Fig. 3.

Activation of apoptosis and the UPR in recessive OI patient fibroblasts. (A) Representative western blot (left) to evaluate the expression of cleaved caspase 3 (CASP3), a terminal marker for apoptosis and the dot plot of the quantitation analysis (right). β-actin was used for normalization. (B) Quantitative analysis of the fraction of apoptotic events in the cell lines following FACS analysis upon cells staining with annexin V (FITC) and propidium iodide (PI). Apoptosis is activated in all tested OI patients' cells. (C) Representative western blots (left) and dot plots of the quantitative analysis (right and bottom) of the collagen chaperone PDI and of proteins involved in the UPR (BIP, PERK, p-PERK, ATF4, ATF6) in control (WT) cells and in cells with mutations in CRTAP, P3H1 or CyPB. The PERK branch of the UPR was upregulated in all patients' fibroblasts with the exception of patient P3H1-2. β-actin was used for normalization. WT values are represented as black dots; CRTAP as gray dots; P3H1 as white dots; CyPB as dark gray dots. *P<0.05. (D) RT-PCR amplification of XBP1 mRNA from control (WT) and patient cells. The spliced XBP1-1s form of XBP1 transcript (XBP-1u) is not detectable in patient cells. Fibroblasts treated with thapsigargin were used as positive control (C+).