Journal: Wellcome Open Research

Article Title: Collagen fibril formation at the plasma membrane occurs independently from collagen secretion

doi: 10.12688/wellcomeopenres.23776.1

Figure Lengend Snippet: A ) Dendra2::Col1a2 NIH3T3 cells were imaged using Airyscan microscopy after 18 h of culture, 1000x magnification, and 2.5 × digital zoom; a maximum intensity projection is shown. Vesicles containing Dendra2 signals were observed at the sites of fibril assembly. The Dendra2 signal was observed at the periphery of the vesicles; the scale bar represents 1 µm. The area outlined by the box is enlarged in the right-hand panel. Small panels show looped structures containing Dendra2-positive collagen. B ) Individual frames taken from Extended data 14 demonstrate that a single fibripositor (highlighted in the red box) is in contact numerous times by both the endoplasmic reticulum and electron-dense vesicles. The Golgi apparatus did not contact fibripositor (Extended data 15). C ) Immunofluorescence imaging of type I collagen in mouse embryonic fibroblasts using the ER marker PDI. Scale bar represents 20 µm. D ) Fractionation of NIH3T3 cells using a sucrose density gradient, type I collagen is co-resident with the ER protein calreticulin (Calr), but also with the lysosomal protein Lamp1 and the collagen chaperone Hp47. E ) Proteomic analysis of lysosomal fractions 6 and 7 revealed significant enrichment of proteins identified by proteomic analysis of fractions 6 and 7 based on GO Cellular component terms. F ) Col1a1 (Red and Magenta) and Col1a2 (Blue and Green)-derived procollagen peptides were present in fractions 6 and 7, suggesting that the newly synthesized collagen transitioned to these compartments. G ) Dendra2::Col1a2 NIH3T3 cells were imaged by Airyscan microscopy 48 h after transfection with LAMP1-YFP. Images were recorded at 1000x magnification using a 2.5 × digital zoom. The maximum intensity projection of the 31 images is shown. LAMP1-YFP vesicles (green) containing photoswitched Dendra2 signals were observed. Three LAMP1-positive areas were also observed. H ) Live super-resolution microscopy of NIH3T3 cells stably transduced with Hsp47-BFP-RDEL lentivirus and transfected with LAMP1-YFP. Scale bar, 20 µm.

Article Snippet: Antibodies used in this study were collagen (Gentaur, OARA02579, dilution 1:2000 (WB), 1:500 (IF)), Dendra2 (Origene, TA180094, dilution 1:500), GAPDH (Sigma, G8795, dilution 1:10,000), Calreticulin (Stressgen; SPA-601, dilution 1:1000), Lamp1 (Santa Cruz, sc-20011, 1:500), vinculin (Chemicon; CBL233, 1:2000), Hp47 (Santa Cruz, sc-398579, 1:1000 (WB), 1:500 (IF)), and syntaxin 5 (Santa Cruz, sc-365124, 1:500(WB)) PDI (Abcam, ab180993, dilution 1:500 (IF)).

Techniques: Microscopy, Immunofluorescence, Imaging, Marker, Fractionation, Derivative Assay, Synthesized, Transfection, Super-Resolution Microscopy, Stable Transfection, Transduction

Journal: BMC Ophthalmology

Article Title: Role of Heat Shock Protein 47 in Transdifferentiation of Human Tenon's Fibroblasts to Myofibroblasts

doi: 10.1186/1471-2415-12-49

Figure Lengend Snippet: Primer sequence for real time RT-PCR

Article Snippet: Small interfering RNA (siRNA) molecules targeting Hsp47 mRNA were purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA, USA) and delivered into cells according to the manufacturer’s instructions.

Techniques: Sequencing

Journal: BMC Ophthalmology

Article Title: Role of Heat Shock Protein 47 in Transdifferentiation of Human Tenon's Fibroblasts to Myofibroblasts

doi: 10.1186/1471-2415-12-49

Figure Lengend Snippet: Quantitative real time RT-PCR for heat shock protein 47 (Hsp47) and α smooth muscle actin (αSMA) after exposure to transforming growth factor-β1 (TGF-β1) for up to 48 hours. The level of target mRNA was calculated using a relative ratio to β-actin of no treatment control and expressed as the mean ± S.E.M. (n = 16 for each group).

Article Snippet: Small interfering RNA (siRNA) molecules targeting Hsp47 mRNA were purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA, USA) and delivered into cells according to the manufacturer’s instructions.

Techniques: Quantitative RT-PCR, Control

Journal: BMC Ophthalmology

Article Title: Role of Heat Shock Protein 47 in Transdifferentiation of Human Tenon's Fibroblasts to Myofibroblasts

doi: 10.1186/1471-2415-12-49

Figure Lengend Snippet: Representative sequential bands of western immunoblots (A) and densitometric data (B) for heat shock protein 47 (Hsp47), α smooth muscle actin (αSMA), and β-actin. Human Tenon’s fibroblasts were exposed to transforming growth factor-β1 (TGF-β1) for up to 48 hours with or without siRNAs targeting Hsp47. The densitometric data are expressed as the mean ± S.E.M. (n = 16 for each group) *p < 0.001.

Article Snippet: Small interfering RNA (siRNA) molecules targeting Hsp47 mRNA were purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA, USA) and delivered into cells according to the manufacturer’s instructions.

Techniques: Western Blot