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: eLife

Article Title: Hsp47 promotes biogenesis of multi-subunit neuroreceptors in the endoplasmic reticulum

doi: 10.7554/eLife.84798

Figure Lengend Snippet: ( A ) Endogenous interactions between GABA A receptor α1 subunits and Hsp47. Mouse brain homogenates from 8 to 10 weeks C57BL/6 J mice were immunoprecipitated with an anti-α1 antibody, and the immunoisolated eluents were blotted with indicated antibodies. IgG was included as a negative control for non-specific binding. Three biological replicates were performed. ( B ) Recombinant Hsp47 binds recombinant α1 subunit and β2 subunit of GABA A receptors in vitro. GST, GST-tagged α1 or GST-tagged β2 recombinant protein was mixed with His-tagged Hsp47 in buffers containing 1% Triton X-100. The protein complex was isolated by immunoprecipitation using an anti-His antibody, and the immunopurified eluents were separated by SDS-PAGE and blotted with indicated antibodies. Three biological replicates were performed. ( C ) MicroScale Thermophoresis (MST) was used to determine the binding affinities between Hsp47, an ER luminal chaperone, to RED-labeled His-α1(ERD) and His-β2(ERD). Increasing concentrations of recombinant Hsp47 proteins (0.2 nM – 10 μM) were incubated with 50 nM RED-labeled His-α1(ERD) or His-β2(ERD) in PBS with Tween-20 (0.05%). Then samples were loaded to the capillaries and measured using a Monolith NT.115 instrument with the settings of 40% LED/excitation and 40% MST power. Three biological replicates were performed. The data were analyzed using the Monolith software for the calculation of the dissociation constant (Kd). IP, immunoprecipitation; IB, immunoblotting. Figure 1—source data 1. Original files for the western blot analysis in . Figure 1—source data 2. PDF containing the original blots in with the relevant bands clearly labeled.

Article Snippet: Peptide, recombinant protein , His-Hsp47 , Novus , Cat#: NBC1-22576 , .

Techniques: Immunoprecipitation, Negative Control, Binding Assay, Recombinant, In Vitro, Isolation, SDS Page, Microscale Thermophoresis, Labeling, Incubation, Software, Western Blot

Journal: eLife

Article Title: Hsp47 promotes biogenesis of multi-subunit neuroreceptors in the endoplasmic reticulum

doi: 10.7554/eLife.84798

Figure Lengend Snippet: ( A ) Recombinant His-tagged Hsp47 protein was mixed with FLAG-tagged ZIP7, hERG, GST-tagged GABA A receptor α1 recombinant proteins, or buffer only in binding buffers (50 mM Tris, pH 7.5, 150 mM NaCl, and 2 mM N-dodecyl-β-D-maltoside (DDM)). The protein complex was isolated by immunoprecipitation using an anti-His antibody, and the immunopurified eluents were separated by SDS-PAGE and blotted with indicated antibodies. Three biological replicates were performed. ( B ) Representative circular dichroism (CD) spectra of α1 subunit ERD domain and β2 subunit ERD domain. Molar ellipticity [θ] was plotted against the wavelength (nm). Each CD Spectrum was measured by accumulating three spectra to obtain the average with the blank correction. Figure 1—figure supplement 2—source data 1. Original files for the western blot analysis in . Figure 1—figure supplement 2—source data 2. PDF containing the original blots in with the relevant bands clearly labeled.

Article Snippet: Peptide, recombinant protein , His-Hsp47 , Novus , Cat#: NBC1-22576 , .

Techniques: Recombinant, Binding Assay, Isolation, Immunoprecipitation, SDS Page, Circular Dichroism, Western Blot, Labeling

Journal: eLife

Article Title: Hsp47 promotes biogenesis of multi-subunit neuroreceptors in the endoplasmic reticulum

doi: 10.7554/eLife.84798

Figure Lengend Snippet:

Article Snippet: Peptide, recombinant protein , His-Hsp47 , Novus , Cat#: NBC1-22576 , .

Techniques: Transfection, Construct, Control, Recombinant, Plasmid Preparation, Cloning, Software

Journal: eLife

Article Title: Targeting A-kinase anchoring protein 12 phosphorylation in hepatic stellate cells regulates liver injury and fibrosis in mouse models

doi: 10.7554/eLife.78430

Figure Lengend Snippet: Mice were administered CCl 4 or mineral oil (control) as in methods. ( A ) Total protein (left panel) was immunoblotted with AKAP12, α-SMA, or GAPDH (control) antibody and blots were quantified by ImageJ densitometry. Data represented by GAPDH normalized densitometry is mean ± SE from three experimental groups. Source data are presented in . P values are calculated in . Total RNA (right panel) from mouse liver was subjected to real-time RT-PCR to evaluate the expression of Akap12, Acta2 , or Gapdh (normalizing control) mRNA (mean ± S.E from four experimental groups). P values are calculated in . ( B ) Sections of control or CCl 4 livers stained with the HSC marker, desmin was overlayed with antibodies to detect ligation of AKAP12 with the phospho-serine antibody by PLA as in methods. 200× magnification, scale bar=50 µm. Total AKAP12 expression was detected by HRP/DAB staining as in Materials and methods. Images were quantified using ImageJ and represented as the proximity ligation/fluorescence/HRP count. Mean ± SE from four experimental groups. Source data are presented in . P values are calculated in . ( C ) Control or CCl 4 liver protein was immunoprecipitated with AKAP12 antibody and probed for HSP47 by western blotting. Normal mouse IgG was a negative control. Data represented by GAPDH normalized densitometry are mean ± SE from three experimental groups. Source data are presented in . P values are calculated in . ( D ) Human tissue arrays were stained with AKAP12 and HSP47 far red PLA probes as in methods. AlexaFluor antibodies (see Key resource table) were used to detect expression of AKAP12 or HSP47 in these arrays. A representative area is shown at 400× magnification, scale bar=100 µm. Each tissue within the array was quantified by densitometry using ImageJ and represented as the proximity ligation/fluorescence count . Mean ± SE, from 11 normal livers and 16 liver fibrosis tissues. P values are calculated in . Figure 1—source data 1. Raw blots for . Figure 1—source data 2. Individual images for . Figure 1—source data 3. Raw blots for . Figure 1—source data 4. Post hoc analysis for .

Article Snippet: Antibody , Anti-HSP47 antibody (clone # 950806, mouse monoclonal) , Novus Biologicals , MAB9166-100 , Western: (1:2000) in 5% milk/TBS-Tween; PLA: (1:250) dilution in PLA buffer.

Techniques: Control, Quantitative RT-PCR, Expressing, Staining, Marker, Ligation, Fluorescence, Immunoprecipitation, Western Blot, Negative Control

Journal: eLife

Article Title: Targeting A-kinase anchoring protein 12 phosphorylation in hepatic stellate cells regulates liver injury and fibrosis in mouse models

doi: 10.7554/eLife.78430

Figure Lengend Snippet: ( A ) Cell extracts from human HSCs cultured for 0, 3, or 6 days (see Materials and methods) were processed for co-immunoprecipitation of AKAP12 and HSP47 or for α-SMA western blotting. Data represented as GAPDH normalized densitometry is mean ± SE from three experiments. Source data are presented in . P values are calculated in . ( B ) Activated human HSCs were transfected with CRISPR reagents and GFAP-SaCas9 vector to cause CRISPR-directed HDR as in Materials and methods. Untransfected (WT) or cells with SaCas9 alone were used as controls. CRISPR editing at the AKAP12 locus (left panel) was confirmed by performing PCR (right panel) using primers that specifically detected the edited region as listed in Key resource table. Four independent experiments are shown. ( C ) CRISPR-edited (HDR) or WT cells as in ( B ) above were assessed for AKAP12-HSP47 co-immunoprecipitation, HSP47, AKAP12, and α-SMA (HSC activation marker) western blotting. Data represented as GAPDH normalized densitometry is mean ± SE from three experiments. Source data are presented in . P values are calculated in . ( D ) Day 0 attached HSCs were culture activated till Day 3 and then transfected with CRISPR vectors till Day 5. The autofluorescence of vitamin A as a marker of HSC quiescence was visualized by fluorescence microscopy and compared to brightfield images of cells as in Materials and methods. Three independent experiments are shown. Scale bar=80 µm. ( E ) AKAP12-HSP47 interaction (left panel) and HSP47-collagen interaction (right panel) in the ER was compared between WT and HDR cells by PLA staining and co-staining with the ER marker, calreticulin as in methods. Magnification at 200×, scale bar=60 µm. Data represented as proximity ligation/fluorescence count are mean ± SE from four experiments. Source data are presented in . P values are calculated in . ER, endoplasmic reticulum; HDR, homology-directed repair; HSC, hepatic stellate cell; PLA, proximity ligation assay; WT, wild-type. Figure 2—source data 1. Source blots for . Figure 2—source data 2. Source blots for . Figure 2—source data 3. Source data for . Figure 2—source data 4. Post-hoc analysis for .

Article Snippet: Antibody , Anti-HSP47 antibody (clone # 950806, mouse monoclonal) , Novus Biologicals , MAB9166-100 , Western: (1:2000) in 5% milk/TBS-Tween; PLA: (1:250) dilution in PLA buffer.

Techniques: Cell Culture, Immunoprecipitation, Western Blot, Transfection, CRISPR, Plasmid Preparation, Activation Assay, Marker, Fluorescence, Microscopy, Staining, Ligation, Proximity Ligation Assay

Journal: eLife

Article Title: Targeting A-kinase anchoring protein 12 phosphorylation in hepatic stellate cells regulates liver injury and fibrosis in mouse models

doi: 10.7554/eLife.78430

Figure Lengend Snippet: Activated mouse HSCs were transfected with CRISPR reagents and GFAP-Cas9 vector to cause CRISPR-directed HDR as described under Materials and methods. Untransfected (WT) or cells with Cas9 alone were used as controls. ( A ) CRISPR editing at the AKAP12 locus (left panel) was confirmed by performing PCR (right panel) using primers that specifically detected the edited region as listed inTable S1key resource table. A representative image from three experiments is shown. ( B ) CRISPR-edited (HDR) or WT cells were assessed for AKAP12-HSP47 interaction by co-immunoprecipitation-immunoblotting. Data represented as GAPDH normalized densitometry are mean ± SE from three experiments. *p=0.03 versus WT. HDR, homology-directed repair; HSC, hepatic stellate cell; WT, wild-type. Figure 2—figure supplement 1—source data 1. Source blots. . Figure 2—figure supplement 1—source data 2. Original gel image.

Article Snippet: Antibody , Anti-HSP47 antibody (clone # 950806, mouse monoclonal) , Novus Biologicals , MAB9166-100 , Western: (1:2000) in 5% milk/TBS-Tween; PLA: (1:250) dilution in PLA buffer.

Techniques: Transfection, CRISPR, Plasmid Preparation, Immunoprecipitation, Western Blot

Journal: eLife

Article Title: Targeting A-kinase anchoring protein 12 phosphorylation in hepatic stellate cells regulates liver injury and fibrosis in mouse models

doi: 10.7554/eLife.78430

Figure Lengend Snippet: ( A ) AKAP12 is phosphorylated by PKCα at its activation-responsive phospho-sites. Recombinant WT or AKAP12 phospho-mutants were in vitro translated from their vectors and subjected to in vitro kinase assay in the presence of active PKCα enzyme as in Materials and methods. The reactions were run on a phostag gel to detect phosphorylated AKAP12 or its mutants. Representative phostag gels from three experiments are shown. Source data are presented in . P values are calculated in . ( B ) Direct Interaction between AKAP12 and HSP47 in recombinant system in the absence or presence of active PKCα enzyme. In vitro translated biotinylated AKAP12 was incubated with recombinant HSP47 antibody column containing bound HSP47 (left) or recombinant HSP47 was incubated with Biotin antibody column containing bound AKAP12-Biotin (right) in the presence or absence of active PKCα as in Materials and methods. Two representative data out of four experiments are shown. Source data are presented in . ( C ) Silencing Prkca in activated human HSCs enhances AKAP12-HSP47 interaction. Culture-activated human HSCs were transfected with a universal negative control siRNA (Neg) or two Prkca siRNAs ( A or B ) as in Materials and methods. Total protein was assessed for AKAP12-HSP47 co-immunoprecipitation or PKCα, HSP47, and GAPDH immunoblotting. Data represented as GAPDH normalized densitometry are mean ± SE from five experiments. Source data are presented in . P values are calculated in . HSC, hepatic stellate cell; WT, wild-type. Figure 3—source data 1. Source blots for . Figure 3—source data 2. Source blots for . Figure 3—source data 3. Source blots for . Figure 3—source data 4. Post hoc analysis for .

Article Snippet: Antibody , Anti-HSP47 antibody (clone # 950806, mouse monoclonal) , Novus Biologicals , MAB9166-100 , Western: (1:2000) in 5% milk/TBS-Tween; PLA: (1:250) dilution in PLA buffer.

Techniques: Activation Assay, Recombinant, In Vitro, Kinase Assay, Incubation, Transfection, Negative Control, Immunoprecipitation, Western Blot

Journal: eLife

Article Title: Targeting A-kinase anchoring protein 12 phosphorylation in hepatic stellate cells regulates liver injury and fibrosis in mouse models

doi: 10.7554/eLife.78430

Figure Lengend Snippet: ( A ) AKAP12-HSP47 co-immunoprecipitation, AKAP12, HSP47, and α-SMA western blotting from liver protein of CR-PDEL experiment. Data represented as GAPDH normalized densitometry are mean±SE from six experiments. Three representatives are shown. Source data are presented in . P values are calculated in . ( B ) AKAP12-HSP47 co-immunoprecipitation, AKAP12, HSP47, and α-SMA western blotting from liver protein of CR-PMUT experiment. Data represented as GAPDH normalized densitometry are mean±SE from six experiments. Three representatives are shown. Source data are presented in . P values are calculated in . ( C ) Co-immunoprecipitation of collagen with HSP47 in CRISPR-edited livers are mean±SE from five PDEL and three PMUT experiments. Source data are presented in . P values are calculated in . ( D ) Col1A1 or Acta2 mRNA levels by real-time PCR from PDEL (left) or PMUT (right) mouse livers. Data are mean±SE from six experimental groups. P values are calculated in . ( E ) Interaction between AKAP12 and HSP47 in desmin-positive HSCs of CRISPR model by PLA staining. Data representative of the AKAP12-HSP47 PLA count per desmin area are mean±SE from four PDEL or PMUT experiments. 200× magnification, scale bar=100 µm. P values are calculated in . HSC, hepatic stellate cell; PLA, proximity ligation assay. Figure 6—source data 1. Source blots for . Figure 6—source data 2. Source blots for . Figure 6—source data 3. Source blots for . Figure 6—source data 4. Post hoc analysis for .

Article Snippet: Antibody , Anti-HSP47 antibody (clone # 950806, mouse monoclonal) , Novus Biologicals , MAB9166-100 , Western: (1:2000) in 5% milk/TBS-Tween; PLA: (1:250) dilution in PLA buffer.

Techniques: Immunoprecipitation, Western Blot, CRISPR, Real-time Polymerase Chain Reaction, Staining, Proximity Ligation Assay

Journal: eLife

Article Title: Targeting A-kinase anchoring protein 12 phosphorylation in hepatic stellate cells regulates liver injury and fibrosis in mouse models

doi: 10.7554/eLife.78430

Figure Lengend Snippet: ( A ) Heat map of total liver and HSCs ER stress/UPR signaling components in four groups, oil+EV, oil+CR, CCl 4 +EV and CCL 4 +CR. Proteomics data utilized to prepare the heatmap are presented in . Alterations in ER stress responsive elements in HSCs from AKAP12 PDEL CRISPR model ( B ) and total liver ( C ). GAPDH normalized densitometry is mean±SE from three experiments. Raw densitometry of each experiment is presented in . Source data are presented in and . P values are calculated in . ( D ) Inflammatory cytokine expression in HSCs from oil+EV, oil+CR, CCl 4 +EV and CCL 4 +CR groups. GAPDH normalized densitometry is mean±SE from four experiments. Raw densitometry of each experiment is presented in . Source data are presented in . P values are calculated in . ( E ) ER stress response in hepatocytes co-cultured with quiescent or activated WT HSCs with or without AKAP12 PDEL CRISPR editing (CR). GAPDH normalized densitometry represented as fold over hepatocytes +WT quiescent HSCs is mean±SE from three experiments. Raw densitometry of each experiment is presented in . Source data are presented in . P values are calculated in . ( F ) Summary of findings. AKAP12 interacts with HSP47 in the ER of normal HSCs and negatively regulates HSP47’s collagen-chaperoning activity and its ability to promote ER stress-directed IRE1α branch of UPR signaling. AKAP12 negatively regulates HSC activation. Pro-fibrogenic stimuli that cause HSC activation allow AKAP12’s PKCα-dependent site-specific phosphorylation. By AKAP12 CRISPR phospho-editing, we show that AKAP12 phosphorylation inhibits AKAP12’s HSP47 scaffolding activity, increases HSP47-collagen chaperoning activity and induces HSP47’s interaction with UPR signals (IRE1α and P-IRE1α). AKAP12 phosphorylation may lead to increased downstream events associated with the UPR signaling such as BIP-collagen chaperoning, phosphorylation of SMADs/P38MAPK and UPR-regulated inflammatory signaling. Blocking AKAP12 phosphorylation in activated HSCs prevents the ER stress response in hepatocytes co-cultured with activated HSCs. ER, endoplasmic reticulum; HSC, hepatic stellate cell; UPR, unfolded protein response; WT, wild-type. Figure 7—source data 1. Source blots for . Figure 7—source data 2. Source blots for . Figure 7—source data 3. Source blots for . Figure 7—source data 4. Source blots for . Figure 7—source data 5. Post hoc analysis for .

Article Snippet: Antibody , Anti-HSP47 antibody (clone # 950806, mouse monoclonal) , Novus Biologicals , MAB9166-100 , Western: (1:2000) in 5% milk/TBS-Tween; PLA: (1:250) dilution in PLA buffer.

Techniques: CRISPR, Expressing, Cell Culture, Activity Assay, Activation Assay, Phospho-proteomics, Scaffolding, Blocking Assay

Journal: eLife

Article Title: Targeting A-kinase anchoring protein 12 phosphorylation in hepatic stellate cells regulates liver injury and fibrosis in mouse models

doi: 10.7554/eLife.78430

Figure Lengend Snippet:

Article Snippet: Antibody , Anti-HSP47 antibody (clone # 950806, mouse monoclonal) , Novus Biologicals , MAB9166-100 , Western: (1:2000) in 5% milk/TBS-Tween; PLA: (1:250) dilution in PLA buffer.

Techniques: Transfection, Construct, Negative Control, Sequencing, Amplification, CRISPR, Recombinant, Expressing, Plasmid Preparation, In Vitro, Cloning, Kinase Assay, Binding Assay, Mutagenesis, Hydroxyproline Assay, Activity Assay, Clinical Proteomics, Immunoprecipitation, Western Blot, Immunostaining, In Situ

Journal: Current protocols

Article Title: Simple, Fast, and Efficient Method for Derivation of Dermal Fibroblasts From Skin Biopsies.

doi: 10.1002/cpz1.714

Figure Lengend Snippet: Figure 3 Skin biopsy–derived cells express fibroblast-specific markers. Upon fixation with 4% PFA, cells were stained with specific antibodies. (A) Anti-vimentin (in red) and anti-Oregon Green 488 Phalloidin (F-actin, in green) staining. (B) Anti-Hsp47 antibody (in green) staining. Nuclei were detected by DAPI (in blue) staining.

Article Snippet: See the T erm s and C onditions (https://onlinelibrary.w iley.com /term s-and-conditions) on W iley O nline L ibrary for rules of use; O A articles are governed by the applicable C reative C om m ons L icense Table 1 List of Antibodies Used for Characterizing Fibroblasts by Immunocytochemistry Species Company Cat # Dilution Primary antibody Human Hsp47 (Serpin H1) Antibody Monoclonal mouse IgG2B R&D Systems MAB91661SP 1:200 to 1:500 Oregon Green 488 Phalloidin (F-actin) (conjugated) Amanita phalloides mushroom Thermo Fisher Scientific O7466 1:50 Vimentin mouse McAb Mouse IgG1 Proteintech 60330-1-Ig 1:500 Secondary antibody F(ab’)2-Goat anti-Mouse IgG (H+L) Cross-Adsorbed Secondary Antibody, Alexa FluorTM 546 Goat anti-Mouse Thermo Fisher Scientific A-11018 1:1000 F(ab’)2-Goat anti-Mouse IgG (H+L) Cross-Adsorbed Secondary Antibody, Alexa FluorTM 488 Goat anti-Mouse Thermo Fisher Scientific A-11017 1:1000 dermal fibroblasts can be expanded by passaging with trypsin (in our hands, fibroblasts still showed the ability to divide up to passage five, but we did not try to passage more than P5).

Techniques: Derivative Assay, Staining

Journal: JCI insight

Article Title: Cell-specific ablation of Hsp47 defines the collagen-producing cells in the injured heart

doi: 10.1172/jci.insight.128722

Figure Lengend Snippet: Figure 1. Cardiomyocyte-specific deletion of Hsp47 in the mouse heart does not block maladaptive fibrosis with TAC. (A) Representative immunostain- ing for Hsp47 (red fluorescence) in cultured cardiac fibroblasts 72 hours after infection with Adβgal and AdCre. Nuclei are stained blue with DAPI. Scale bar: 100 μm. (B) Western blot showing levels of secreted collagen type I in the culture media of heart fibroblasts 72 hours after infection with either Adβgal or AdCre. White arrows show collagen isoforms. Molecular weight migration standard and sizes are also shown. Nonspecific (n.s.) Ponceau staining (pink) is shown as a processing and loading control. (C) Schematic representation of breeding βMHC-Cre–transgenic mice with Hsp47-loxP–targeted mice. (D) West- ern blot analysis for Hsp47 isolated from fractionated cardiomyocytes of the 2 genotypes of mice shown. Gapdh is shown as the loading control. (E and F) Picrosirius red–stained histological heart sections, and quantitation of the area of fibrosis (red) in hearts from the indicated genotypes of mice with the βMHC-Cre transgene after 4 weeks of TAC injury. Average fibrotic area ± SEM, n = 5–8 mice in each group, *P < 0.05 versus sham-operated βMHC-Cre mice. P values were calculated by 1-way ANOVA with Tukey’s post hoc test. Scale bar: 200 μm (G) Heart-weight-to-body-weight (HW/BW) ratio in mice after 4 weeks of TAC. n = 5–8 in each group. *P < 0.05 versus βMHC-Cre sham mice. (H) Breeding scheme of αMHC-MCM–transgenic mice with Hsp47-loxP–target- ed mice. (I) Experimental regimen whereby mice were subjected to TAC injury or sham procedure for 4 weeks along with tamoxifen treatment by injection (vertical red arrows). (J) Western blot analysis for Hsp47 isolated from adult heart fractionated cardiomyocytes of the 2 genotypes shown. Gapdh is shown as a loading control. (K, L, and M) Quantitation of collagens type I, III, and V, respectively, from immunohistochemical heart images from WT αMHC-MCM mice versus Hsp47 cardiomyocyte-specific mice, as shown in Supplemental Figure 3. Ten random histological sections from each mouse heart were imaged and quantified from 5–10 mice each per group. *P < 0.05 versus αMHC-MCM Sham. P values were calculated with 1-way ANOVA with Tukey’s post hoc test.

Article Snippet: The membranes were incubated with antibodies against Hsp47 (1:000, Novus Biologicals, M16.10A1), Gapdh (1:10000, Fitzgerald, 10R-G109a), or α-tubulin (1:1000, Santa Cruz, sc-8035).

Techniques: Blocking Assay, Fluorescence, Cell Culture, Infection, Staining, Western Blot, Molecular Weight, Migration, Control, Transgenic Assay, Isolation, Quantitation Assay, Injection, Immunohistochemical staining

Journal: JCI insight

Article Title: Cell-specific ablation of Hsp47 defines the collagen-producing cells in the injured heart

doi: 10.1172/jci.insight.128722

Figure Lengend Snippet: Figure 2. Endothelial-specific deletion of Hsp47 in the heart. (A) Schematic of breeding tamoxifen-inducible Tie2-CreERT2–transgenic mice with Hsp47- loxP–targeted mice. (B) Experimental regimen for mice subjected to TAC or a sham procedure for 4 weeks. Mice were injected at 6 weeks of age 2 times with tamoxifen and then put on tamoxifen chow before 8 weeks of age through harvesting at 12 weeks. (C) Western blot analysis for Hsp47 from endothe- lial cells isolated by FACS from the genotypes shown. Gapdh is a loading control. (D–G) Immunohistochemical heart images stained (scale bar: 100 μm) and quantified for collagen type I, III, and V from Tie2-CreERT2–transgenic mice and Hsp47 endothelial-specific deleted mice. Mice were subjected to TAC as shown in B. Quantitation shows mean intensity of immunoreactivity taken from 10 random histological sections from 5–10 mice in each group. *P < 0.05 versus Tie2-CreERT2 Sham. #P < 0.05 versus Tie2-CreERT2 TAC. P values were calculated with 1-way ANOVA with Tukey’s post hoc test.

Article Snippet: The membranes were incubated with antibodies against Hsp47 (1:000, Novus Biologicals, M16.10A1), Gapdh (1:10000, Fitzgerald, 10R-G109a), or α-tubulin (1:1000, Santa Cruz, sc-8035).

Techniques: Transgenic Assay, Injection, Western Blot, Isolation, Control, Immunohistochemical staining, Staining, Quantitation Assay

Journal: JCI insight

Article Title: Cell-specific ablation of Hsp47 defines the collagen-producing cells in the injured heart

doi: 10.1172/jci.insight.128722

Figure Lengend Snippet: Figure 3. Myofibroblast-specific deletion of Hsp47 in the heart reduces myocardial fibrosis after TAC. (A) Schematic representation of Postn-Mer- CreMer–targeted (MCM-targeted) mice crossed with Hsp47-loxP–targeted mice. (B) Experimental regimen of tamoxifen injections (red vertical arrows) and feed treatment (red horizontal line) in mice subjected to TAC for 4 weeks. (C) Western blot analysis for Hsp47 and α-tubulin from 500,000 EGFP+ cells isolated by FACS from hearts of the 2 genotypes of mice shown (R26eGFP reporter was also present). (D–G) Representative immunohistochemistry (scale bar: 100 μm) of heart tissue sections and quantitation for collagen type I, III, and V from hearts of Postn-MCM control mice and Hsp47 myofibroblast-specific deleted mice using the Postn-MCM allele after 4 weeks of TAC. (H) Quantitation from Picrosirius red–stained histological sections in hearts from the indicated genotypes of mice after 4 weeks of TAC injury. *P < 0.05 versus Postn-MCM Sham. #P < 0.05 versus Postn-MCM TAC. n = 5–10 mice in each group. P values were calculated using a 1-way ANOVA with Tukey’s post hoc test. (I) Western blot analysis for collagen type I from heats of sham and TAC-operated mice using cardiac extracellular matrix–specific protein preparations from the indicated genotypes of mice. The red arrows show collagen isoforms. Position of molecular weight standards (kDa) are shown on the left.

Article Snippet: The membranes were incubated with antibodies against Hsp47 (1:000, Novus Biologicals, M16.10A1), Gapdh (1:10000, Fitzgerald, 10R-G109a), or α-tubulin (1:1000, Santa Cruz, sc-8035).

Techniques: Western Blot, Isolation, Immunohistochemistry, Quantitation Assay, Control, Staining, Molecular Weight

Journal: JCI insight

Article Title: Cell-specific ablation of Hsp47 defines the collagen-producing cells in the injured heart

doi: 10.1172/jci.insight.128722

Figure Lengend Snippet: Figure 4. Myofibroblast-specific but not myofiber-specific deletion of Hsp47 in skeletal muscle reduces muscular dystrophy–dependent tissue fibrosis. (A) Schematic of the MCM cDNA driven by the human skeletal α-actin promoter (myofiber-specific) or the myofibroblast-specific Postn genetic locus to delete the Hsp47 gene with tamoxifen treat- ment. These lines were crossed into the δ-sarcoglycan–null (Sgcd–/–) background. (B) Experimental tamoxifen dosing regimen administered in the feed. (C) Western blot analysis for total Hsp47 protein using whole muscle protein lysates from the quadriceps of mice of the indicated genotypes. n = 6 mice per group. Gapdh is shown as a loading control. (D) Representative Picrosirius red–stained histological sections from quadriceps and diaphragm from 4-month-old mice of the indicated genotypes. Scale bar: 200 μm. (E and F) Quantitation of fibrosis from Picrosirius red–stained histological sections from quadriceps and diaphragm of 4-month-old mice of the indicated genotypes. n = 7 mice in each group. (G) Average time spent running on a treadmill of 4-month-old mice of the indicated genotypes. n = 6–10 in each group. Significance was determined using P values calculated by 1-way ANOVA with Tukey’s post hoc test. *P < 0.05 versus Sgcd–/–-control. #P < 0.05 versus Sgcd–/– Hsp47fl/fl-Ska-MCM. The legend applies to E, F and G.

Article Snippet: The membranes were incubated with antibodies against Hsp47 (1:000, Novus Biologicals, M16.10A1), Gapdh (1:10000, Fitzgerald, 10R-G109a), or α-tubulin (1:1000, Santa Cruz, sc-8035).

Techniques: Western Blot, Control, Staining, Quantitation Assay

Journal: JCI insight

Article Title: Cell-specific ablation of Hsp47 defines the collagen-producing cells in the injured heart

doi: 10.1172/jci.insight.128722

Figure Lengend Snippet: Figure 5. Myofibroblast-specific Hsp47 deletion alters acute scar formation and the hypertrophic response. (A) Experimental scheme whereby αMHC-Mer- CreMer–transgenic mice or Postn-MerCreMer allele–containing mice were subjected to myocardial infarction injury for 4 weeks with 2 injections (vertical red arrows) of tamoxifen treatment and then tamoxifen in the feed for 4 weeks (horizontal red arrow). (B) Kaplan-Meier plot of survival of the indicated geno- types of mice after MI injury. n = 11–13 mice in each group. (C) Quantitation of fibrosis from Picrosirius red–stained histological sections from hearts after 4 week of I/R injury of the indicated genotypes. n = 6 mice in each group. (D) Gravimetric assessed heart-weight-to-body-weight (HW/BW) ratios in mice of the indicated genotypes after 4 weeks of TAC. n = 5 sham mice, n = 9–10 TAC mice in each group. *P < 0.05 versus sham; #P < 0.05 versus Postn-MCM TAC. P values were calculated by 2-way ANOVA and Bonferroni post hoc test. (E–G) Echocardiographic assessment of ventricular (LV) calculated mass, left ventric- ular fractional shortening (FS%) percentage, and early mitral inflow velocity to mitral annular early diastolic velocity ratio (E/e) in the indicated genotypes of mice after 4 weeks of TAC injury or a sham procedure. *P < 0.05 versus Postn-MCM sham. #P < 0.05 versus Postn-MCM TAC. P values were calculated with 1-way ANOVA with Tukey’s post hoc test. Number of mice used is shown in the scatter plots.

Article Snippet: The membranes were incubated with antibodies against Hsp47 (1:000, Novus Biologicals, M16.10A1), Gapdh (1:10000, Fitzgerald, 10R-G109a), or α-tubulin (1:1000, Santa Cruz, sc-8035).

Techniques: Transgenic Assay, Quantitation Assay, Staining

Journal: JCI insight

Article Title: Cell-specific ablation of Hsp47 defines the collagen-producing cells in the injured heart

doi: 10.1172/jci.insight.128722

Figure Lengend Snippet: Figure 6. Myofibroblast-specific Hsp47 deletion reduces myofibroblasts in vivo and their proliferation. (A) Experimental scheme whereby mice were subjected to TAC injury for 7 days. Mice received 2 i.p injections of tamoxifen and were fed tamoxifen-laden chow 48 hours before surgery and were then maintained on this chow until harvesting. Mice also received a single i.p EdU injection 4 hours before sacrifice at day 7 after TAC. (B) Representative flow cytometry plots of isolated EGFP+ interstitial cells (plotted as EGFP fluorescence signal on the x axis versus side scatter on the y axis) from hearts of the indicated genotypes of mice; 100,000 cells are displayed in the blots. (C) The ratio of total EGFP+ myofibroblasts normal- ized to CD31+ cells from the hearts of the indicated genotypes of mice after 1 week of TAC. Error bars represent SEM; n = 4 mice in each group. *P < 0.05 versus Postn-MCM; R26eGFP. P values were calculated with a Student’s t test. (D) Relative number of CD31+ cells in the interstitial fractions in hearts of the indicated genotypes of mice after 1 week of TAC. (E) Representative immunohistological images (scale bar: 100 μm) of EdU+ and EGFP+ interstitial cells at the time of harvest for mice treated, as shown in A. DAPI was used to show nuclei (blue). n = 5 mice in each group. (F) Quantita- tion of GFP+ cells that were also EdU+ in heart histological sections from mice subjected to TAC of the indicated genotype. P values were calculated with Student’s t test. #P < 0.05 versus R26eGFP Postn-MCM controls. (G) Quantitation of EdU+ Hsp47fl/fl cardiac fibroblasts over 24 hours in culture previously treated with AdCre or Adβgal infection. A total of 6 images were analyzed per group. P values were calculated with Student’s t test. #P < 0.05 versus Adβgal infection. Data shown are the mean ± SEM.

Article Snippet: The membranes were incubated with antibodies against Hsp47 (1:000, Novus Biologicals, M16.10A1), Gapdh (1:10000, Fitzgerald, 10R-G109a), or α-tubulin (1:1000, Santa Cruz, sc-8035).

Techniques: In Vivo, Injection, Flow Cytometry, Isolation, Fluorescence, Quantitation Assay, Infection

Journal: JCI insight

Article Title: Cell-specific ablation of Hsp47 defines the collagen-producing cells in the injured heart

doi: 10.1172/jci.insight.128722

Figure Lengend Snippet: Figure 7. Hsp47 deletion in myofibroblasts reduces ECM-related gene expression and promotes an altered differentiated state. (A) Adult primary heart fibroblasts were isolated from Hsp47-loxP–targeted mice infected with Adβgal (WT) or AdCre (deleted samples). Seventy-two hours after infection cells were washed and incubated in 2% serum containing DMEM media with 20 μM ascorbic acid for 24 hours before RNA isolation. The data are real-time PCR results showing the expression levels of the indicated genes. n = 4 separate experiments. *P < 0.05 versus Adβgal WT. (B) Schematic representation of the Postn- MCM mouse line crossed with the Hsp47-loxP site–containing gene–targeted line and the Rosa26 reporter line (R26eGFP). (C) Experimental scheme with TAC stimulation and tamoxifen with injection and laden food. (D and E) Quantification of select- ed mRNAs in Hsp47-deleted EGFP+ myofibroblasts isolated from hearts of Hsp47fl/flPostn-MCM/+R26eGFP/+ allele containing mice, 4 weeks after TAC injury. n = 3, *P < 0.5. P values were calculated with Student’s t test. Data shown are the mean ± SEM.

Article Snippet: The membranes were incubated with antibodies against Hsp47 (1:000, Novus Biologicals, M16.10A1), Gapdh (1:10000, Fitzgerald, 10R-G109a), or α-tubulin (1:1000, Santa Cruz, sc-8035).

Techniques: Gene Expression, Isolation, Infection, Incubation, Real-time Polymerase Chain Reaction, Expressing, Injection