Journal: Cell Death & Disease

Article Title: CBAP promotes thymocyte negative selection by facilitating T-cell receptor proximal signaling

doi: 10.1038/cddis.2014.474

Figure Lengend Snippet: Reduced TCR-induced BIM accumulation in CBAP-deficient thymocytes. ( a ) Eight-week-old littermates were injected with 20 μ g anti-CD3 Ab. Total thymocytes were harvested without treatment or 24 h post injection. BIM and heat shock protein 70 (HSP70) were detected in cell lysates by western blotting. BIM expression in each group was quantified relative to HSP70 and plotted as mean±S.D. ( n =4). ( b ) Thymocytes were cultured on plates coated with anti-CD3 (10 μ g/ml) and anti-CD28 (20 μ g/ml) Abs. After the indicated times, cells were collected, and BIM and actin were detected by western blotting. Expression of BIM was quantified relative to actin ( n =4). ( c ) Thymocytes from indicated genotypes were cultured onto plates coated with anti-CD3 (1 μ g/ml) and anti-CD28 (5 μ g/ml) Abs or uncoated plates. After 24 h, thymocytes were collected and stained for CD8, CD4, annexin V, and PI. Survival of DP thymocytes was assessed by flow cytometry with statistical analysis. Specific TCR-induced apoptosis (▵) was calculated as the percentage of dead DP thymocytes in test cultures minus that in untreated cultures. Results were plotted as mean±S.D. ( n =4)

Article Snippet: Abs were obtained as follows: (p-)MAPK Family Ab Sampler Kit (Cell Signaling Technology, Danvers, MA, USA); ZAP70, GRB2, and pPLC γ 1(Y783) (Abcam, Cambridge, UK); PLC γ 1 (Abcam and Merck Millipore, Billerica, MA, USA); LAT, pLAT(Y191/Y195), and pZAP70(Y319) (Merck Millipore); BIM (ProSci, Poway, CA, USA); HSP70 (Santa Cruz Biotechnology, Dallas, TX, USA); and actin (Sigma-Aldrich).

Techniques: Injection, Western Blot, Expressing, Cell Culture, Staining, Flow Cytometry

Journal: PLoS ONE

Article Title: Increased liver-specific proteins in circulating extracellular vesicles as potential biomarkers for drug- and alcohol-induced liver injury

doi: 10.1371/journal.pone.0172463

Figure Lengend Snippet: Wild-type male BALB/c mice (6 weeks old) received a single ip injection of APAP (300 mg/kg) or saline control (CON) and were sacrificed after 24 h (n = 10/group). (A) Representative hematoxylin and eosin (H&E) staining of formalin-fixed liver sections. (B) Plasma ALT and AST levels. (C) Representative electron microscopic images of EVs purified from mouse plasma. (D) Analysis of the protein amounts in circulating EVs. The amounts of a liver-specific protein ALB were measured by ELISA specific for mouse ALB. (E) Wild-type male BALB/c mice (6 weeks old) were injected with a single dose of 75, 150, or 300 mg/kg APAP, and the protein amounts in circulating EVs were measured after 1 or 3 h (n = 10/group). The ALT levels are shown on the left while the circulating EV protein amounts are indicated on the right side of each Figure. (F) The amounts of albumin (ALB) in circulating EVs, isolated from plasma of the indicated groups, were measured by ELISA. * P < 0.05.

Article Snippet: The ELISA microplate provided in the commercial kit was pre-coated with an antibody specific to ALB (Abcam) and STN1 (Uscn Life Science, Houston, TX).

Techniques: Injection, Saline, Control, Staining, Clinical Proteomics, Purification, Enzyme-linked Immunosorbent Assay, Isolation

Journal: PLoS ONE

Article Title: Increased liver-specific proteins in circulating extracellular vesicles as potential biomarkers for drug- and alcohol-induced liver injury

doi: 10.1371/journal.pone.0172463

Figure Lengend Snippet: Acute liver injury was induced in BALB/c male mice by a single i.p. injection of APAP (300 mg/kg), and some of the mice received 10 mL/kg saline, 100 mg/kg NAC, or 200 mg/kg GSH intravenously 1.5 h after APAP administration (n = 10/group). (A) Representative H&E staining of formalin-fixed liver sections. (B) Plasma ALT levels. (C) Protein amounts in circulating EVs derived from each indicated group were calculated. (D) Detection of liver-specific proteins in circulating EVs by immunoblot analyses, as indicated. (E) The amounts of mouse ALB in EVs from each group were measured by ELISA. (F) Hep3B and primary hepatocyte cells were exposed to APAP (IC 50 ) without or with NAC (5 mM) for 24 h (n = 4/group). The amounts of EV proteins isolated from Hep3B and primary hepatocyte cells treated with APAP alone or APAP+NAC were measured. * P < 0.05.

Article Snippet: The ELISA microplate provided in the commercial kit was pre-coated with an antibody specific to ALB (Abcam) and STN1 (Uscn Life Science, Houston, TX).

Techniques: Injection, Saline, Staining, Clinical Proteomics, Derivative Assay, Western Blot, Enzyme-linked Immunosorbent Assay, Isolation

Journal: PLoS ONE

Article Title: Increased liver-specific proteins in circulating extracellular vesicles as potential biomarkers for drug- and alcohol-induced liver injury

doi: 10.1371/journal.pone.0172463

Figure Lengend Snippet: Wild-type male BALB/c mice (6 weeks old) were treated with a single ip injection of APAP (300 mg/kg) or intramuscular injection with 0% or 0.5% BPVC into the right and left tibialis anterior muscles and were sacrificed at 24 h post treatment (n = 10/group). (A) Representative H&E staining of formalin-fixed muscle sections for all indicated groups. (B) Plasma ALT and AST levels. (C) Analysis of the total protein amounts in circulating EVs isolated from the indicated groups. (D) Immunoblot analyses of liver-specific or muscle-specific proteins in circulating EVs from each group, as indicated. (E) The amounts of ALB or STN1 in circulating EVs from each group were respectively measured by ELISA specific for mouse proteins. * P < 0.05.

Article Snippet: The ELISA microplate provided in the commercial kit was pre-coated with an antibody specific to ALB (Abcam) and STN1 (Uscn Life Science, Houston, TX).

Techniques: Injection, Muscles, Staining, Clinical Proteomics, Isolation, Western Blot, Enzyme-linked Immunosorbent Assay

Journal: PLoS ONE

Article Title: Increased liver-specific proteins in circulating extracellular vesicles as potential biomarkers for drug- and alcohol-induced liver injury

doi: 10.1371/journal.pone.0172463

Figure Lengend Snippet: Wild-type male BALB/c mice (6 weeks old) were treated a single ip injection with PBS, DGAL (1,000 mg/kg), TAA (200 mg/kg), or PCN (2,400 mg/kg, control group) for 24 h (n = 10/group). (A) Representative H&E staining of formalin-fixed liver sections. (B) Plasma ALT level. (C) Protein amounts in circulating EVs were measured. (D) Detection of liver-specific proteins in circulating EVs by immunoblot analyses for each target protein. (E) The amounts of liver-specific mouse ALB in EVs from the indicated groups were measured by ELISA. * P < 0.05.

Article Snippet: The ELISA microplate provided in the commercial kit was pre-coated with an antibody specific to ALB (Abcam) and STN1 (Uscn Life Science, Houston, TX).

Techniques: Injection, Control, Staining, Clinical Proteomics, Western Blot, Enzyme-linked Immunosorbent Assay

Journal: PLoS ONE

Article Title: Increased liver-specific proteins in circulating extracellular vesicles as potential biomarkers for drug- and alcohol-induced liver injury

doi: 10.1371/journal.pone.0172463

Figure Lengend Snippet: (A) Wild-type male BALB/c mice (6 weeks old) were treated twice with saline (CON) or 6 g ethanol/kg via oral gavage at 12-h interval and were sacrificed 1 h after the second dose (n = 10/group). Representative H&E staining of formalin-fixed liver sections. (B) Protein amounts of the circulating EVs isolated from control or alcohol-exposed mice, as indicated. * P < 0.05. (C) Detection of the liver-specific proteins in circulating EVs from the indicated mouse plasma by immunoblot analysis. (D) Analysis of the protein amounts in circulating EVs isolated from the sera of healthy controls (n = 9), who never drank alcohol due to religious reasons, and alcoholics (n = 13) with hepatitis. (E) The amounts of liver-specific protein ALB in circulating EVs from the indicated groups were measured by ELISA for human ALB protein. * P < 0.05. (F) Detection of the liver-specific proteins in circulating EVs from the different groups by immunoblot analyses, as indicated. * P < 0.05.

Article Snippet: The ELISA microplate provided in the commercial kit was pre-coated with an antibody specific to ALB (Abcam) and STN1 (Uscn Life Science, Houston, TX).

Techniques: Saline, Staining, Isolation, Control, Clinical Proteomics, Western Blot, Enzyme-linked Immunosorbent Assay

Journal: bioRxiv

Article Title: Human CST complex protects replication fork stability by directly blocking MRE11 degradation of nascent strand DNA

doi: 10.1101/797647

Figure Lengend Snippet: (A) Scheme of SIRF assay. Nascent DNA was pulse-labelled with EdU. Upon fork stalling, proteins located at stalled forks are in close proximity to EdU. EdU was then biotinylated using click reaction, followed by incubating with biotin and target protein antibodies. PLA amplification was used to visualize the localization of target proteins at EdU-labeled stalled forks. (B) Detection of CST at forks with SIRF assay. Cells stably expressing 13xMyc-CTC1 or 14xMyc-STN1 were used. To detect CST at stalled forks, cells were pulse-labeled with EdU for 8 min, followed by 4 mM HU treatment for 3 h. To detect CST at active forks, SIRF was performed without HU treatment. Representative images of vector only, 13Myc-CTC1, 14Myc-STN1 and PCNA on normal or stalled replication forks by SIRF analysis are shown. Scale bars: 20 µm. (C) Scatter plot of relative CTC1-SIRF, STN1-SIRF, and PCNA-SIRF fluorescence intensity with and without HU treatment. Relative SIRF fluorescence intensity was calculated by normalizing to respective untreated sample. In all SIRF experiments, three independent treatments were performed and results from one set of experiment are shown in each graph. P values were calculated by unpaired t-test with Welch’s correction. Red lines indicate the mean values. Error bars: SEM.

Article Snippet: The pCL-CTC1Δ700N-13xMyc plasmid was constructed by PCR amplifying CTC1Δ700 cDNA from pCL-CTC1-13xMyc, and then cloned into pCL-puro-FLAG-MYC plasmid using the In-Fusion® HD cloning kit (Takara). pBabe-puro-STN1-14xMyc was constructed by GenScript USA Inc. pCI-neo-His 6 -STN1-His 6 plasmid was constructed by PCR amplifying STN1 cDNA from pCI-neo-Myc-STN1 using primers containing His 6 sequences, and then cloning into the Xhol and Not1 sites of the pCI-neo vector.

Techniques: Amplification, Labeling, Stable Transfection, Expressing, Plasmid Preparation, Fluorescence

Journal: bioRxiv

Article Title: Human CST complex protects replication fork stability by directly blocking MRE11 degradation of nascent strand DNA

doi: 10.1101/797647

Figure Lengend Snippet: (A) Western blot showing STN1 and CTC1 knockdown. (B) CST deficiency causes ssDNA accumulation upon fork stalling. HeLa cells with STN1 knockdown were incubated with 10 µM BrdU for 48 h, followed by 2 mM HU treatment for 6 h, and subsequently stained with BrdU antibody under a non-denaturing condition to detect ssDNA. Scale bars: 20 µm. Amplified images showing distinct BrdU foci are included in Suppl. Figure S1. Relative BrdU fluorescence intensity was quantitated by Image J. P values were calculated by one-way ANOVA analysis with post hoc Tukey from three independent experiments. Error bars, SEM. (C) CST deficiency delays fork recovery. HeLa cells with CST knockdown were enriched in the mid-S phase as described in Methods, treated with 2 mM HU for 3 hrs to stall replication, and then released to no HU media for indicated time, fixed, and stained with RPA32 antibody. Scale bars in each image: 10 μm. Mean RPA32 nuclear foci fluorescence was quantified and relative foci fluorescence was calculated by normalizing to shLUC at 0 time point. Over all time points of P values were calculated by two-way ANOVA analysis with post hoc Tukey, p**** ≤ 0.0001. Error bars, SEM.

Article Snippet: The pCL-CTC1Δ700N-13xMyc plasmid was constructed by PCR amplifying CTC1Δ700 cDNA from pCL-CTC1-13xMyc, and then cloned into pCL-puro-FLAG-MYC plasmid using the In-Fusion® HD cloning kit (Takara). pBabe-puro-STN1-14xMyc was constructed by GenScript USA Inc. pCI-neo-His 6 -STN1-His 6 plasmid was constructed by PCR amplifying STN1 cDNA from pCI-neo-Myc-STN1 using primers containing His 6 sequences, and then cloning into the Xhol and Not1 sites of the pCI-neo vector.

Techniques: Western Blot, Incubation, Staining, Amplification, Fluorescence

Journal: bioRxiv

Article Title: Human CST complex protects replication fork stability by directly blocking MRE11 degradation of nascent strand DNA

doi: 10.1101/797647

Figure Lengend Snippet: (A) Scheme of DNA fiber analysis and representative fiber images. (B) DNA fiber analysis of BJ cells with STN1 knockdown with and without mirin treatment. In all DNA fiber experiments in this study, at least two independent treatments and fiber experiments were performed to ensure reproducibility. Results from one set of experiment are shown in each graph. Scatter plots indicates IdU/CIdU tract length ratios for individual replication forks. P values were calculated by one-way ANOVA analysis with post hoc Tukey. Red lines indicate the mean values. Western blots showing protein knockdown in DNA fiber assays are included. (C) DNA fiber analysis of U2OS cells with STN1 knockdown with and without mirin treatment. (D) DNA fiber analysis of HCT116 cells with STN1 knockdown with and without mirin treatment. (E) DNA fiber analysis of U2OS cells showing that SMARCAL1-mediated fork reversal is needed for nascent stand degradation observed in CST-deficient cells. (F) DNA fiber analysis of U2OS cells showing that ZRANB3-mediated fork reversal is needed for nascent strand degradation observed in CST-deficient cells. (G) Colony formation assay showing that STN1 depletion decreases cell viability in U2OS cells after exposure to CPT and MMS. Results are the average of three independent experiments, with triplicate in each experiment. P values were calculated by one-way ANOVA analysis with post hoc Tukey from three independent experiments. Error bars, SEM.

Article Snippet: The pCL-CTC1Δ700N-13xMyc plasmid was constructed by PCR amplifying CTC1Δ700 cDNA from pCL-CTC1-13xMyc, and then cloned into pCL-puro-FLAG-MYC plasmid using the In-Fusion® HD cloning kit (Takara). pBabe-puro-STN1-14xMyc was constructed by GenScript USA Inc. pCI-neo-His 6 -STN1-His 6 plasmid was constructed by PCR amplifying STN1 cDNA from pCI-neo-Myc-STN1 using primers containing His 6 sequences, and then cloning into the Xhol and Not1 sites of the pCI-neo vector.

Techniques: Western Blot, Colony Assay

Journal: bioRxiv

Article Title: Human CST complex protects replication fork stability by directly blocking MRE11 degradation of nascent strand DNA

doi: 10.1101/797647

Figure Lengend Snippet: (A) Illustration of the full-length CTC1 gene and Δ700N. STN1/TEN1 interacts with CTC1 via the predicted OB4 domain at the C-terminus, whereas the predicted N-terminal OB1 and OB2 domains are needed for DNA binding. (B) Deletion of the N-terminal 700 aa of CTC1 abolishes CTC1 localization at telomeres. U2OS stably expressing WT Myc-CTC1 and Myc-Δ700N were treated with or without HU and co-stained with Myc (red) and TRF2 (green) antibodies. Boxed areas are amplified in inserts to indicate CTC1/TRF2 colocalization (yellow). Scale bars: 10 μm. (C) Δ700N does not affect CST complex formation. HEK293T cells were co-transfected with Myc-CTC1, His 6 -STN1 and HA-TEN1. Co-IP was performed with Myc antibody to pulldown His 6 -STN1 and HA-TEN1. Full-length Myc-CTC1 was prone to degradation during immunoprecipitation. (D) Δ700N retains RAD51 interaction. HEK293T cells were co-transfected with Myc-CTC1, His 6 -STN1, HA-TEN1 and Flag-RAD51, and treated with HU (2 mM, 16 h). Co-IP was performed with Flag antibody to pulldown Myc-CTC1. (E) DNA fiber analysis of U2OS CTC1 knockdown cells rescued by RNAi-resistant WT and Δ700N. P values were calculated by one-way ANOVA analysis with post hoc Tukey. Red lines indicate the mean values.

Article Snippet: The pCL-CTC1Δ700N-13xMyc plasmid was constructed by PCR amplifying CTC1Δ700 cDNA from pCL-CTC1-13xMyc, and then cloned into pCL-puro-FLAG-MYC plasmid using the In-Fusion® HD cloning kit (Takara). pBabe-puro-STN1-14xMyc was constructed by GenScript USA Inc. pCI-neo-His 6 -STN1-His 6 plasmid was constructed by PCR amplifying STN1 cDNA from pCI-neo-Myc-STN1 using primers containing His 6 sequences, and then cloning into the Xhol and Not1 sites of the pCI-neo vector.

Techniques: Binding Assay, Stable Transfection, Expressing, Staining, Amplification, Transfection, Co-Immunoprecipitation Assay, Immunoprecipitation

Journal: bioRxiv

Article Title: Human CST complex protects replication fork stability by directly blocking MRE11 degradation of nascent strand DNA

doi: 10.1101/797647

Figure Lengend Snippet: (A) Micronuclei (MN) formation in BRCA2- and STN1-deficient cells. BRCA2 and STN1 were co-depleted in U2OS cells with siRNA or shRNA. Representative DAPI staining images are shown. Arrows point to MN. Scale bars: 20 µm. Average percentage of nuclei containing MN are shown. P values were calculated by one-way ANOVA analysis with post hoc Tukey from three independent experiments. Error bars: SEM. (B) Anaphase bridges (arrows) in BRCA2- and STN1-deficient U2OS cells. Results were from three independent knockdown experiments. Scale bar: 10 µm. P values were calculated by one-way ANOVA analysis with post hoc Tukey from three independent experiments. Error bars: SEM. (C) γH2AX induced by BRCA2 knockdown and STN1 knockdown in U2OS cells. Results were from three independent knockdown experiments. P values were calculated by one-way ANOVA analysis with post hoc Tukey from three independent experiments. Error bars: SEM. (D) Co-depletion of STN1 and BRCA2 significantly impairs DNA replication. Results were from three independent knockdown experiments. Scale bar 50 µm. P values were calculated by one-way ANOVA analysis with post hoc Tukey from three independent experiments. Error bars: SEM. (E) BRCA2 and CST form non-overlapping foci after HU treatment. U2OS cells stably expressing Myc-CTC1 were treated with or without HU (2 mM) and stained with anti-Myc, anti-BRCA2, and anti-pS33. Boxed areas are amplified and shown at the right side of images. White arrows point to colocalizations. Scale bars: 10 µm.

Article Snippet: The pCL-CTC1Δ700N-13xMyc plasmid was constructed by PCR amplifying CTC1Δ700 cDNA from pCL-CTC1-13xMyc, and then cloned into pCL-puro-FLAG-MYC plasmid using the In-Fusion® HD cloning kit (Takara). pBabe-puro-STN1-14xMyc was constructed by GenScript USA Inc. pCI-neo-His 6 -STN1-His 6 plasmid was constructed by PCR amplifying STN1 cDNA from pCI-neo-Myc-STN1 using primers containing His 6 sequences, and then cloning into the Xhol and Not1 sites of the pCI-neo vector.

Techniques: shRNA, Staining, Stable Transfection, Expressing, Amplification