Journal: The Journal of Biological Chemistry

Article Title: Limited proteolysis as a tool to probe the tertiary conformation of dysferlin and structural consequences of patient missense variant L344P

doi: 10.1074/jbc.M117.790014

Figure Lengend Snippet: Sucrose density ultracentrifugation is consistent with C2A as a solitary domain with evidence for multimerization of midregion and mini-dysferlinC72 modules. A, primary human myotubes differentiated for 4 days were harvested via scrape injury, solubilized in lysis buffer, and sedimented on a 5–20% linear sucrose gradient. 13 fractions were removed from top (5%) to bottom (20%) and analyzed via SDS-PAGE. Western blots were probed with dysferlin N- and C-terminal antibodies and syntaxin-4. Mouse brain lysate (strain A/J) and human serum were also run on a separate 5–20% gradient and stained with GelCode or the membrane was probed with syntaxin-1 for known sedimentation standards. The graph shows quantification of sedimentation profiles for dysferlin (blue), mini-dysferlinC72 (mDF) (aqua), and known standards albumin (4S), IgG (7S), and fibrinogen (9S). Full-length (FL) dysferlin is detected in all of the dense fractions of the gradient; in contrast, mini-dysferlinC72 shows a clear sedimentation peak in fraction 4, consistent with formation of a discreet oligomeric species. B, C-terminal regions of dysferlin mediate oligomerization, whereas the C2A domain appears to exist as a monomeric species. HEK293 cells transiently transfected with DYSF40a were harvested via scrape injury 24 h post-transfection and subjected to limited proteolysis with 125 μg/ml trypsin and sucrose gradient ultracentrifugation. A total of 13 fractions were removed from top (5%) to bottom (20%) and analyzed via SDS-PAGE. Romeo displayed peak sedimentation of the excised C2A domain in fractions 2 and 3, similar to sedimentation of syntaxin-4 (34 kDa; see Fig. 4A) and broadly consistent with migration as a monomeric species. Midregion fragments detected by Hamlet-2 and FerA antibodies show clear sedimentation peaks in fractions 5 and 6, similar to IgG (150–180 kDa; 7S). C-terminal tryptic fragments were reproducibly difficult to detect, often with smeared banding, suggesting that once extracted from membrane bilayers into solution they may be susceptible to proteolysis.

Article Snippet: Antibodies used included Romeo (α-rabbit, 1:1000; Abcam {"type":"entrez-nucleotide","attrs":{"text":"Ab124684","term_id":"55166618","term_text":"AB124684"}} Ab124684 ), Hamlet-2 (α-mouse, 1:500; Leica Microsystems HAMLET-2-CE), anti-Fer-A (α-rabbit, 1:1000; Abcam HPA021945), anti-C2DE (α-rabbit, 1:500; Sigma-Aldrich SAB2100636), Hamlet-1 (α-mouse, 1:5000; Leica Microsystems HAMLET-CE), anti-Myc 9E10 (α-mouse, 1:1000; Santa Cruz Biotechnology SC-40), anti-Myc (α-rabbit, 1:5000; Abcam Ab9106), anti-GAPDH (α-mouse, 1:10,000; Merck Millipore MAB374), anti-syntaxin-4 (α-rabbit, 1:250; Synaptic Systems 110042), anti-syntaxin-1 (α-mouse, 1:250; Synaptic Systems 110011), anti-myosin MY32 (1:10,000; Sigma-Aldrich M4276), spectrin (1:100; Leica Biosystems SPEC1-CE), and α-actinin-2 4A3 (1:300,000; a gift from A. Beggs, Children's Hospital Boston, Boston, MA).

Techniques: Lysis, SDS Page, Western Blot, Staining, Sedimentation, Transfection, Migration

Journal: Lipids in Health and Disease

Article Title: Coenzyme Q10 protects against hyperlipidemia-induced cardiac damage in apolipoprotein E-deficient mice

doi: 10.1186/s12944-018-0928-9

Figure Lengend Snippet: Primer oligonucleotide sequences

Article Snippet: Sections were treated with 3% H 2 O 2 in methanol for 15 min to inactivate endogenous peroxidases and then incubated with a primary antibody against p62 (rabbit anti-p62 antibody, 1:200; Proteintech); LC3 (rabbit anti-LC3 antibody, 1:200; Proteintech); CD68 (rabbit anti-CD68 antibody, 1:250; Abcam) at room temperature for 1 h. All sections were examined under an Olympus BX40 upright light microscope (Olympus, Tokyo, Japan).

Techniques:

Journal: Lipids in Health and Disease

Article Title: Coenzyme Q10 protects against hyperlipidemia-induced cardiac damage in apolipoprotein E-deficient mice

doi: 10.1186/s12944-018-0928-9

Figure Lengend Snippet: P62 and LC3 expression in cardiac tissues of the four groups after 16 weeks under different treatments. a Representative immunohistochemistry for p62 and LC3 in cardiac tissues. Scale bar = 100 μm. Arrows indicate positively stained cells. b Relative mRNA expression of p62 and LC3 in cardiac tissue of each group after 16 weeks under different treatments. c Immunoblotting for p62 0061nd LC3 in cardiac tissues. d Bar graph showing quantification of p62 and LC3 protein expression. Data are given as the means ± SEM; n = 5–6 in each group. * P < 0.05 vs. ApoE −/− HD

Article Snippet: Sections were treated with 3% H 2 O 2 in methanol for 15 min to inactivate endogenous peroxidases and then incubated with a primary antibody against p62 (rabbit anti-p62 antibody, 1:200; Proteintech); LC3 (rabbit anti-LC3 antibody, 1:200; Proteintech); CD68 (rabbit anti-CD68 antibody, 1:250; Abcam) at room temperature for 1 h. All sections were examined under an Olympus BX40 upright light microscope (Olympus, Tokyo, Japan).

Techniques: Expressing, Immunohistochemistry, Staining, Western Blot

Journal: Cell Research

Article Title: Tissue-specific transcription reprogramming promotes liver metastasis of colorectal cancer

doi: 10.1038/s41422-019-0259-z

Figure Lengend Snippet: Variations in enhancer and super-enhancer landscape between primary and liver metastatic CRC cells. a Venn diagram indicating overlap and specificity of enhancers marked by H3K4me2 and H3K27ac in SW480 and SW620 cells. b Heatmap showing the densities of enhancers marked by H3K4me2 and H3K27ac at the nearest differentially expressed genes between non-metastatic SW480 and liver metastatic CRC SW620 cells. Rows are ordered the same for all plots. RNA-seq and ChIP-seq signals are shown as log2 of tag counts normalized to 1 × 107 uniquely mapped tags. c Boxplots showing log2 ratios of SW620 to SW480 tag densities for genomic regions marked by H3K4me2 (left) and H3K27ac (right) around genes that are colon-specific genes with high expression in SW480 cells and liver-specific genes with high expression in SW620 cells. Statistically significant P value is indicated with asterisks (***P < 0.005, by t-test). spec., specific; reg., regulation. d Boxplots showing log2 ratios of PC-3 (bone metastatic prostate cancer cells) to 22Rv1 (primary prostate cancer cells) (left), T84 (lung metastatic CRC cells) to HCT116 (primary CRC cells) (middle) and Capan-1 (liver metastatic pancreas cancer cells) to Capan-2 (primary pancreas cancer cells) (right) tag densities for genomic regions marked by H3K27ac around tissue-specific genes that are relatively highly expressed in corresponding cells. Statistically significant P value is indicated with asterisks (***P < 0.005, by t-test). spec., specific; reg., regulation. e Enhancers are ranked by increasing H3K27ac ChIP-seq signal in primary (SW480) and liver-prone metastasis CRC cells (SW620). Points in red indicated super-enhancers, which are past the point where the slope is greater than 1. f Venn diagram indicating overlap and specificity of super-enhancers in SW480 and SW620 cells. g Scatterplot of the relationship between the ratio of SW480 to SW620 H3K27ac tag density at super-enhancers (x axis) and the ratio of nearest gene expression (y axis). The RNA-seq and ChIP-seq signals are shown as log2 of tag counts normalized to 1 × 107 uniquely mapped tags. Red dots represent some liver-specific genes (FOXA2, SARDH, ENO3, SLC9A3R2, ABHD2, LRP5 and PROX1) associated with super-enhancers. The Pearson correlation coefficient is 0.807 and P < 2.2e-16. h UCSC genome browser images of super-enhancers around a liver-specific gene SARDH and a colon-specific gene GLI3 in SW620 and SW480 covered genomic regions marked by H3K27ac. Bars labeled with SE indicate super-enhancers

Article Snippet: 61 After crosslinking, chromatin was fragmented by sonication, and the mixture was purified with magnetic beads (Millipore Cat. No. 16–157) conjugated with 1 ng of the antibodies against H3K4me1 (Abcam Cat. No. ab8895), H3K4me2 (Millipore Cat. No. 07–030), H3K4me3 (Millipore Cat. No. 07–473), H3K27ac (Abcam Cat. No. ab4729) or FOXA2 (Proteintech Cat. No. 22474–1-AP).

Techniques: RNA Sequencing Assay, ChIP-sequencing, Expressing, Labeling

Journal: Cell Research

Article Title: Tissue-specific transcription reprogramming promotes liver metastasis of colorectal cancer

doi: 10.1038/s41422-019-0259-z

Figure Lengend Snippet: FOXA2 is required for the activation of liver-specific genes and liver colonization of CRC cells. a Top enriched DNA binding motifs with significant P values, identified in a de novo motif analysis of SW620-unique enhancers (non-promoter regions marked by H3K4me2, far from 3 kb up- or downstream of TSS). b The blue and red bars in the chart show the expression of FOXA2 in normal colon and liver tissues, respectively. The Y axis shows the mean RPKM value. RNA-seq data were obtained from the GTEx project database. c UCSC genome browser images of a super-enhancer around the liver-specific gene FOXA2 in SW620 and SW480 covered genomic regions marked by H3K27ac and H3K4me2, with corresponding RNA-seq data for SW480 and SW620 cells. Bars labeled with SE indicate super-enhancers. d Immunoblot analysis was used to assess the efficiency of FOXA2 knockdown in SW620 cells. GAPDH served as a loading control (Left panel). GSEA of liver-specific signatures as defined from the GTEx project database is shown. Genes are ranked by the log2 fold change of the FPKM values in SW620 FOXA2-knockdown versus shNC cells. The NES and FDR are shown (Right two panels). e Immunoblot analysis was used to assess efficiency of FOXA2 knockdown in Colo205 cells. GAPDH served as a loading control (Left panel). GSEA of liver-specific signatures as defined from the GTEx project database is shown. Genes are ranked by the log2 fold change of the FPKM values in Colo205 FOXA2-knockdown versus shNC cells. The NES and FDR are shown (Right two panels). f Immunoblot analysis was used to assess efficiency of FOXA2 overexpression in SW480 cells. GAPDH served as a loading control (Left panel). GSEA of liver-specific signatures as defined from the GTEx project database is shown. Genes are ranked by the log2 fold change of the FPKM values in SW480 FOXA2-overexpression versus Control cells. The NES and FDR are shown (Right panel). g Immunoblot analysis was used to assess efficiency of FOXA2 overexpression in HCT116 cells. GAPDH served as a loading control (Left panel). GSEA of liver-specific signatures as defined from the GTEx project database is shown. Genes are ranked by the log2 fold change of the FPKM values in HCT116 FOXA2-overexpression versus Control cells. The NES and FDR are shown (Right panel). h FOXA2 is highly expressed in human liver metastatic CRC tumors as compared with primary tumors. The expression data are from the GSE41258 dataset. Each dot represents one primary or liver metastatic CRC tumor, and the red bar represents the mean value. Statistically significant P values are indicated with asterisks (***P < 0.001, by t-test). i Plots showing the IHC scores for the nuclear FOXA2 in human primary CRC tumors and liver metastatic CRC tumors. Each dot represents one primary or liver metastatic CRC tumor, and the red bar represents the mean value. Statistically-significant P values are indicated with asterisks (**P < 0.005, by t-test). j Representative IHC images of FOXA2 expression in paired CRC primary tumors and liver metastatic tumors. Scale bars, 500 µm. k Representative images of the liver colonies upon intrahepatic injection of SW620 shNC and shFOXA2 cells. l The stacked bars indicate the percentage of mice with liver colonies or with no liver colonies relative to the total number of mice in each section. Data were analyzed using Pearson’s Chi-square test. *P < 0.05, ***P < 0.001. The hepatic colonization rate is indicated at the bottom. m Representative images of the liver colonies (Top panel) upon intrahepatic injection of SW480 Control and FOXA2-overexpressing cells. The stacked bars indicate the percentage of mice with liver colonies and with no liver colonies relative to the total number of mice in each section. Data were analyzed using Pearson’s Chi-square test. n.s., not significant. The hepatic colonization rate is indicated at the bottom (Bottom panel). n Representative images of the liver colonies (Top panel) upon intracecal injection of HCT116 Control and FOXA2-overexpressing cells. The stacked bars indicate the percentage of mice with liver colonies and with no liver colonies relative to the total number of mice in each section. Data were analyzed using Pearson’s Chi-square test. n.s., not significant. The hepatic metastasis rate is indicated at the bottom (Bottom panel)

Article Snippet: 61 After crosslinking, chromatin was fragmented by sonication, and the mixture was purified with magnetic beads (Millipore Cat. No. 16–157) conjugated with 1 ng of the antibodies against H3K4me1 (Abcam Cat. No. ab8895), H3K4me2 (Millipore Cat. No. 07–030), H3K4me3 (Millipore Cat. No. 07–473), H3K27ac (Abcam Cat. No. ab4729) or FOXA2 (Proteintech Cat. No. 22474–1-AP).

Techniques: Activation Assay, Binding Assay, Expressing, RNA Sequencing Assay, Labeling, Western Blot, Over Expression, Injection

Journal: PLoS Biology

Article Title: Neuron Specific Rab4 Effector GRASP-1 Coordinates Membrane Specialization and Maturation of Recycling Endosomes

doi: 10.1371/journal.pbio.1000283

Figure Lengend Snippet: Immunogold EM of hippocampal neurons labeled with 10 nm protein A gold for Rab4 and with 15 nm protein A gold for GRASP-1 (A), with 10 nm protein A gold for syntaxin 13 and with 15 nm protein A gold for GRASP-1 (B), with 10 nm protein A gold for syntaxin 13 and with 15 nm protein A gold for Rab4 (C), or with 15 nm protein A gold for GRASP-1, with 5 nm protein gold for syntaxin 13, and with 10 nm protein A gold for rab4 (D). Arrow denotes tubular endosomal membrane to which GRASP-1, syntaxin 13, and Rab4 localized. EE indicates early endosomes and scale bar is 100 nm.

Article Snippet: The following antibodies were obtained from commercial sources: rabbit anti-GRASP-1 (AB96361), mouse anti-β-actin, mouse anti-GluR2 (Chemicon), mouse anti-Rab4, mouse anti-EEA1 (BD Biosciences), mouse anti-FLAG, mouse anti-MAP2, mouse anti-αtubulin (Sigma), mouse anti-GFP (Roche), mouse anti-bassoon (Stressgen), rabbit anti-β-galactosidase (MP Biomedicals), mouse anti-β-galactosidase (Promega), rabbit anti-GluR1 (Calbiochem), mouse anti-HA (Roche), rabbit anti-myc (Upstate Biotechnology), mouse LAMP-1 (Stressgen), mouse anti-myc, rabbit anti-Rab5 (Santa Cruz Biotechnology), rabbit anti-syntaxin 13 (Synaptic Systems), human anti-EEA1, and mouse anti-human TfR (ATCC).

Techniques: Labeling

Journal: PLoS Biology

Article Title: Neuron Specific Rab4 Effector GRASP-1 Coordinates Membrane Specialization and Maturation of Recycling Endosomes

doi: 10.1371/journal.pbio.1000283

Figure Lengend Snippet: (A) Lysates of COS-7 cells cotransfected with GFP-GRASP-1 and myc-syntaxins were immunoprecipitated with anti-GFP antibody and analyzed by Western blot. (B) Lysates of COS-7 cells cotransfected with GFP-syntaxin 13 and full-length myc-GRASP-1 (1–837) or truncated myc-GRASP-1 constructs (1–695 or 695–837) were immunoprecipitated with anti-GFP antibody and analyzed by Western blot. Asterisk indicates background band. Arrows point to co-precipitated GRASP-1 proteins. (C) Binding assay using lysates of COS-7 cells expressing myc-syntaxin 13 with or without GFP-GRASP-1 and GMP-PNP-charged GST-rab4. Note that myc-syntaxin 13 is only isolated on the beads in the presence of GRASP-1. (D) Binding assay using lysate of COS-7 cells transfected with GFP-GRASP-1(594–837) and GST-syntaxins without transmembrane domain (ΔTM). GRASP-1 was analyzed by Western blot with antibody against GFP. (E) Binding assay of 35 S-labeled GRASP-1 and immobilized GST-syntaxin 13ΔTM.

Article Snippet: The following antibodies were obtained from commercial sources: rabbit anti-GRASP-1 (AB96361), mouse anti-β-actin, mouse anti-GluR2 (Chemicon), mouse anti-Rab4, mouse anti-EEA1 (BD Biosciences), mouse anti-FLAG, mouse anti-MAP2, mouse anti-αtubulin (Sigma), mouse anti-GFP (Roche), mouse anti-bassoon (Stressgen), rabbit anti-β-galactosidase (MP Biomedicals), mouse anti-β-galactosidase (Promega), rabbit anti-GluR1 (Calbiochem), mouse anti-HA (Roche), rabbit anti-myc (Upstate Biotechnology), mouse LAMP-1 (Stressgen), mouse anti-myc, rabbit anti-Rab5 (Santa Cruz Biotechnology), rabbit anti-syntaxin 13 (Synaptic Systems), human anti-EEA1, and mouse anti-human TfR (ATCC).

Techniques: Immunoprecipitation, Western Blot, Construct, Binding Assay, Expressing, Isolation, Transfection, Labeling

Journal: PLoS Biology

Article Title: Neuron Specific Rab4 Effector GRASP-1 Coordinates Membrane Specialization and Maturation of Recycling Endosomes

doi: 10.1371/journal.pbio.1000283

Figure Lengend Snippet: (A) Representative image of hippocampal neuron triple transfected at DIV13 for 4 d with GFP-Rab4, HA-GRASP-1, and myc-syntaxin 13 and labeled with anti-HA (blue) or anti-myc (red) antibodies. Magnified region of the cell body is shown to indicate the strong colocalization of GRASP-1, Rab4, and syntaxin 13. (B) Representative images of dendrites of hippocampal neurons transfected at DIV13 with GFP-GRASP-1 for 4 d and labeled with anti-syntaxin 13 (red). (C) Representative images of dendrites of hippocampal neurons transfected at DIV13 with GFP-GRASP-1 for 4 d and labeled with anti-Neep21 (red). (D) Representative images of dendrites of hippocampal neurons cotransfected at DIV13 for 4 d with myc-syntaxin 13 and control vector or HA-GRASP-1 and labeled with anti-myc (green), anti-HA (blue), and anti-Neep21 (red). (E) Representative images of dendrites of hippocampal neurons cotransfected at DIV13 for 4 d with GFP-Rab4, HA-Rab11, and control vector or myc-syntaxin 13ΔTM and labeled with anti-myc (blue) and anti-HA (red). (F) Percentage of colocalization between HA-GRASP-1 and myc-syntaxin 1 or myc-syntaxin 13 in neurons. (G) Percentage of colocalization between myc-syntaxin 13 and Neep21 in dendrites as indicated in (D). (H) Percentage of colocalization between GFP-Rab4 and HA-Rab11 domains in dendrites expressing myc-syntaxin 13ΔTM as indicated in (E). Error bars indicate S.E.M. ** p <0.005. *** p <0.0005. Bar in A is 10 µm; Bar in (B–E) is 1 µm.

Article Snippet: The following antibodies were obtained from commercial sources: rabbit anti-GRASP-1 (AB96361), mouse anti-β-actin, mouse anti-GluR2 (Chemicon), mouse anti-Rab4, mouse anti-EEA1 (BD Biosciences), mouse anti-FLAG, mouse anti-MAP2, mouse anti-αtubulin (Sigma), mouse anti-GFP (Roche), mouse anti-bassoon (Stressgen), rabbit anti-β-galactosidase (MP Biomedicals), mouse anti-β-galactosidase (Promega), rabbit anti-GluR1 (Calbiochem), mouse anti-HA (Roche), rabbit anti-myc (Upstate Biotechnology), mouse LAMP-1 (Stressgen), mouse anti-myc, rabbit anti-Rab5 (Santa Cruz Biotechnology), rabbit anti-syntaxin 13 (Synaptic Systems), human anti-EEA1, and mouse anti-human TfR (ATCC).

Techniques: Transfection, Labeling, Plasmid Preparation, Expressing

Journal: PLoS Biology

Article Title: Neuron Specific Rab4 Effector GRASP-1 Coordinates Membrane Specialization and Maturation of Recycling Endosomes

doi: 10.1371/journal.pbio.1000283

Figure Lengend Snippet: Endosomes can be viewed as mosaic distribution of Rab4, Rab5, and Rab11 domains that dynamically interact via effector proteins and SNAREs. The Rab5 domain allows entry into the early/sorting endosome, whereas the Rab4 and Rab11 domains contain the machinery that is necessary for sorting and recycling membranes and receptors back to the plasma membrane. (A) GRASP-1 binds to Rab4 and syntaxin 13 and couples Rab4 and Rab11 recycling endosomes. The complex formed between GRASP-1 and t-SNARE syntaxin 13 might mediate fusion between Rab4 and Rab11 endosomes. (B) Absence of GRASP-1 interferes with complex formation at the recycling step, causing cargo accumulation in early endosomes, impairment of receptor expression, and changes in spine morphology. (C) Overexpression of GRASP-1 leads to recruitment of syntaxin 13 and strongly couples Rab4 and Rab11 domains, causing accumulation of internalized receptors in recycling endosomes. Consistent with the observed decrease in AMPAR clusters , Caspase-3 cleavage of GRASP-1 might separate the N-terminal Rab4 domain from the C-terminal syntaxin 13 binding site and disrupt the coupling between Rab4 and Rab11 domains.

Article Snippet: The following antibodies were obtained from commercial sources: rabbit anti-GRASP-1 (AB96361), mouse anti-β-actin, mouse anti-GluR2 (Chemicon), mouse anti-Rab4, mouse anti-EEA1 (BD Biosciences), mouse anti-FLAG, mouse anti-MAP2, mouse anti-αtubulin (Sigma), mouse anti-GFP (Roche), mouse anti-bassoon (Stressgen), rabbit anti-β-galactosidase (MP Biomedicals), mouse anti-β-galactosidase (Promega), rabbit anti-GluR1 (Calbiochem), mouse anti-HA (Roche), rabbit anti-myc (Upstate Biotechnology), mouse LAMP-1 (Stressgen), mouse anti-myc, rabbit anti-Rab5 (Santa Cruz Biotechnology), rabbit anti-syntaxin 13 (Synaptic Systems), human anti-EEA1, and mouse anti-human TfR (ATCC).

Techniques: Expressing, Over Expression, Binding Assay