Journal: International journal of molecular sciences

Article Title: Novel Identification of Ankyrin-R in Cardiac Fibroblasts and a Potential Role in Heart Failure.

doi: 10.3390/ijms25158403

Figure Lengend Snippet: Figure 2. Fibroblasts express AnkR at the membrane and cytoplasm. (A) Isolated cardiac fibroblasts (CFs) from wild-type mice were stained with αSMA and AnkR, identifying strong cytoplasmic and perinuclear staining, n = 3. (B) Subcellular fractionation of isolated CFs displayed canonical AnkR in most cellular compartments, but predominantly from the membrane and cytosolic fractions, n = 3, scale bar = 50 µm. (C) Isolated CFs co-immunostained with TGN38 and AnkR showing colocalization in white, n = 3, scale bar = 10 µm.

Article Snippet: Isolated cardiac fibroblasts were cultured on glass coverslips and fixed with 1:1 acetone:methanol for 10 min and incubated in blocking solution (PBS with 3% fish gelatin, 0.75% Triton-X 100 (10%), 1% DMSO) for 1 h. Cells were then incubated in primary antibody (custom AnkR antibody (1:300); WGA-488 5 μg/mL Thermo Fisher, W11261; TGN38 1:1000, Novus Biologicals, Centennial, CO USA NB300-575; αSMA, 1:500 Sigma-Aldrich A2547-100UL) at 4 ◦C overnight.

Techniques: Membrane, Isolation, Staining, Fractionation

Journal: Function

Article Title: ER Redox Homeostasis Regulates Proinsulin Trafficking and Insulin Granule Formation in the Pancreatic Islet β-Cell

doi: 10.1093/function/zqac051

Figure Lengend Snippet: Delayed ER export of proinsulin in animal and cell culture models of hyperglycemia. (A–C) INS1 832/3 cells stably expressing proCpepSNAP were cultured for up to 72 h in control media supplemented with BSA or media containing oleate/palmitate (2:1, 1 m m ) and elevated glucose (20 m m ; OPG) as indicated. Cells were pulse-labeled with SNAP-505 (green), chased for 15 min, immunostained for GRP94 (red), TGN38 (magenta), and counterstained with DAPI (blue). Representative images (A) are shown (scale bar = 5 μm) and the ratio of proCpepSNAP fluorescence coincident with the Golgi compared to ER quantified (B) ( n = 6–8 independent experiments; 48–64 cells per condition). (C) Total SNAP fluorescence per cell was quantified. (D and E) Isolated mouse islets (C57BLKS/J db/+ vs. db/db) were treated with AdRIP-proCpepSNAP. 48 h post-infection, islets were pulse-labeled with SNAP-505 (green), chased for 10 min, immunostained for GM130 (magenta), and counterstained with DAPI (blue). Representative images (E) are shown (scale bar = 3 μ m ) and the ratio of proCpepSNAP fluorescence coincident with the Golgi compared to ER quantified (D) ( n = 4 mice per group; 6–36 cells/mouse). (B and D) Data represent the mean ± SD * P < 0.05, *** P < 0.0005, or not significant (ns) by Student’s t -test.

Article Snippet: For immunostaining, cells were incubated overnight with antibodies raised against GM130 (mouse, BD Transduction 610 823, 1:200), GRP94 (rabbit, kind gift of Dr. Christopher Nicchita, Duke University, 1:500), or TGN38 (mouse, Novus Biologicals NB300-575, 1:200) as indicated.

Techniques: Cell Culture, Stable Transfection, Expressing, Control, Labeling, Fluorescence, Isolation, Infection

Journal: Function

Article Title: ER Redox Homeostasis Regulates Proinsulin Trafficking and Insulin Granule Formation in the Pancreatic Islet β-Cell

doi: 10.1093/function/zqac051

Figure Lengend Snippet: Proinsulin ER export delay can be rescued by chemical reducing agent. INS1 832/3 cells stably expressing proCpepSNAP were cultured in control media supplemented with BSA or media containing oleate: palmitate (2:1 and 1 m m ) and elevated glucose (20 m m ; OPG) as indicated. (A-C) Prior to SNAP labeling, cells were treated with vehicle (control) or DTT (0.5 m m ) for 4 h. Cells were then pulse-labeled with SNAP-505 (green), fixed, and immunostained for GRP94 (red), TGN38 (magenta), and counterstained with DAPI (blue). Representative images (A) are shown (scale bar = 5 μ m ) and Mander’s correlation coefficient (B) was used to determine the colocalization of labeled proCpepSNAP (SNAP) with TGN38 ( n = 3–4 independent experiments; 71–91 cells/condition). Total SNAP fluorescence was quantified (C). (D) Cells expressing ERroGFP (AdRIP) were cultured overnight as indicated with vehicle (control) or ebselen (Eb, 10 μ m ) and imaged at 470/535 nm and 395/510 nm. Normalized ratiometric intensities were quantified to determine ER oxidation ( n = 4 independent experiments). (E) Cells were cultured overnight as indicated with vehicle (-) or ebselen (Eb, 10 μM). GSIS was measured by static incubation in media containing 2.5 m m Glc followed by 12 m m Glc for 1 h each. (B–E) Data represent the mean ± SD * P < 0.05 by two-way ANOVA with Sidak post-test analysis (B, D, and E), or ns by two-way ANOVA (C).

Article Snippet: For immunostaining, cells were incubated overnight with antibodies raised against GM130 (mouse, BD Transduction 610 823, 1:200), GRP94 (rabbit, kind gift of Dr. Christopher Nicchita, Duke University, 1:500), or TGN38 (mouse, Novus Biologicals NB300-575, 1:200) as indicated.

Techniques: Stable Transfection, Expressing, Cell Culture, Control, Labeling, Fluorescence, Incubation

Journal: eLife

Article Title: The ESCRT protein CHMP5 restricts bone formation by controlling endolysosome-mitochondrion-mediated cell senescence

doi: 10.7554/eLife.101984

Figure Lengend Snippet: ( A ) Representative confocal fluorescence images and quantification of the fluorescence intensity of LysoTracker Red DND-99 in Ctsk Cre ;Chmp5 fl/fl compared to wild-type periskeletal progenitors. n=11 replicates per group for quantitative analysis; repeated three times using cells from three mice. Scale bars, 50 μm. ( B ) Isotype controls for immunofluorescence staining. Scale bars, 20 μm. ( C ) Co-localization of LAMP1 and RAB7 in Chmp5 -sufficient or Chmp5 -deficient ATDC5 cells. Scale bars, 10 μm. ( D ) Colocalization analysis of LAMP1 and RAB7 performed by the ImageJ Coloc2 programme and Pearson’s R value (above threshold) shown, n=20 cells per group. ( E ) Representative confocal images of immunostaining for early endosome marker EEA1, cycling endosome marker RAB11, and trans-Golgi network marker TGN38 in Ctsk Cre ;Chmp5 fl/fl vs. wild-type periskeletal progenitors. n=30 cells per group. Scale bars, 10 μm (EEA1), 20 μm (RAB11), 25 μm (TGN38). ( F ) RNA-seq data showing Vps4a mRNA expression in Ctsk Cre ;Chmp5 fl/fl vs. Ctsk Cre ;Chmp5 fl/+ periskeletal progenitors. n=3 with cells from three different animals for each group. ( G ) Western blot showing that CHMP5 and VPS4A protein expression after transfection of CHMP5 or empty vector in HEK-293T cells. Experiments repeated three times with independent cells and transfections at different times. ( H ) Quantitative PCR showing the expression of CHMP5 and VPS4A mRNA after transfecting CHMP5 or empty vector in HEK-293T cells. n=3 replicates for each group, experiments repeated three times with independent cells and transfections at different times. All data shown as mean ± s.d.; two-tailed unpaired Student’s t -test. Figure 6—figure supplement 1—source data 1. Data of LysoTracker fluorescence intensity, colocalization of LAMP1 and RAB7, and gene expression. For , Data of quantifying LysoTracker Red DND-99 fluorescence intensity; , Pearson’s R value (above threshold) of colocalization analysis of LAMP1 and RAB7 by ImageJ Coloc2 programme; , Vps4a mRNA expression in Ctsk Cre ;Chmp5 fl/fl vs. Ctsk Cre ;Chmp5 fl/+ periskeletal progenitors (FPKM by RNA-seq); , Relative gene expression of CHMP5 and VPS4A (2 -ΔΔCt ). Figure 6—figure supplement 1—source data 2. Original files for western blot analysis displayed in . Figure 6—figure supplement 1—source data 3. PDF file containing original western blots for , indicating the relevant bands.

Article Snippet: Antibody , Anti-TGN38 antibody (rat monoclonal) , R and D Systems , Cat# MAB7944, RRID: AB_2713953 , IF (1:250).

Techniques: Fluorescence, Immunofluorescence, Staining, Immunostaining, Marker, RNA Sequencing, Expressing, Western Blot, Transfection, Plasmid Preparation, Real-time Polymerase Chain Reaction, Two Tailed Test, Gene Expression