Journal: Disease Models & Mechanisms

Article Title: Cellular stress due to impairment of collagen prolyl hydroxylation complex is rescued by the chaperone 4-phenylbutyrate

doi: 10.1242/dmm.038521

Figure Lengend Snippet: Loss of mutant CRTAP , P3H1 and CyPB in OI patient fibroblasts. (A) Quantitation of CRTAP , P3H1 and PPIB expression evaluated by qPCR. Mutations in CRTAP , P3H1 and PPIB caused a close to complete absence of the mutated transcripts in CRTAP-1, CRTAP-2 and P3H1-2 patients, and a reduced mRNA level in P3H1-1 and P3H1-3. * P <0.05. WT values are represented as black dots; CRTAP as gray dots; P3H1 as white dots; CyPB as dark gray dots. (B) Amplification of the exon 6-exon 8 region of P3H1 transcript generated the expected 217 bp amplicon in control cells (WT), whereas, in the P3H1-2 patient, the presence of a higher molecular weight (∼400 bp) band compatible with intronic retention was detected. C-, RT-PCR negative control. (C) Representative western blot to evaluate the expression of CRTAP, P3H1 and CyPB in control (WT) and mutant cell lysate fractions (CRTAP-1, CRTAP-2, CRTAP-3, P3H1-1, P3H1-2, P3H1-3, CyPB). Loss of the mutated protein in patient's cells was demonstrated. Patients with mutations in CRTAP showed also no P3H1 expression and patients with mutations in P3H1 showed no CRTAP expression, as a consequence of their mutual protection in the complex.

Article Snippet: The membranes were then blocked with 5% (w/v) BSA in 20 mM Tris-HCl, 500 mM NaCl, pH 7.5 (TBS), 0.05% (v/v) Tween-20 (Sigma-Aldrich) (TBS-T) at room temperature (RT) for 1 h. After washing with TBS-T, the membranes were incubated with 1:1000 primary antibody against the specific proteins CRTAP (generously provided by Dr Lee Brendan, Baylor College of Medicine,TX, USA), P3H1 (NovusBio), CyPB (Proteintech), BIP (Cell Signaling), PERK (Cell Signaling), PDI (Cell Signaling), p-PERK (Thr980; Cell Signaling), LC3A/B (Cell Signaling), cleaved caspase-3 (Cell Signaling), ATF4 (Novus Biological), ATF6 (Abcam) in 5% BSA in TBS-T overnight at 4°C.

Techniques: Mutagenesis, Quantitation Assay, Expressing, Amplification, Generated, Control, Molecular Weight, Reverse Transcription Polymerase Chain Reaction, Negative Control, Western Blot

Journal: Disease Models & Mechanisms

Article Title: Cellular stress due to impairment of collagen prolyl hydroxylation complex is rescued by the chaperone 4-phenylbutyrate

doi: 10.1242/dmm.038521

Figure Lengend Snippet: Mutations in the collagen prolyl-3-hydroxylation complex lead to collagen overmodifications and collagen intracellular retention. (A) Representative SDS-urea-PAGE fluorographies of 3 H-labeled collagen extracted from the cell layer and medium of control (WT) and patient (CRTAP-1, CRTAP-2, CRTAP-3, P3H1-1, P3H1-2, P3H1-3, CyPB) fibroblasts. In mutant samples, broader and slower α(I) bands demonstrated the overglycosylation of type I collagen. (B) The percentage of intracellular collagen retention was evaluated as a ratio between the CPM in the cell layer and in medium plus cell layer. Collagen molecules in mutant cells were more intracellularly retained compared to WT. * P <0.05. WT values are represented as black dots; CRTAP as gray dots; P3H1 as white dots; CyPB as dark gray dots.

Article Snippet: The membranes were then blocked with 5% (w/v) BSA in 20 mM Tris-HCl, 500 mM NaCl, pH 7.5 (TBS), 0.05% (v/v) Tween-20 (Sigma-Aldrich) (TBS-T) at room temperature (RT) for 1 h. After washing with TBS-T, the membranes were incubated with 1:1000 primary antibody against the specific proteins CRTAP (generously provided by Dr Lee Brendan, Baylor College of Medicine,TX, USA), P3H1 (NovusBio), CyPB (Proteintech), BIP (Cell Signaling), PERK (Cell Signaling), PDI (Cell Signaling), p-PERK (Thr980; Cell Signaling), LC3A/B (Cell Signaling), cleaved caspase-3 (Cell Signaling), ATF4 (Novus Biological), ATF6 (Abcam) in 5% BSA in TBS-T overnight at 4°C.

Techniques: Labeling, Control, Mutagenesis

Journal: Disease Models & Mechanisms

Article Title: Cellular stress due to impairment of collagen prolyl hydroxylation complex is rescued by the chaperone 4-phenylbutyrate

doi: 10.1242/dmm.038521

Figure Lengend Snippet: 4-PBA ameliorates recessive OI fibroblast homeostasis. (A) Representative western blots (top) and dot plot of the quantitative analysis (bottom) of p-PERK and cleaved caspase 3 (CASP3) in the absence (−) or presence (+) of 4-PBA incubation in control (WT) cells and in cells with mutations in CRTAP, P3H1 or CyPB. The levels of these proteins were compared in treated versus untreated cells and in treated OI cells versus untreated controls. β-actin was used for normalization. * P <0.05 mutant fibroblasts with respect to control fibroblasts. # P <0.05 treated mutant fibroblasts with respect to untreated mutant fibroblasts. § P <0.05 treated mutant fibroblasts with respect to untreated control fibroblasts. p-PERK:PERK and cleaved caspase 3 levels were decreased to or even less than control values in all cases, with the exception of P3H1-2. WT untreated values are shown as black dots, mutants untreated as white dots and mutants treated as gray dots. (B) Transmission electron microscopy representative images of OI patient fibroblasts in the absence (−) or presence (+) of 4-PBA. The analyses revealed ER enlargement (*) and cellular vacuolization (arrow) in mutant cells with the exception of P3H1-2. 4-PBA treatment reduced the ER cisternae enlargement. Magnification 20,000×. (C) Representative western blot of CRTAP and P3H1 in control (WT) and CRTAP-3 cell lysates, and of CyPB in WT and CyPB lysates. No protein rescue was detected after the treatment in the two mutant cell lines, in which normal transcript level was detected. β-actin was used for normalization. Scale bar: 2 µm.

Article Snippet: The membranes were then blocked with 5% (w/v) BSA in 20 mM Tris-HCl, 500 mM NaCl, pH 7.5 (TBS), 0.05% (v/v) Tween-20 (Sigma-Aldrich) (TBS-T) at room temperature (RT) for 1 h. After washing with TBS-T, the membranes were incubated with 1:1000 primary antibody against the specific proteins CRTAP (generously provided by Dr Lee Brendan, Baylor College of Medicine,TX, USA), P3H1 (NovusBio), CyPB (Proteintech), BIP (Cell Signaling), PERK (Cell Signaling), PDI (Cell Signaling), p-PERK (Thr980; Cell Signaling), LC3A/B (Cell Signaling), cleaved caspase-3 (Cell Signaling), ATF4 (Novus Biological), ATF6 (Abcam) in 5% BSA in TBS-T overnight at 4°C.

Techniques: Western Blot, Incubation, Control, Mutagenesis, Transmission Assay, Electron Microscopy

Journal: Disease Models & Mechanisms

Article Title: Cellular stress due to impairment of collagen prolyl hydroxylation complex is rescued by the chaperone 4-phenylbutyrate

doi: 10.1242/dmm.038521

Figure Lengend Snippet: 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+).

Article Snippet: The membranes were then blocked with 5% (w/v) BSA in 20 mM Tris-HCl, 500 mM NaCl, pH 7.5 (TBS), 0.05% (v/v) Tween-20 (Sigma-Aldrich) (TBS-T) at room temperature (RT) for 1 h. After washing with TBS-T, the membranes were incubated with 1:1000 primary antibody against the specific proteins CRTAP (generously provided by Dr Lee Brendan, Baylor College of Medicine,TX, USA), P3H1 (NovusBio), CyPB (Proteintech), BIP (Cell Signaling), PERK (Cell Signaling), PDI (Cell Signaling), p-PERK (Thr980; Cell Signaling), LC3A/B (Cell Signaling), cleaved caspase-3 (Cell Signaling), ATF4 (Novus Biological), ATF6 (Abcam) in 5% BSA in TBS-T overnight at 4°C.

Techniques: Activation Assay, Western Blot, Expressing, Marker, Quantitation Assay, Staining, Control, Reverse Transcription Polymerase Chain Reaction, Amplification, Positive Control

Journal: Disease Models & Mechanisms

Article Title: Cellular stress due to impairment of collagen prolyl hydroxylation complex is rescued by the chaperone 4-phenylbutyrate

doi: 10.1242/dmm.038521

Figure Lengend Snippet: Recessive OI cells react to cellular stress by activating autophagy. (A) Representative western blot (left) and dot plot of the quantitative analysis (right) of the terminal autophagic marker LC3 in control (WT) and in cells with mutations in CRTAP, P3H1 or CyPB. LC3-II is upregulated in all cases except in patient P3H1-2. β-actin was used for normalization. (B) Representative LC3 western blot (left) performed on cell lysates obtained following chloroquine incubation from WT and mutant samples, and dot plot of the quantitative analysis (right). The terminal marker of autophagy evaluated in dynamic conditions is increased in CRTAP-2, CRTAP-3, P3H1-2 and CyPB. β-actin was used for normalization. (C) Representative LC3 immunofluorescence images of WT and mutant fibroblasts treated with chloroquine. Quantitation of the total area of punctate signal per cell confirms the activation of autophagy. DAPI (nuclei) in blue and LC3 in green. Magnification 40×, zoom 4×. WT values are represented as black dots; CRTAP as gray dots; P3H1 as white dots; CyPB as dark gray dots. * P <0.05. Scale bar: 40 µm.

Article Snippet: The membranes were then blocked with 5% (w/v) BSA in 20 mM Tris-HCl, 500 mM NaCl, pH 7.5 (TBS), 0.05% (v/v) Tween-20 (Sigma-Aldrich) (TBS-T) at room temperature (RT) for 1 h. After washing with TBS-T, the membranes were incubated with 1:1000 primary antibody against the specific proteins CRTAP (generously provided by Dr Lee Brendan, Baylor College of Medicine,TX, USA), P3H1 (NovusBio), CyPB (Proteintech), BIP (Cell Signaling), PERK (Cell Signaling), PDI (Cell Signaling), p-PERK (Thr980; Cell Signaling), LC3A/B (Cell Signaling), cleaved caspase-3 (Cell Signaling), ATF4 (Novus Biological), ATF6 (Abcam) in 5% BSA in TBS-T overnight at 4°C.

Techniques: Western Blot, Marker, Control, Incubation, Mutagenesis, Immunofluorescence, Quantitation Assay, Activation Assay

Journal: PLoS ONE

Article Title: A Novel Mutation in LEPRE1 That Eliminates Only the KDEL ER- Retrieval Sequence Causes Non-Lethal Osteogenesis Imperfecta

doi: 10.1371/journal.pone.0036809

Figure Lengend Snippet: A: A partial sequence of PCR product of Patient II-3 is shown. Compound heterozygous frame shift mutations (c.484delG, p.A162LfsX22 and c.2155dupC, p.E719RfsX11) are indicated by arrows. The mutations have been confirmed by the subsequent sequencing of subcloned products of normal and mutant alleles. B: Schematic presentation of the positions of the mutation. LEPRE1 cDNA encodes the tetratricopeptide repeat domain (four black regions), the Prolyl/Lysyl/hydroxylase domain (green region), and the KDEL ER- retrieval motif (red region). LEPRE1 with a p.E719RfsX11 change results in the lack of only the KDEL ER-retrieval sequence, whereas other functional domains remain intact.

Article Snippet: Cells were then blocked in 10% goat serum and incubated with primary antibody (LEPRE1 MaxPab, Abnova, Taipei, Taiwan) for 2.5 h. Secondary staining and imaging was done as above.

Techniques: Sequencing, Mutagenesis, Functional Assay

Journal: PLoS ONE

Article Title: A Novel Mutation in LEPRE1 That Eliminates Only the KDEL ER- Retrieval Sequence Causes Non-Lethal Osteogenesis Imperfecta

doi: 10.1371/journal.pone.0036809

Figure Lengend Snippet: A: Patient II-3 LEPRE1 transcripts are about one-half the control level, by real-time RT-PCR. B: Steady-state type I collagen protein from fibroblasts of Patient II-3 and a normal control is shown. In both the cell layer and media, overmodification, detected as back-streaking of collagen alpha chains (α1 (I) and α2 (I)) on gel electrophoresis, is present in Patient II-3. We also detected mild overmodification of type V collagen (α1 (V)). C: Western blots of fibroblast P3H1 in Patient II-3 and control cells confirm absence of intracellular P3H1. D: Immunofluorescent staining of fibroblasts from Patient II-3 and a normal control show colocalization of P3H1 and CRTAP with GRP94 in control cells. Both P3H1 and CRTAP proteins are absent in fibroblasts from Patient II-3.

Article Snippet: Cells were then blocked in 10% goat serum and incubated with primary antibody (LEPRE1 MaxPab, Abnova, Taipei, Taiwan) for 2.5 h. Secondary staining and imaging was done as above.

Techniques: Control, Quantitative RT-PCR, Nucleic Acid Electrophoresis, Western Blot, Staining