 3 independent experiments. *p < 0.05; **p < 0.01. (E,F) Representative Western blot analysis of cathepsin L (CTSL1) and FANCD2 in IMR90* (E) or WI38* (F) cells depleted or not depleted for FANCD2. Cells were exposed to CTSL1 inhibitor (CTSLi, Z-FF-FMK, dose 2.5 µM) during the OIS kinetic. The three forms of CTSL1 could be observed. ERK1, actin and vinculin were used as loading controls. (G,H) Analysis of CTSL1 expression and activation (revealed by its processing) in IMR90* or WI38* cells depleted or not depleted of FANCD2 in absence or presence of a CTSL1 inhibitor. The data were shown as a fold change compared with the value obtained for siLacZ transfected cells. Bars represent the mean +/− SEM of at least 3 independent experiments. *p < 0.05; **p < 0.01. (I) Analysis of SAHF-positive cells in IMR90* or WI38* cells depleted or not depleted of FANCD2 in the absence or presence of a CTSL1 inhibitor. The data were shown as a fold change compared with the value obtained for siLacZ transfected cells. Bars represent the mean +/− SEM of at least 3 independent experiments. *p < 0.05. (J) Quantitative analysis of FANCD2 expression in IMR90* cells in the absence or presence of a CTSL1 inhibitor. Bars represent the mean +/− SEM of at least 3 independent experiments.
