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: Molecular Cancer Research

Article Title: Heat Shock Protein 47 Maintains Cancer Cell Growth by Inhibiting the Unfolded Protein Response Transducer IRE1α

doi: 10.1158/1541-7786.mcr-19-0673

Figure Lengend Snippet: Figure 1. Expression of HSP47 and collagens in human cancer cells. A, Expression of HSP47, collagen type 1a1, collagen type 1a2, collagen type 2a1, collagen type 3a1, collagen type 4a1, and collagen type 4a2 mRNAs in 13 human cancer cell lines, human hepatic stellate cell line LX-2, and NHDF. B, Expression of HSP47, collagen type 1 (Col1), and collagen type 4 (Col4) proteins in the 13 human cancer cell lines, LX-2 cells, and NHDF.

Article Snippet: Establishment of cells reconstituting HSP47 expression HSP47 knockout (KO) cancer cells were transfected with the pCMV6-HSP47 plasmid (Origene).

Techniques: Expressing

Journal: Molecular Cancer Research

Article Title: Heat Shock Protein 47 Maintains Cancer Cell Growth by Inhibiting the Unfolded Protein Response Transducer IRE1α

doi: 10.1158/1541-7786.mcr-19-0673

Figure Lengend Snippet: Figure 2. Silencing of HSP47 induces impaired cancer cell growth. A, Expression of HSP47 protein in cancer cells (SW480 and HCT116 cells) at day 3 after transfection with siControl and three batches of siHSP47. B, Proliferation of cancer cells after transfection with siControl and siHSP47 (siHSP47-A, siHSP47-B, siHSP47-C). C, Expression of HSP47 protein in HSP47 KO cancer cells [clone 1 (c1) and c2], HSP47 KO cells with reconstituted HSP47 (reHSP47; c1 and c2). D, Proliferation of HSP47 KO cancer cells (c1 and c2), HSP47 KO cancer cells with reHSP47 (c1 and c2). E, Tumor growth of cancer cells (mock, n ¼ 6), HSP47 KO cancer cells (c1 and c2, n ¼ 6, respectively), HSP47 KO cancer cells with reHSP47 (c1 and c2, n ¼ 6, respectively; , P < 0.05; n.s., not significant).

Article Snippet: Establishment of cells reconstituting HSP47 expression HSP47 knockout (KO) cancer cells were transfected with the pCMV6-HSP47 plasmid (Origene).

Techniques: Expressing, Transfection

Journal: Molecular Cancer Research

Article Title: Heat Shock Protein 47 Maintains Cancer Cell Growth by Inhibiting the Unfolded Protein Response Transducer IRE1α

doi: 10.1158/1541-7786.mcr-19-0673

Figure Lengend Snippet: Figure 3. Silencing of HSP47 stimulates the UPR in cancer cells. A, Transmission electron microscopy in cancer cells at 48 hours after treatment with siControl (SW480, n ¼ 25; HCT116, n ¼ 28) and siHSP47 (SW480, n ¼ 31; HCT116, n ¼ 29). As a control, cancer cells were treated with tunicamycin (Tm). Red lines indicate the dilated ER. N, nucleus; scale bars, 2 mm. Quantification of area (pixels/ER) was determined by NIH ImageJ software. B, Expression of unspliced Xbp-1 (Xbp1u) mRNA and spliced Xbp1 (Xbp1s) mRNA in cancer cells treated with or not treated with siControl or siHSP47. C, Expression of mRNAs of UPR-associated genes (ERdj4, p58IPK, EDEM, and PDIA6) in cancer cells at 48 hours after transfection with siControl and siHSP47. D, Expression of ERdj4, EDEM, p58IPK, and PDIA6 proteins in cancer cells at day 2 after transfection with siControl or siHSP47. E, Activation status of IRE1a signaling pathways in cancer cells at 48 hours after transfection with siControl and siHSP47. F, Activation of the PERK pathway and cleavage of ATF6a in cancer cells at 96 hours after transfection with siControl and siHSP47. G, Activation status of IRE1a and PERK and cleavageof ATF6a in HSP47 KO cancer cells (c1 and c2), HSP47 KO cancer cellswith reconstitutedHSP47 (reHSP47, c1, and c2). H, Activation status of IRE1a and PERK and cleavage of ATF6a in NHDF at 48 hours (for RE1a) and at 96 hours (for PERK and ATF6a) after transfection with siControl and siHSP47 (, P < 0.05; n.s., not significant).

Article Snippet: Establishment of cells reconstituting HSP47 expression HSP47 knockout (KO) cancer cells were transfected with the pCMV6-HSP47 plasmid (Origene).

Techniques: Transmission Assay, Electron Microscopy, Control, Software, Expressing, Transfection, Activation Assay, Protein-Protein interactions

Journal: Molecular Cancer Research

Article Title: Heat Shock Protein 47 Maintains Cancer Cell Growth by Inhibiting the Unfolded Protein Response Transducer IRE1α

doi: 10.1158/1541-7786.mcr-19-0673

Figure Lengend Snippet: Figure 4. HSP47 forms a complex with IRE1a and BiP in cancer cells. A, Immunoprecipitation of HSP47 in cancer cells (SW480 and HCT116 cells) not treated or treated with siControl andsiHSP47 was carried out, and then immunoblotting againstHSP47, IRE1a,PERK, and ATF6awas performed. B, Interaction of IRE1a with HSP47 in cancer cells at 72 hours after transfection with siControl or siHSP47 was determined by immunoprecipitation (IP), and phosphorylated IRE1a (p-IRE1a), IRE1a, HSP47, and BiP were detected by immunoblotting. C, Complex formation of HSP47 with IRE1a and BiP in SW480 cells was determined by native-PAGE and immunoblotting (IB). Arrowheads indicate a complex of HSP47 with IRE1a and BiP.

Article Snippet: Establishment of cells reconstituting HSP47 expression HSP47 knockout (KO) cancer cells were transfected with the pCMV6-HSP47 plasmid (Origene).

Techniques: Immunoprecipitation, Western Blot, Transfection, Clear Native PAGE

Journal: Molecular Cancer Research

Article Title: Heat Shock Protein 47 Maintains Cancer Cell Growth by Inhibiting the Unfolded Protein Response Transducer IRE1α

doi: 10.1158/1541-7786.mcr-19-0673

Figure Lengend Snippet: Figure 5. Silencing of HSP47 induces activation of the IRE1a/Xbp1 axis followed by activation of PERK and ATF6a. A, Temporal changes in activation status of the three branches of the UPR in cancer cells (SW480 cells) at each indicated time point after transfection (TF) with siControl and siHSP47. B, Immunoblot of IRE1a protein in shControl SW480 cells (c1 and c2) and shIRE1a SW480 cells (c1, c2, and c3). C, Expression of Xbp1u and Xbp1s mRNAs in shControl SW480 cells (c1 and c2) and shXbp1 SW480 cells (c1 and c2) treated with tunicamycin (Tm, 250 ng/mL). D, Activation status of the UPR in shControl SW480 cells, shIRE1a SW480 cells and shXbp1 SW480 cells at 96 hours after transfection with siControl and siHSP47. E, Phosphorylation level of PERK in shControl SW480 cells, shIRE1a SW480 cells, and shXbp1 SW480 cells at 96 hours after transfection with siControl and siHSP47 were determined by calculating the ratio of phosphorylated PERK to total PERK. F, Immunoblots of HSP47, Xbp1s, phosphorylated PERK (p-PERK), PERK, and cleaved ATF6a in SW480 cells treated with 4m8 (10 mmol/L) at day 2 (for Xbp1s) and at day 4 after transfection with siControl and siHSP47. Proliferation of shControl SW480 cells (siControl and siHSP47), shIRE1a SW480 cells (siControl and siHSP47; G) and shXbp1 SW480 cells (siControl and siHSP47; H) after transfection with siRNA (, P < 0.05; n.s., not significant).

Article Snippet: Establishment of cells reconstituting HSP47 expression HSP47 knockout (KO) cancer cells were transfected with the pCMV6-HSP47 plasmid (Origene).

Techniques: Activation Assay, Transfection, Western Blot, Expressing, Phospho-proteomics

Journal: Molecular Cancer Research

Article Title: Heat Shock Protein 47 Maintains Cancer Cell Growth by Inhibiting the Unfolded Protein Response Transducer IRE1α

doi: 10.1158/1541-7786.mcr-19-0673

Figure Lengend Snippet: Figure 6. Activation of the IRE1a/Xbp1 axis by silencing of HSP47 induces increment of ROS in cancer cells. A, Levels of ROS in cancer cells (SW480 and HCT116 cells) treated with siControl or siHSP47 was determined by calculating the fluorescent intensity of CM-H2DCFDA per cell. Representative images and quantification were shown in left panel and right panel, respectively. B, Levels of ROS in cancer cells not treated (siControl and siHSP47) or treated with cycloheximide (CHX; 10 mmol/L; siControl and siHSP47). C, Levels of ROS in shControl cancer cells (siControl and siHSP47), shIRE1a cancer cells (siControl and siHSP47), and shXbp1 cancer cells (siControl and siHSP47). D, Levels of ROS in cancer cells not treated or treated with NAC (50 mmol/L) at 48 hours after transfection with siControl and siHSP47. E, Phosphorylation of PERK and cleavage of ATF6a in SW480 cells treated with or without NAC at 48 hours (for p-IRE1a, IRE1a, and Xbp1s) and at 96 hours after transfection with siControl and siHSP47. F, Immunoblots of HSP47, phosphorylated PERK (p-PERK), PERK, and cleaved ATF6a in SW480 cells treated with cycloheximide at day 4 after transfection with siControl and siHSP47 (, P < 0.05).

Article Snippet: Establishment of cells reconstituting HSP47 expression HSP47 knockout (KO) cancer cells were transfected with the pCMV6-HSP47 plasmid (Origene).

Techniques: Activation Assay, Transfection, Phospho-proteomics, Western Blot

Journal: Molecular Cancer Research

Article Title: Heat Shock Protein 47 Maintains Cancer Cell Growth by Inhibiting the Unfolded Protein Response Transducer IRE1α

doi: 10.1158/1541-7786.mcr-19-0673

Figure Lengend Snippet: Figure 7. Silencing of HSP47 stimulates the PERK and ATF6a axes via increment of ROS-mediated 4-HNE–protein adducts. A, Accumulation of intracellular lipid in cancer cells (SW480 and HCT116 cells) after transfection with siControl and siHSP47. B, Content of 4-HNE–protein adducts in cancer cells not treated or treated with NAC (siControl and siHSP47). C, Dotblot of 4-HNE–protein adducts in SW480 cells not treated or treated with hydralazine after transfection with siControl and siHSP47. D, Content of 4-HNE–protein adducts in SW480 cells not treated or treated with hydralazine (siControl and siHSP47). E, Phosphorylation of PERK and cleavage of ATF6a in SW480 cells not treated or treated with hydralazine at 48 hours (for p-IRE1a, IRE1a, and Xbp1s) and at 96 hours after transfection with siControl and siHSP47 (, P < 0.05; n.s., not significant).

Article Snippet: Establishment of cells reconstituting HSP47 expression HSP47 knockout (KO) cancer cells were transfected with the pCMV6-HSP47 plasmid (Origene).

Techniques: Transfection, Phospho-proteomics