ALMS1-Deficient Fibroblasts Over-Express Extra-Cellular Matrix Components, Display Cell Cycle Delay and Are Resistant to Apoptosis
Figure 5
ALMS fibroblasts are resistant to cell death induced by apoptotic stimuli.
(a) Fibroblasts of healthy controls and ALMS patients were stimulated with thapsigargin (THAP, 100 nM for 48 hours), C2-ceramide (C2-C, 100 µM for 24 hours), cycloheximide (CX, 100 µg/ml for 24 hours), staurosporine (STP, 100 nM for 48 hours) and TNF-α (100 ng/ml for 48 hours) in 10% FBS SM. The % of viable cells was determined by MTT assay and shown as mean values ± SEM with respect to unstimulated cells (−) indicated as 100%. *P<0.05, ***P<0.001 cell viability of ALMS fibroblasts versus controls. (b) Control (C1) and ALMS fibroblast (PT4) were treated with THAP (100 nM for 48 hours), stained with PI and analyzed by flow cytometry. Results are presented as histograms of cell cycle phase distribution and the reported % represents the increase in sub-G1/G0 population (M1 region). (c) Control (C1) and ALMS fibroblast (PT1) were grown on glass coverslips and stimulated with CX (100 µg/ml for 24 hours) and THAP (100 nM for 48 hours). Cells were fixed and stained for fluorescence in situ DNA end-labelling (TUNEL) (green stain); nuclei were counter-stained with DAPI (blue stain) (magnification = 20×). (d) Control (C1) and ALMS fibroblast (PT1) were treated with THAP (100 nM for 48 and 72 hours). Cells were labelled by TUNEL and PI and analyzed by flow cytometry. Each cytogram shows the TUNEL positivity (ordinate) with respect to DNA content (abscissa), upon 48 and 72 hours of THAP-treatment, respectively. A representative control and patient are reported in panel b, c and d; see also Figure S9 and S10.