Journal: Methods in molecular biology (Clifton, N.J.)

Article Title: Detecting Purinosome Metabolon Formation with Fluorescence Microscopy

doi: 10.1007/978-1-4939-7759-8_17

Figure Lengend Snippet: Colocalization of endogenous FGAMS and GART for visualization of purinosomes by immunofluorescence. Purine-depleted HeLa cells were fixed and permeabilized prior to being probed for with FGAMS rabbit polyclonal antibody and GART mouse monoclonal antibody. Fluorescently labeled secondary antibodies CF488A-conjugated donkey anti-rabbit and CF568-conjugated donkey anti-mouse were used to visualize the expression and localization of FGAMS and GART, respectively. A representative image of an individual cell was captured using a 100× oil objective on an Olympus Fluoview 1000 confocal laser scanning microscope. Sequential imaging of CF488A and CF568 showed colocalization of (a) FGAMS with (b) GART as represented by the yellow puncta present in (c) the merged image. Scale bar: 10 μm

Article Snippet: Primary antibody solution: 1:500 dilution of PFAS rabbit polyclonal antibody (Bethyl Laboratories) and 1:1000 dilution of GART mouse monoclonal antibody (Novus Biologicals) prepared in blocking buffer.

Techniques: Immunofluorescence, Labeling, Expressing, Laser-Scanning Microscopy, Imaging

Journal: Oncotarget

Article Title: N -acetylcysteine negatively regulates Notch3 and its malignant signaling

doi: 10.18632/oncotarget.8806

Figure Lengend Snippet: A. Time- and dose-dependent inhibition of NAC on the intracellular domain of Notch3 (N3IC), but not Notch1 (N1IC). HeLa cells were treated with NAC (2-10 mM) for 0-24 h. B. Dose-dependent inhibition by NAC (0-10 mM, 6 h) on the protein expression of N3IC in HeLa cells. C. NAC treatment (5 mM, 0-12 h) reduces protein levels of N3IC and extracellular domain of Notch 3 (N3EC) but not full length Notch 3 precursor (N3FL) in HeLa cells. Densitometry quantifications of the protein bands were shown after normalization with their respective β-actin levels. Data are presented as means ± SE, n=3. *, p < 0.05 compared with their respective non-treated group.

Article Snippet: Mouse anti-Notch3 NECD antibody (H00004854-M01, Novus Biologicals, Littleton, CO, USA) recognizes the extracellular fragments and full length Notch3 precursor.

Techniques: Inhibition, Expressing

Journal: Oncotarget

Article Title: N -acetylcysteine negatively regulates Notch3 and its malignant signaling

doi: 10.18632/oncotarget.8806

Figure Lengend Snippet: A. NAC treatment (2-10 mM, 0-24 h) decreases Hes1 and HRT1 protein levels in HeLa cells. B. NAC treatment (0-10 mM for 6 h or 5 mM for 0-12 h) decreases Hes1 and HRT1 mRNA expression in HeLa cells. The mRNA expression of NAC-treated cells was normalized to that of non-treated cells whose value was set as 1. C. NAC treatment (0-10 mM, 12 h) inhibits Hes1 reporter activity in HeLa cells. The luciferase activity in NAC-treated cells was normalized to that of non-treated cells whose value was set as 1. D. Notch3 siRNA knockdown reduces Hes1 and HRT1 levels in HeLa cells. Protein levels were determined 2 days after siRNA transfection. siCtrl, scramble siRNA; siNotch3, Notch3 siRNA. Protein densitometry quantifications were shown after normalization with β-actin levels. Data are presented as means ± SE, n=3-4. *, p < 0.05 compared with their respective non-treated group.

Article Snippet: Mouse anti-Notch3 NECD antibody (H00004854-M01, Novus Biologicals, Littleton, CO, USA) recognizes the extracellular fragments and full length Notch3 precursor.

Techniques: Expressing, Activity Assay, Luciferase, Knockdown, Transfection

Journal: Oncotarget

Article Title: N -acetylcysteine negatively regulates Notch3 and its malignant signaling

doi: 10.18632/oncotarget.8806

Figure Lengend Snippet: A. Pre-treatment with a γ-secretase inhibitor, DAPT (20 μM, 30 min), had no effect on NAC-induced (5 mM, 0-12 h) decrease in N3IC protein expression in HeLa cells. B. NAC treatment (5 mM, 0-12 h) did not affect Notch3 mRNA expression in HeLa cells. C. Pre-treatment with NH 4 Cl (25 mM, 1 h), but not lactacystin (10 μM, 30 min), reversed NAC-induced (5 mM, 0-12 h) decrease of N3IC protein levels in HeLa cells. D. NAC treatment did not affect levels of exogenously expressed Notch3 active intracellular domain (N3ICD). HeLa cells were transfected with vectors expressing N3ICD or N3FL for 24 h, followed by treatment with NAC (5 mM, 0-12 h). E. Subcellular analysis of Notch3 protein levels following NAC treatment (5 mM, 6 h) in HeLa cells. Protein levels of N3FL, N3EC and N3IC in cytosolic, nuclear and membrane fractions were determined. Successful fractionation was evidenced by using the marker proteins GAPDH, cyclin B1, and Na + , K + -ATPase. N3FL, N3EC and N3IC denoted Notch3 full length, extracellular domain and intracellular domain, respectively. Protein densitometry quantifications were shown after normalization with β-actin (A, C, D) or their respective cellular compartment markers (E). Data are presented as means ± SE, n=3-4. *, p < 0.05 compared with their respective non-treated group.

Article Snippet: Mouse anti-Notch3 NECD antibody (H00004854-M01, Novus Biologicals, Littleton, CO, USA) recognizes the extracellular fragments and full length Notch3 precursor.

Techniques: Expressing, Transfection, Membrane, Fractionation, Marker

Journal: Oncotarget

Article Title: N -acetylcysteine negatively regulates Notch3 and its malignant signaling

doi: 10.18632/oncotarget.8806

Figure Lengend Snippet: N3ICD overexpression rescues NAC-induced inhibition of proliferation (A), migration (B), and invasion (C). A. Numbers of EV- and N3ICD-transfected cells were counted at 12-48 h after NAC treatment (0-10 mM, left panel). *, p < 0.05 compared with the EV-transfected cells within the same treatment and time point. B. Results of the wound healing assay (left panels) were expressed as the migration index (the distance migrated relative to the initial scraped gap) and that of EV-transfected cells without NAC treatment was set as 100% (middle panel). C. Cells per field on the insert membrane were imaged (left panels) and counted (middle panel). B and C: *, p < 0.05 compared with no NAC treatment; #, p < 0.05 compared with the EV-transfected cells within the same treatment. Percent rescue (A-C, right panels) after N3ICD expression was calculated by dividing the net change after NAC treatment in N3ICD-transfected cells by that in EV-transfected cells. Notch3 siRNA knockdown inhibits cell proliferation D. , migration E. , and invasion F. as assessed by the same approaches described above. Representative images for migration and invasion were shown. *, p < 0.05 compared with the siCtrl-transfected cells. All data are presented as mean ±SE, n=3. I, the initial seeded cell number. EV, empty vector; N3ICD, Notch3 active intracellular domain; siCtrl, scrambled siRNA; siNotch3, Notch3 siRNA.

Article Snippet: Mouse anti-Notch3 NECD antibody (H00004854-M01, Novus Biologicals, Littleton, CO, USA) recognizes the extracellular fragments and full length Notch3 precursor.

Techniques: Over Expression, Inhibition, Migration, Transfection, Wound Healing Assay, Membrane, Expressing, Knockdown, Plasmid Preparation

Journal: Oncotarget

Article Title: N -acetylcysteine negatively regulates Notch3 and its malignant signaling

doi: 10.18632/oncotarget.8806

Figure Lengend Snippet: A. NAC treatment (5 and 10 mM, 0-24 h) decreases N3IC protein levels in HCC1937 cells. Expression of exogenous N3ICD rescues NAC-induced inhibition of proliferation B. , migration C. , and invasion D. , and Notch3 siRNA knockdown inhibits proliferation E. , migration F. , and invasion G. in HCC1937 cells. Quantifications, sample size, statistics, and abbreviations for protein levels, proliferation, migration, and invasion assays were as described in Figure & legends.

Article Snippet: Mouse anti-Notch3 NECD antibody (H00004854-M01, Novus Biologicals, Littleton, CO, USA) recognizes the extracellular fragments and full length Notch3 precursor.

Techniques: Expressing, Inhibition, Migration, Knockdown

Journal: Journal of the American Society of Nephrology

Article Title: Chordin-like 1 and Twisted Gastrulation 1 Regulate BMP Signaling following Kidney Injury

doi: 10.1681/asn.2008070768

Figure Lengend Snippet: Figure 4. CHRDL1 functions as a general BMP-signaling amplifier but specifically antagonizes BMP7 in the presence of TWSG1. (A) P19 embryonal carcinoma cells transfected with the BMP transcriptional reporter pBRE-Luc were incubated overnight with BMP4 (5 ng/ml) or BMP7 (10 ng/ml) and increasing amounts of CHRDL1 (50 to 400 ng/ml) or Chordin (100 to 800 ng/ml). Both CHRDL1 and Chordin amplify BMP4 signaling; however, in contrast to Chordin, which antagonizes BMP7 signaling, CHRDL1 amplifies BMP7 signaling in a dosage-responsive manner. (B) In the presence of TWSG1 (100 to 400 ng/ml), CHRDL1 (200 ng/ml) continues to act as an amplifier of BMP4 signaling unlike Chordin (400 ng/ml), which becomes a potent BMP4 antagonist. In contrast, CHRDL1 amplification of BMP7 signaling is converted to antagonism by addition of TWSG1 in a dosage-dependent manner. (C) In the P19 pBRE-luc reporter assay, the addition of TWSG1, CHRDL1, or Chordin (400 ng/ml) alone does not affect transcriptional activation. (D) CHRDL1 binds BMP7 and TWSG1 only in a trimolecular complex. A co-immunoprecipitation experiment in which CHRDL1 (500 ng/ml) is incubated in the presence of TWSG1 (500 ng/ml) and/or BMP7 (250 ng/ml) demonstrates that CHRDL1 binds TWSG1 and BMP7 only when all three proteins are present. No evidence of binding is detected when TWSG1 or BMP7 alone is incubated with CHRDL1. Recombinant proteins were immunoprecipitated with a goat polyclonal antibody against CHRDL1, and blots of goat IgG serve as loading controls. The last panel, a CHRDL1 immunoblot of an identical immunoprecipitation substituting an irrelevant goat antibody, demonstrates the absence of nonspecific binding by CHRDL1 to either the goat antibody or Protein G beads used in the experiment. The first lane of all blots contains recombinant protein as a positive control. (E) TWSG1 is strongly expressed in tubule epithelia of the mouse and human kidney. (Top) Strong immunohistochemical staining for TWSG1 in tubule epithelia of adult mouse kidney but weak staining in the glomerulus (G) and interstitial cell population (inset). (Right) Negative control using an antibody of the same species specific for macrophage. (Bottom) Immunofluorescent micro- graphs of an adult human kidney showing TWSG1 expression (red) in both proximal tubules (PT), marked green with Lotus lectin, and other tubules. As in the mouse, glomerular expression of TWSG1 is much weaker than seen in tubular epithelia. (Right) Negative control for TWSG1.

Article Snippet: BMP7 was detected with mouse anti- BMP7 (R&D Systems) and a sheep anti-mouse HRP conjugate (Am- ersham).

Techniques: Transfection, Incubation, Amplification, Reporter Assay, Activation Assay, Immunoprecipitation, Binding Assay, Recombinant, Western Blot, Positive Control, Immunohistochemical staining, Staining, Negative Control, Expressing

Journal: Journal of the American Society of Nephrology

Article Title: Chordin-like 1 and Twisted Gastrulation 1 Regulate BMP Signaling following Kidney Injury

doi: 10.1681/asn.2008070768

Figure Lengend Snippet: Figure 5. CHRDL1 reduces BMP7-stimulated Smad activation and ID gene expression in the presence of TWSG1 but has no effect on BMP4 signaling. (A and B) In the presence of TWSG1, CHRDL1 inhibits Smad phosphorylation by BMP7 but not by BMP4. P19 cells were serum starved for 2 h and then incubated with BMPs (10 ng/ml) and antagonists for 2 h before lysis and Western blotting. Blots were probed for phosphorylated Smads 1, 5, and 8 and -tubulin. In lanes 3, 4, and 5, Noggin (200 ng/ml), TWSG1, and CHRDL1 alone (400 ng/ml) were added. In lanes 6 through 9 CHRDL1 (400 ng/ml) was added together with increasing concentrations of TWSG1 (50, 100, 200, and 400 ng/ml). BMPs were incubated at 37 C for 1 h with or without antagonists before application. Note that the arrow points to the band corresponding to pSmad1/5/8. The top band (arrowhead) is a contaminating band specific to P19 lysates and is not present in lysates from MDCK cells (compare C and D). (C and D) The effects of CHRDL1 and TWSG1 on BMP signaling can be reproduced in the MDCK kidney cell line. (E) RT-PCR showing expression of ID1, 2, and 3 genes in HK-2 cells stimulated with BMP4 or 7 (25 ng/ml) for 6 h in the presence of varying concentrations (100 to 400 ng/ml) of CHRDL1 and/or TWSG1. ID expression by HK-2 cells in response to BMP4 is unaffected by the presence of TWSG1. In cells incubated with BMP7, the addition of TWSG1 reduces ID gene expression.

Article Snippet: BMP7 was detected with mouse anti- BMP7 (R&D Systems) and a sheep anti-mouse HRP conjugate (Am- ersham).

Techniques: Activation Assay, Gene Expression, Phospho-proteomics, Incubation, Lysis, Western Blot, Reverse Transcription Polymerase Chain Reaction, Expressing

Journal: Journal of the American Society of Nephrology

Article Title: Chordin-like 1 and Twisted Gastrulation 1 Regulate BMP Signaling following Kidney Injury

doi: 10.1681/asn.2008070768

Figure Lengend Snippet: Figure 6. Overexpression of mouse Chrdl1 in the collecting duct substan- tially reduces BMP signaling in vivo. (A) Diagram of mouse Chrdl1 transgene construct driven by a collecting duct–specific enhancer element from intron 1 of the Bmp7 gene; quantitative PCR assay shows Chrdl1 overexpression in kidneys of two embryonic day 17.5 transgenic embryos (150 and 151) com- pared with wild-type. (B through D) Immunolocalization of phosphorylated Smads (red) in the embryonic day 17.5 wild-type kidney (B), and kidneys from transgenic embryos 150 and 151 (C and D) show that BMP signaling is substantially reduced by expression of the Chrdl1 transgene. Collecting ducts (CD) were localized by staining with the lectin Dolichos Biflorus Agglu- tinin (green), and nuclei were counterstained with DAPI (blue).

Article Snippet: BMP7 was detected with mouse anti- BMP7 (R&D Systems) and a sheep anti-mouse HRP conjugate (Am- ersham).

Techniques: Over Expression, In Vivo, Construct, Real-time Polymerase Chain Reaction, Transgenic Assay, Expressing, Staining

Journal: Redox Biology

Article Title: Targeting feed-forward signaling of TGFβ/NOX4/DHFR/eNOS uncoupling/TGFβ axis with anti-TGFβ and folic acid attenuates formation of aortic aneurysms: Novel mechanisms and therapeutics

doi: 10.1016/j.redox.2020.101757

Figure Lengend Snippet: TGFβ blocking antibody recouples eNOS and attenuates ROS production via inhibition of NOX4 and restoration of DHFR expression in Fbn1 C1039G/+ mice. The aortas were isolated from 12 weeks old Fbn1 +/+ and Fbn1 C1039G/+ mice and subjected to Western blotting, immunohistochemistry (IHC) and electron spin resonance (ESR) analyses. (A) Representative Western blots and grouped densitometric data of aortic protein expression levels of inactive and mature TGFβ indicating upregulation of mature TGFβ in Fbn1 C1039G/+ mice. Data are presented as Mean ± SEM, n = 5–9. (B) Representative Western blots and grouped densitometric data of aortic NOX4 protein expression indicating upregulation of NOX4 in Fbn1 C1039G/+ mice. Data are presented as Mean ± SEM, n = 4. (C) Representative Western blots and grouped densitometric data of endothelial DHFR protein expression indicating downregulation of DHFR in Fbn1 C1039G/+ mice. Data are presented as Mean ± SEM, n = 5. (D) Representative IHC images of aortic roots, indicating downregulation of endothelial DHFR in 12 weeks Fbn1 C1039G/+ mice. Arrows indicating highly expressed DHFR in the endothelial layer of the control animals, which was however reduced in Fbn1 C1039G/+ mice. In parallel experiments, 4 weeks old Fbn1 C1039G/+ mice were treated with TGFβ blocking antibody for 4 weeks prior to analyses of NOX4 expression and eNOS uncoupling activity. (E) Representative Western blots and grouped densitometric data of aortic NOX4 protein expression in Fbn1 C1039G/+ mice treated with TGFβ blocking antibody, indicating abrogated NOX4 expression. Data are presented as Mean ± SEM, n = 4. (F) Representative Western blots and grouped densitometric data of endothelial DHFR protein expression in Fbn1 C1039G/+ mice treated with TGFβ blocking antibody, indicating restored DHFR expression. Data are presented as Mean ± SEM, n = 5. G) Superoxide production was determined by ESR in the aortic tissues of Fbn1 C1039G/+ mice after TGFβ blocking antibody treatment for 4 weeks. The results indicate that TGFβ blocking antibody reduced ROS production. Data are presented as Mean ± SEM, n = 5–7. (H) Total superoxide production in the presence or absence of l -NAME was determined by ESR in the aortic tissues of Fbn1 C1039G/+ mice after TGFβ blocking antibody treatment for 4 weeks. The results indicate recoupling of eNOS by treatment with TGFβ blocking antibody. Data are presented as Mean ± SEM, n = 5. Bar = 30 μm, *p < 0.05, **p < 0.01.

Article Snippet: Primary antibodies and their dilutions used were: TGFβ (1:500, Abcam, ab92486), NOX4 (1:500, Novus Biologicals, NB110–58849SS), DHFR (1:500, Novus Biologicals, H00001719-M01), eNOS (1:2000, BD Transduction Laboratories, 610297), GTPCHI (1:400, Sigma-Aldrich, SAB1410516), Fibrillin (1:500, Invitrogen, MA5-12770), and β-actin (1:3000, Sigma-Aldrich, A2066).

Techniques: Blocking Assay, Inhibition, Expressing, Isolation, Western Blot, Immunohistochemistry, Electron Paramagnetic Resonance, Control, Activity Assay

Journal: Redox Biology

Article Title: Targeting feed-forward signaling of TGFβ/NOX4/DHFR/eNOS uncoupling/TGFβ axis with anti-TGFβ and folic acid attenuates formation of aortic aneurysms: Novel mechanisms and therapeutics

doi: 10.1016/j.redox.2020.101757

Figure Lengend Snippet: Folic acid diet restores endothelial DHFR protein expression and activity in Fbn1 C1039/+ mice. Fbn1 +/+ and Fbn1 C1039G/+ mice of 4 weeks age were treated with FA diet (15 mg/kg/day) for 4 and 8 weeks, after which Western blotting was used to examine endothelial DHFR and eNOS expression levels, while IHC was used to detect in situ DHFR protein expression. In parallel experiments, HPLC was used to determine DHFR activity in both isolated aortic endothelial cells (ECs) and the EC-denuded aortas. (A) Representative Western blots of endothelial DHFR and eNOS protein expression with β-actin serving as an internal control. (B) Grouped densitometric data of endothelial DHFR protein expression, indicating that FA diet upregulated endothelial DHFR protein abundance in both Fbn1 +/+ and Fbn1 C1039G/+ mice. Data are presented as Mean ± SEM, n = 4. (C) Grouped densitometric data of eNOS expression, indicating that FA diet decreased endothelial eNOS protein expression in both Fbn1 +/+ and Fbn1 C1039G/+ mice. When uncoupled, upregulation of eNOS represents a deteriorating response, which has been previously shown in apoE null mice and hph-1 mice where eNOS is modestly uncoupled at baseline. Hence, a reduction in eNOS while uncoupled is beneficial on the contrary. Data are presented as Mean ± SEM, n = 4. (D) Representative IHC images of aortic roots, indicating upregulation of DHFR protein abundance in 12 weeks Fbn1 C1039G/+ mice fed FA diet. Arrows indicate reduced DHFR expression in the endothelial layer which was however restored by oral FA administration. In parallel experiments, DHFR activity was determined from both isolated ECs. Data are presented as Mean ± SEM, n = 4. (E) and the EC-denuded aortas. Data are presented as Mean ± SEM, n = 4–8. (F), indicating increased activity in both fractions. However, only the endothelial DHFR activity is relevant to eNOS coupling/uncoupling activity. Bar = 30 μm, *p < 0.05, **p < 0.01, ***p < 0.001.

Article Snippet: Primary antibodies and their dilutions used were: TGFβ (1:500, Abcam, ab92486), NOX4 (1:500, Novus Biologicals, NB110–58849SS), DHFR (1:500, Novus Biologicals, H00001719-M01), eNOS (1:2000, BD Transduction Laboratories, 610297), GTPCHI (1:400, Sigma-Aldrich, SAB1410516), Fibrillin (1:500, Invitrogen, MA5-12770), and β-actin (1:3000, Sigma-Aldrich, A2066).

Techniques: Expressing, Activity Assay, Western Blot, In Situ, Isolation, Control, Quantitative Proteomics

Journal: Redox Biology

Article Title: Targeting feed-forward signaling of TGFβ/NOX4/DHFR/eNOS uncoupling/TGFβ axis with anti-TGFβ and folic acid attenuates formation of aortic aneurysms: Novel mechanisms and therapeutics

doi: 10.1016/j.redox.2020.101757

Figure Lengend Snippet: Targeting feed-forward signaling of TGFβ/NOX4/DHFR/eNOS uncoupling/TGFβ axis with anti-TGFβ and folic acid attenuates formation of aortic aneurysms. Our data for the first time establish a novel feed-forward mechanism of TGFβ/NOX4/DHFR/eNOS uncoupling/TGFβ axis in the development of TAA in MFS mice, targeting of which with anti-TGFβ or FA diet (via DHFR/H 4 B/eNOS recoupling/NO pathway) abrogates aneurysm formation in MFS mice. Notably, FA diet downregulated TGFβ and NOX4 protein expression, further disrupting the feed-forward loop to attenuate aneurysm formation. Correction of fibrillin is additionally beneficial in preserving GTPCHI protein abundance to maintain eNOS coupling activity.

Article Snippet: Primary antibodies and their dilutions used were: TGFβ (1:500, Abcam, ab92486), NOX4 (1:500, Novus Biologicals, NB110–58849SS), DHFR (1:500, Novus Biologicals, H00001719-M01), eNOS (1:2000, BD Transduction Laboratories, 610297), GTPCHI (1:400, Sigma-Aldrich, SAB1410516), Fibrillin (1:500, Invitrogen, MA5-12770), and β-actin (1:3000, Sigma-Aldrich, A2066).

Techniques: Expressing, Preserving, Quantitative Proteomics, Activity Assay