Journal: Neoplasia (New York, N.Y.)

Article Title: Glycogen Synthase Kinase 3 Regulates Cell Death and Survival Signaling in Tumor Cells under Redox Stress 1

doi: 10.1016/j.neo.2014.07.012

Figure Lengend Snippet: 4HPR sustains GSK3β Ser9 phosphorylation in Y79 cells. GSK3β phosphorylation correlates with PARP cleavage and AMPK activation in cell death induced by 4HPR (2.5 μM). IGF-1 (100 ng/ml) does not affect PARP cleavage. 4HPR inhibits IGF-1–induced AKT phosphorylation at 24 hours.

Article Snippet: Human retinoblastoma Y79 cells (ATCC HTB-18), androgen-independent PC3 (ATCC CRL-1435), and DU145 (ATCC HTB-81) human prostate adenocarcinoma cells were obtained from American Type Culture Collection (ATCC) (Manassas, VA) and certified by the Interlab Cell Line Collection of the Biological Bank and Cell Factory core facility (member of the European BioBanking and Molecular Resources Research Infrastructure), IRCCS AOU S. Martino-IST, (Genoa, Italy).

Techniques: Phospho-proteomics, Activation Assay

Journal: Neoplasia (New York, N.Y.)

Article Title: Glycogen Synthase Kinase 3 Regulates Cell Death and Survival Signaling in Tumor Cells under Redox Stress 1

doi: 10.1016/j.neo.2014.07.012

Figure Lengend Snippet: (A) Induction of antioxidant enzymes is associated with PARP cleavage induced by 4HPR in Y79 cells. Densitometric analysis was carried out by normalizing protein expression versus GAPDH and calculation of fold increase over control samples. The dividing vertical line indicates the junction between control and lanes spliced from the blot. (B) Dose-dependent increase of GSK3β phosphorylation and HO-1 and Nrf2 expression in 4HPR-treated Y79 cells. (C) Time-dependent GSK3β phosphorylation, HO-1 expression, and PARP processing in PC3 cells treated with 4HPR. (D) Dose-dependent GSK3β phosphorylation, HO-1 and Nrf2 expression, PARP and caspase-8 processing in PC3 and DU145 cells treated with CDDO-Me (24 hours). pAMPK induction indicates ATP decline, in line with previous results . Pretreatment for 2 hours with the antioxidant NAC (10 mM) (E) or DFX (200 μM) (F) decreases GSK3β phosphorylation, PARP cleavage, and HO-1 expression induced by 4HPR (2.5 μM, 24 hours). (G) Similar modulation by NAC of the molecular pattern analyzed under the effect of PEITC. HO-1 overexpression is visible with the combination of As 2 O 3 and NAC. In all panels, results from representative experiments are shown.

Article Snippet: Human retinoblastoma Y79 cells (ATCC HTB-18), androgen-independent PC3 (ATCC CRL-1435), and DU145 (ATCC HTB-81) human prostate adenocarcinoma cells were obtained from American Type Culture Collection (ATCC) (Manassas, VA) and certified by the Interlab Cell Line Collection of the Biological Bank and Cell Factory core facility (member of the European BioBanking and Molecular Resources Research Infrastructure), IRCCS AOU S. Martino-IST, (Genoa, Italy).

Techniques: Expressing, Control, Phospho-proteomics, Over Expression

Journal: Neoplasia (New York, N.Y.)

Article Title: Glycogen Synthase Kinase 3 Regulates Cell Death and Survival Signaling in Tumor Cells under Redox Stress 1

doi: 10.1016/j.neo.2014.07.012

Figure Lengend Snippet: (A) Determination of GSH with mBCl distinguishes Y79 cell subpopulations with different GSH content. Representative flow cytometry histogram of cell subpopulations (bright fluorescence, dim fluorescence, and dying cells; left) and fluorescence microscope image (right). The analysis of mBCl fluorescence was done on 10,000 gated cells. (B) The percentage of subpopulations as mean ± SD was calculated from three independent experiments run in duplicate. (C) Relative GSH content in the bright fluorescence population at 24 hours. (D) Relative GSH in the whole-cell population at 24 hours. (E) GSH levels determined with DTNB in cells treated with 4HPR (2.5 μM, 24 hours) and effects of 2-hour pretreatment with NAC (10 mM) or CHX (0.125 μg/ml). The relative GSH content was calculated from MFI data normalized versus controls set as 1 from three independent experiments run in duplicate. Results are expressed as means ± SD. *** P < .001 and ** P < .01, statistically significant difference versus control samples at 24 hours; ### P < .001, statistically significant difference versus samples treated with 4HPR alone.

Article Snippet: Human retinoblastoma Y79 cells (ATCC HTB-18), androgen-independent PC3 (ATCC CRL-1435), and DU145 (ATCC HTB-81) human prostate adenocarcinoma cells were obtained from American Type Culture Collection (ATCC) (Manassas, VA) and certified by the Interlab Cell Line Collection of the Biological Bank and Cell Factory core facility (member of the European BioBanking and Molecular Resources Research Infrastructure), IRCCS AOU S. Martino-IST, (Genoa, Italy).

Techniques: Flow Cytometry, Fluorescence, Microscopy, Control

Journal: Neoplasia (New York, N.Y.)

Article Title: Glycogen Synthase Kinase 3 Regulates Cell Death and Survival Signaling in Tumor Cells under Redox Stress 1

doi: 10.1016/j.neo.2014.07.012

Figure Lengend Snippet: (A) Y79 retinoblastoma cells resistant to 4HPR show increased GSK3β phosphorylation that correlates with lack of PARP cleavage. (B) GSH and NAD(P)H levels are increased in 4HPR-resistant Y79 cells (R2.5). Increased GSK3β phosphorylation and high basal HO-1 and GCLC expression in PC3 (C) and DU145 (E) cells resistant to 4HPR (5 μM). GSH and NAD(P)H levels in PC3 (D) and DU145 (F) cells resistant to 5 μM 4HPR (R5). Data represent means ± SD from two flow cytometry analyses run in duplicate. *** P < .001 versus wt cells.

Article Snippet: Human retinoblastoma Y79 cells (ATCC HTB-18), androgen-independent PC3 (ATCC CRL-1435), and DU145 (ATCC HTB-81) human prostate adenocarcinoma cells were obtained from American Type Culture Collection (ATCC) (Manassas, VA) and certified by the Interlab Cell Line Collection of the Biological Bank and Cell Factory core facility (member of the European BioBanking and Molecular Resources Research Infrastructure), IRCCS AOU S. Martino-IST, (Genoa, Italy).

Techniques: Phospho-proteomics, Expressing, Flow Cytometry

Journal: Neoplasia (New York, N.Y.)

Article Title: Glycogen Synthase Kinase 3 Regulates Cell Death and Survival Signaling in Tumor Cells under Redox Stress 1

doi: 10.1016/j.neo.2014.07.012

Figure Lengend Snippet: Interference with glucose metabolism hinders GSK3β phosphorylation induced by 4HPR and As 2 O 3 . (A) Y79 cells were pretreated with 2DG (50 mM) for 2 hours and then treated with 2.5 μM 4HPR for 24 hours. AMPK activation indicates energy depletion. (B) Effects of 2DG on GSK3β phosphorylation induced by As 2 O 3 . (C) Transient elevation at 16 hours and decreased activity of G6PD at 24 hours after 4HPR administration. Results expressed as absorbance per minute per milligram of protein were normalized relative to controls. *** P < .001, statistical significant differences between control and 4HPR-treated samples at 16 and 24 hours. (D) Effects of G6PD inhibition by 6AN (1 mM). (E) Decreased MTT reduction and ATP depletion in Y79 cells pretreated with 2DG or 6AN for 2 hours and treated with 4HPR for 24 hours (left). *** P < .001, statistically significant difference versus samples treated with 4HPR alone. (F) Glucose uptake, as determined with the fluorescent glucose analogue 2-NBDG or 6-NBDG and flow cytometry analysis, in wt and 4HPR-resistant Y79 cells. *** P < .001 and ** P < .01.

Article Snippet: Human retinoblastoma Y79 cells (ATCC HTB-18), androgen-independent PC3 (ATCC CRL-1435), and DU145 (ATCC HTB-81) human prostate adenocarcinoma cells were obtained from American Type Culture Collection (ATCC) (Manassas, VA) and certified by the Interlab Cell Line Collection of the Biological Bank and Cell Factory core facility (member of the European BioBanking and Molecular Resources Research Infrastructure), IRCCS AOU S. Martino-IST, (Genoa, Italy).

Techniques: Phospho-proteomics, Activation Assay, Activity Assay, Control, Inhibition, Flow Cytometry

Journal: Neoplasia (New York, N.Y.)

Article Title: Glycogen Synthase Kinase 3 Regulates Cell Death and Survival Signaling in Tumor Cells under Redox Stress 1

doi: 10.1016/j.neo.2014.07.012

Figure Lengend Snippet: Analysis of signaling pathways regulating GSK3β phosphorylation in Y79 cells. (A) The MEK inhibitor UO126 decreases GSK3β phosphorylation, PARP cleavage, HO-1, and p90RSK phosphorylation induced by 4HPR at 24 hours. (B) UO126 rescues loss of MTT reduction and cell membrane damage, as measured by propidium iodide (PI) permeability, by 4HPR (2.5 μM) at 48 hours. The data shown are means ± SD of three independent experiments run in duplicate. (C) UO126 preserves GSH levels. Data were collected at 48 hours of treatment with 4HPR (2.5 μM) from three independent measurements run in duplicate. Means ± SD are shown. *** P < .001, statistical significance of differences versus samples treated with 4HPR alone. (D) The antioxidant NAC inhibits sustained ERK1/2 phosphorylation induced by 4HPR at 24 hours, an indication of redox control of ERK1/2 during oxidative stress. (E) The p90RSK inhibitor BI-D1780 inhibits GSK3β phosphorylation, but not ERK1/2 phosphorylation, suggesting GSK3β regulation by p90RSK downstream ERK1/2. (F) BI-D1780 (10 μM) exacerbates 4HPR toxicity in Y79 cells (2.5 μM, 24 hours). *** P < .001, statistical significance of differences versus samples treated with 4HPR alone.

Article Snippet: Human retinoblastoma Y79 cells (ATCC HTB-18), androgen-independent PC3 (ATCC CRL-1435), and DU145 (ATCC HTB-81) human prostate adenocarcinoma cells were obtained from American Type Culture Collection (ATCC) (Manassas, VA) and certified by the Interlab Cell Line Collection of the Biological Bank and Cell Factory core facility (member of the European BioBanking and Molecular Resources Research Infrastructure), IRCCS AOU S. Martino-IST, (Genoa, Italy).

Techniques: Protein-Protein interactions, Phospho-proteomics, Membrane, Permeability, Control

Journal: Frontiers in Microbiology

Article Title: The dual action of poly(ADP-ribose) polymerase -1 (PARP-1) inhibition in HIV-1 infection: HIV-1 LTR inhibition and diminution in Rho GTPase activity

doi: 10.3389/fmicb.2015.00878

Figure Lengend Snippet: Inhibition of Rho or Rac GTPase decreases HIV-1 reverse transcriptase (RT) activity in HIV-1 infected human macrophages (MDM) . RT activity assays performed on HIV-infected MDM treated with Rho and Rac GTPase inhibitors, CT04, NSC, or AZT either for 24 h prior to HIV infection (A) or immediately after (B) HIV infection. The results are shown as the mean value ± SEM from three independent experiments utilizing MDM prepared from different donors. Treatments with Rho and Rac GTPase inhibitors or AZT were replenished every 12 h. ( *** p = 0.005, **** p < 0.0001).

Article Snippet: Rho A GTPase-specific inhibitor, CT04, and Rho GTPase switch™ activator, CN04, were purchased from Cytoskeleton Inc. (Denver, CO).

Techniques: Inhibition, Activity Assay, Infection

Journal: Frontiers in Microbiology

Article Title: The dual action of poly(ADP-ribose) polymerase -1 (PARP-1) inhibition in HIV-1 infection: HIV-1 LTR inhibition and diminution in Rho GTPase activity

doi: 10.3389/fmicb.2015.00878

Figure Lengend Snippet: PARP inhibition decreases Rac1 activation in primary MDM. (A) RhoA and (B) Rac1 GTPases activity was measured in primary MDMs. GTPases were activated by cross-linking (x-linking) of the CXCR4 receptor with α-CXCR4 antibody (mimicking HIV-1 engagement with macrophage cells). The level of RhoA or Rac1 activity in non-treated cross-linked cells was assigned a value of 1 to calculate the relative ratio of activation. The results are shown as the mean value ± SEM from three independent experiments. ( * p < 0.05, ** p < 0.005, and *** p < 0.0001 vs. cross-linked but inhibitor untreated cells).

Article Snippet: Rho A GTPase-specific inhibitor, CT04, and Rho GTPase switch™ activator, CN04, were purchased from Cytoskeleton Inc. (Denver, CO).

Techniques: Inhibition, Activation Assay, Activity Assay

Journal: Frontiers in Microbiology

Article Title: The dual action of poly(ADP-ribose) polymerase -1 (PARP-1) inhibition in HIV-1 infection: HIV-1 LTR inhibition and diminution in Rho GTPase activity

doi: 10.3389/fmicb.2015.00878

Figure Lengend Snippet: PARP inhibition increases inhibitory site Ser3-cofilin phosphorylation. (A) Representative contour plots from flow cytometry analysis of phospho-Ser3-cofilin and total cofilin in MDM stimulated by addition of HIV ADA for 5 min. MDM were treated with or without PARPi for 1 h prior to HIV ADA introduction. Rho and Rac GTPase inhibitors, CT04, NSC, and Rho/Rac activator, CN04, were used as controls. (B) Quantitation of phosphor-Ser3-cofilin cells in human MDM. The results are shown as the mean value ± SEM ( * p < 0.05 vs. HIV-stimulated but inhibitor untreated cells) from three independent experiments utilizing MDM prepared from different donors.

Article Snippet: Rho A GTPase-specific inhibitor, CT04, and Rho GTPase switch™ activator, CN04, were purchased from Cytoskeleton Inc. (Denver, CO).

Techniques: Inhibition, Flow Cytometry, Quantitation Assay

Journal: Frontiers in Microbiology

Article Title: The dual action of poly(ADP-ribose) polymerase -1 (PARP-1) inhibition in HIV-1 infection: HIV-1 LTR inhibition and diminution in Rho GTPase activity

doi: 10.3389/fmicb.2015.00878

Figure Lengend Snippet: PARP inhibitors suppress activation of small GTPases in MDM and affect actin rearrangements. (A) Representative histograms from flow cytometry analysis of F-actin and G-actin in MDM stimulated by addition of HIV ADA for 5 min. MDM were treated with or without PARPi for 1 h prior to HIV ADA introduction. Rho and Rac GTPase inhibitors, CT04, NSC, and Rho/Rac activator, CN04, used as controls. (B) Graphical representation of F-actin to G-actin ratios. The results are shown as the mean value ± SEM ( * p < 0.05 vs. HIV-stimulated but inhibitor untreated cells) from three independent experiments utilizing MDM prepared from different donors.

Article Snippet: Rho A GTPase-specific inhibitor, CT04, and Rho GTPase switch™ activator, CN04, were purchased from Cytoskeleton Inc. (Denver, CO).

Techniques: Activation Assay, Flow Cytometry

Journal: Acta Pharmaceutica Sinica. B

Article Title: A tactical nanomissile mobilizing antitumor immunity enables neoadjuvant chemo-immunotherapy to minimize postsurgical tumor metastasis and recurrence

doi: 10.1016/j.apsb.2022.09.017

Figure Lengend Snippet: Mit elicited the immunogenic death of B16–F10 cells and the preparation diagram of mitoxantrone@anti-PD-L1/azobenzene-lipo (MPAL). (A) CRT detections on B16–F10 cells after being treated with different concentrations of Mit using flow cytometry. (B) CRT expression on B16–F10 cells in response to Mit was observed by confocal microscopy. Scale bar = 20 μm. Release of (C) HMGB1 and (D) ATP by B16–F10 cells after Mit treatment. ( ∗∗∗ P < 0.001 indicates the statistical difference between each group and the group without Mit) (E) The expression of CD86 on BMDCs after co-cultured with Mit-treated B16–F10 cells. (F) The vaccination method was used to identify Mit-induced immunogenic B16–F10 cell death. (G) The synthesis routes of AZO. (H) Schematic illustration of the preparation steps of MPAL. Data are presented as mean ± SD ( n = 3). ∗∗∗ P < 0.001.

Article Snippet: For HMGB1 detection, cells were permeabilized, fixed, and then stained with FITC conjugated anti-mouse HMGB1 antibody (Bioss) before flow cytometric detection.

Techniques: Flow Cytometry, Expressing, Confocal Microscopy, Cell Culture

Journal: International Journal of Molecular Medicine

Article Title: Tanshinone IIA confers protection against myocardial ischemia/reperfusion injury by inhibiting ferroptosis and apoptosis via VDAC1

doi: 10.3892/ijmm.2023.5312

Figure Lengend Snippet: TSN alleviates ferroptosis of A/R-induced H9c2 cardiomyocytes via downregulation of VDAC1. (A) Cell Counting Kit-8 detection of viability in A/R-induced cells following TSN or Fer-1 pretreatment. (B) LDH, (C) MDA, (D) total iron, (E) GSH, GSSG, GSH/GSSG and (F) ROS were determined by quantitative kits in A/R-induced cells following TSN or Fer-1 treatment (magnification, ×200; scale bar, 50 μ m). (G) Expression of (H) ferroptosis-related proteins and VDAC1 were detected by western blot analysis in A/R-induced cells following TSN or Fer-1 pretreatment. Data are expressed as the mean ± SD (n=3). *** P<0.05. TSN, Tanshinone IIA; A/R, Anoxia/reoxygenation; VDAC1, Voltage-dependent anion channel 1; Fer-1, ferrostatin-1; LDH, lactate dehydrogenase; MDA, malondialdehyde; GSH, Glutathione; GSSG, Glutathione disulfide; ROS, reactive oxygen species; PTGS2, Prostaglandin endoperoxide synthase 2; GPX, Glutathione peroxidase 4.

Article Snippet: An equal amount of total protein (40 μ g/lane) in each sample was separated by 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis, transferred to polyvinylidene fluoride membranes and blocked with 5% non-fat dry milk in Tris-buffered saline with 0.1% Tween-20 buffer at room temperature for 2 h. Subsequently, membranes were incubated with primary antibodies against PTGS2 (ProteinTech Group, Inc.; cat. no. #12375-1-AP; 1:1,000), VDAC1 (ProteinTech Group, Inc.; cat. no. #55259-1-AP; 1:1,000), GPX4 (ZENBIO; cat. no. #381958; 1:1,000), Bcl-2 (ZENBIO; cat. no. #250412; 1:1,000), Bax (ZENBIO; cat. no. #380709; 1:1,000) and β-actin (ProteinTech Group, Inc.; cat. no. #20536-1-AP; 1:1,000) at 4°C overnight in a shaker.

Techniques: Cell Counting, Expressing, Western Blot

Journal: International Journal of Molecular Medicine

Article Title: Tanshinone IIA confers protection against myocardial ischemia/reperfusion injury by inhibiting ferroptosis and apoptosis via VDAC1

doi: 10.3892/ijmm.2023.5312

Figure Lengend Snippet: TSN binds to VDAC1. (A) Chemical structure of TSN. (B) Molecular structure of VDAC1. (C) 3D and (D) 2D diagram of the interaction between TSN and VDAC1. TSN, Tanshinone IIA; VDAC1, Voltage-dependent anion channel 1.

Article Snippet: An equal amount of total protein (40 μ g/lane) in each sample was separated by 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis, transferred to polyvinylidene fluoride membranes and blocked with 5% non-fat dry milk in Tris-buffered saline with 0.1% Tween-20 buffer at room temperature for 2 h. Subsequently, membranes were incubated with primary antibodies against PTGS2 (ProteinTech Group, Inc.; cat. no. #12375-1-AP; 1:1,000), VDAC1 (ProteinTech Group, Inc.; cat. no. #55259-1-AP; 1:1,000), GPX4 (ZENBIO; cat. no. #381958; 1:1,000), Bcl-2 (ZENBIO; cat. no. #250412; 1:1,000), Bax (ZENBIO; cat. no. #380709; 1:1,000) and β-actin (ProteinTech Group, Inc.; cat. no. #20536-1-AP; 1:1,000) at 4°C overnight in a shaker.

Techniques:

Journal: International Journal of Molecular Medicine

Article Title: Tanshinone IIA confers protection against myocardial ischemia/reperfusion injury by inhibiting ferroptosis and apoptosis via VDAC1

doi: 10.3892/ijmm.2023.5312

Figure Lengend Snippet: TSN inhibits ferroptosis of A/R-induced H9c2 cardiomyocytes by downregulating VDAC1. (A) Cell Counting Kit-8 detection of viability in A/R-induced cells after TSN, pAd/VDAC1 and pAd/NC pretreatment. (B) LDH, (C) MDA, (D) total iron, (E) GSH, GSSG, GSH/GSSG and (F) ROS were determined by quantitative kits in A/R-induced cells following TSN, pAd/VDAC1 and pAd/NC treatment (magnification, x200; scale bar, 50 μ m). (G) Expression of (H) ferroptosis-associated proteins and VDAC1 were detected by western blot analysis in A/R-induced cells after TSN, pAd/VDAC1 and pAd/NC pretreatment. Data are expressed as the mean ± SD (n=3). *** P<0.05. TSN, tanshinone IIA; A/R, anoxia/reoxygenation; VDAC1, voltage-dependent anion channel 1; NC, negative control; LDH, lactate dehydrogenase; MDA, malondialdehyde; GSH, Glutathione; GSSG, Glutathione disulfide; ROS, reactive oxygen species; PTGS2, Prostaglandin endoperoxide synthase 2; GPX4, Glutathione peroxidase 4.

Article Snippet: An equal amount of total protein (40 μ g/lane) in each sample was separated by 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis, transferred to polyvinylidene fluoride membranes and blocked with 5% non-fat dry milk in Tris-buffered saline with 0.1% Tween-20 buffer at room temperature for 2 h. Subsequently, membranes were incubated with primary antibodies against PTGS2 (ProteinTech Group, Inc.; cat. no. #12375-1-AP; 1:1,000), VDAC1 (ProteinTech Group, Inc.; cat. no. #55259-1-AP; 1:1,000), GPX4 (ZENBIO; cat. no. #381958; 1:1,000), Bcl-2 (ZENBIO; cat. no. #250412; 1:1,000), Bax (ZENBIO; cat. no. #380709; 1:1,000) and β-actin (ProteinTech Group, Inc.; cat. no. #20536-1-AP; 1:1,000) at 4°C overnight in a shaker.

Techniques: Cell Counting, Expressing, Western Blot, Negative Control

Journal: International Journal of Molecular Medicine

Article Title: Tanshinone IIA confers protection against myocardial ischemia/reperfusion injury by inhibiting ferroptosis and apoptosis via VDAC1

doi: 10.3892/ijmm.2023.5312

Figure Lengend Snippet: TSN improves mitochondrial function and integrity in H9c2 cardiomyocytes exposed to A/R by downregulating VDAC1. (A) Fluorescent probe BBcellProbe M61 indicating mPTP opening was detected by flow cytometry with the FL1-A: B525-FITC channel. (B) mPTP flow cytometry. (C) Flameng score and (D) transmission electron microscopy of H9c2 cells (magnification, ×8,000; scale bar, 2 μ m). Data are expressed as the mean ± SD (n=3). *** P<0.05. TSN, tanshinone IIA; A/R, Anoxia/reoxygenation; VDAC1, Voltage-dependent anion channel 1; mPTP, Mitochondrial permeability transition pore; NC, negative control.

Article Snippet: An equal amount of total protein (40 μ g/lane) in each sample was separated by 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis, transferred to polyvinylidene fluoride membranes and blocked with 5% non-fat dry milk in Tris-buffered saline with 0.1% Tween-20 buffer at room temperature for 2 h. Subsequently, membranes were incubated with primary antibodies against PTGS2 (ProteinTech Group, Inc.; cat. no. #12375-1-AP; 1:1,000), VDAC1 (ProteinTech Group, Inc.; cat. no. #55259-1-AP; 1:1,000), GPX4 (ZENBIO; cat. no. #381958; 1:1,000), Bcl-2 (ZENBIO; cat. no. #250412; 1:1,000), Bax (ZENBIO; cat. no. #380709; 1:1,000) and β-actin (ProteinTech Group, Inc.; cat. no. #20536-1-AP; 1:1,000) at 4°C overnight in a shaker.

Techniques: Flow Cytometry, Transmission Assay, Electron Microscopy, Permeability, Negative Control

Journal: International Journal of Molecular Medicine

Article Title: Tanshinone IIA confers protection against myocardial ischemia/reperfusion injury by inhibiting ferroptosis and apoptosis via VDAC1

doi: 10.3892/ijmm.2023.5312

Figure Lengend Snippet: TSN inhibits apoptosis of A/R-induced H9c2 cardiomyocytes by downregulating VDAC1. (A) Expression of (B) apoptosis-associated proteins was detected by western blot analysis in A/R-induced cells following TSN, pAd/VDAC1 and pAd/NC pretreatment. (C) Caspase-3 activity was measured using a Caspase-3 kit in A/R-induced cells after TSN, pAd/VDAC1 and pAd/NC treatment. (D) MMP and (E) apoptosis were detected by flow cytometry. (F) MMP levels detected by JC-1 in H9c2 cells indicated by the red/green fluorescence ratio. (G) Apoptotic rate measured by Annexin V-FITC/PI flow cytometry. Data are expressed as the mean ± SD (n=3). *** P<0.05. TSN, Tanshinone IIA; A/R, Anoxia/reoxygenation; VDAC1, Voltage-dependent anion channel 1; NC, negative control; MMP, mitochondrial membrane potential.

Article Snippet: An equal amount of total protein (40 μ g/lane) in each sample was separated by 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis, transferred to polyvinylidene fluoride membranes and blocked with 5% non-fat dry milk in Tris-buffered saline with 0.1% Tween-20 buffer at room temperature for 2 h. Subsequently, membranes were incubated with primary antibodies against PTGS2 (ProteinTech Group, Inc.; cat. no. #12375-1-AP; 1:1,000), VDAC1 (ProteinTech Group, Inc.; cat. no. #55259-1-AP; 1:1,000), GPX4 (ZENBIO; cat. no. #381958; 1:1,000), Bcl-2 (ZENBIO; cat. no. #250412; 1:1,000), Bax (ZENBIO; cat. no. #380709; 1:1,000) and β-actin (ProteinTech Group, Inc.; cat. no. #20536-1-AP; 1:1,000) at 4°C overnight in a shaker.

Techniques: Expressing, Western Blot, Activity Assay, Flow Cytometry, Fluorescence, Negative Control, Membrane

Journal: International Journal of Molecular Medicine

Article Title: Tanshinone IIA confers protection against myocardial ischemia/reperfusion injury by inhibiting ferroptosis and apoptosis via VDAC1

doi: 10.3892/ijmm.2023.5312

Figure Lengend Snippet: Potential mechanism of TSN in myocardial ischemia/reperfusion injury. TSN pretreatment upregulates the expression of VDAC1, thereby decreasing the accumulation of ROS and iron and abnormal lipid metabolism, maintaining mitochondrial function and protecting the myocardium against anoxia/reoxygenation-induced ferroptosis and apoptosis. TSN, tanshinone IIA; VDAC1, Voltage-dependent anion channel 1; ROS, reactive oxygen species; MDA, malondialdehyde; GSH, Glutathione; GSSG, Glutathione disulfide; LDH, lactate dehydrogenase; MMP, Mitochondrial membrane potential.

Article Snippet: An equal amount of total protein (40 μ g/lane) in each sample was separated by 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis, transferred to polyvinylidene fluoride membranes and blocked with 5% non-fat dry milk in Tris-buffered saline with 0.1% Tween-20 buffer at room temperature for 2 h. Subsequently, membranes were incubated with primary antibodies against PTGS2 (ProteinTech Group, Inc.; cat. no. #12375-1-AP; 1:1,000), VDAC1 (ProteinTech Group, Inc.; cat. no. #55259-1-AP; 1:1,000), GPX4 (ZENBIO; cat. no. #381958; 1:1,000), Bcl-2 (ZENBIO; cat. no. #250412; 1:1,000), Bax (ZENBIO; cat. no. #380709; 1:1,000) and β-actin (ProteinTech Group, Inc.; cat. no. #20536-1-AP; 1:1,000) at 4°C overnight in a shaker.

Techniques: Expressing, Membrane

Journal: Biofabrication

Article Title: A biopsy-sized 3D skin model with a perifollicular vascular plexus enables studying immune cell trafficking in the skin

doi: 10.1088/1758-5090/ad5d1a

Figure Lengend Snippet: Characterization of the vascular plexus in 3D-SoC. (A) The COMSOL model displays the calculated range of shear stresses in the vascular pattern. The design recapitulates the physiological range of shear stress levels found in the cutaneous capillaries, venules, and arterioles. We divide the vasculature into three shear rate zones as low-shear (vertical interconnecting channels), mid-shear (two outermost channels, top and bottom) and high-shear (two innermost, horizontal channels). (B) Imaging of the vascular network seeded with GFP-HDBECs confirms uniform coverage of the microchannel walls. Scale bar: 1 mm; (C) Immunofluorescent staining of primary HDBECs in 3D-SoC with VE-cadherin (VECAD; white). Scale bar: 5 µ m; (D) confocal imaging of the 3D-SoC seeded with HDBECs perfused with both 20 kDa and 40 kDa dextran at time zero and sixty minutes allowing for comparison of the permeability characteristics. Scale bar: 2 mm; (E) the graph shows increased leakage of dextran in the model without HDBECs (acellular control) for both molecular weights. (F) Time-lapse transport data integrated into a COMSOL model enabled the estimation of the average permeability of the vasculature. The permeability values were determined to be 0.62 µ m s −1 for 20 kDa and 0.41 µ m s −1 for 40 kDa respectively (** = p < 0.01).

Article Snippet: Human dermal blood endothelial cells (HDBECs) (PromoCell #C-12211) and GFP-tagged HDBECs (Angio-Proteomie #cAP-0005GFP-PM) were cultured up to passage 3 in Microvascular EC Growth Medium (PromoCell #C-22020).

Techniques: Shear, Imaging, Staining, Comparison, Permeability, Control

Journal: Biofabrication

Article Title: A biopsy-sized 3D skin model with a perifollicular vascular plexus enables studying immune cell trafficking in the skin

doi: 10.1088/1758-5090/ad5d1a

Figure Lengend Snippet: Incorporation and real-time monitoring of circulating T cells in 3D-SoC. (A) Schematic representation of the stages of T cell infiltration into human skin. (B) Live immunofluorescent images showing the naïve T cells labelled with CellTracker (red) on HDBECs in the first 1–2 min (left panel; the round morphology resembles the tethering/rolling stage); between 2–5 min (middle panel; the spread morphology resembles the firm adhesion stage); and between 5–15 min (right panel; the morphology and location relative to ECs resembles the diapedesis stage). The first two images show the top view, and the right-most image shows a cross-section of the 3D-SoC. Scale bars: 5 µ m; (C) High magnification image capturing a T cell (red) with its lamellipodia squeezing between two endothelial cells (green), resembling the morphology of T cells in vivo during their movement through capillary walls (namely diapedesis). Scale bar: 2 µ m; (D) characterization of Th1 cells polarized from Naïve T cells in vitro through flow cytometry showing expression of both Interferon γ and TNFα. (E) Comparison of the attachment of the T cells to the shear stress analysis for naive and Th1 cell population. (F) Total percentage of naïve T cells and Th1 cells retained after 5 and 10 mins of flow. (G) Percentage of cells retained for distinct shear zones; HS: high-shear, MS: mid-shear, LS: low-shear. (* = p < 0.05, ** = p < 0.01, *** = p < 0.005).

Article Snippet: Human dermal blood endothelial cells (HDBECs) (PromoCell #C-12211) and GFP-tagged HDBECs (Angio-Proteomie #cAP-0005GFP-PM) were cultured up to passage 3 in Microvascular EC Growth Medium (PromoCell #C-22020).

Techniques: In Vivo, In Vitro, Flow Cytometry, Expressing, Comparison, Shear

Journal: Life Science Alliance

Article Title: Inflammatory risk contributes to post-COVID endothelial dysfunction through anti-ACKR1 autoantibody

doi: 10.26508/lsa.202402598

Figure Lengend Snippet: (A) Gene expressions of endothelial antigens upon TNFα (10 ng/ml) treatment on human umbilical artery and vein cells for 24 h. Data represent mean ± s.d.; n = 3 biological replicates; two-way ANOVA; * P < 0.05, ** P < 0.01, *** P < 0.001, and **** P < 0.0001. (B) Flow cytometry analysis of ACKR1 protein expression on artery and vein cells after 48 h of stimulation with TNFα (10 ng/ml). Data represent mean ± s.d.; n = 3 biological replicates; two-way ANOVA; **** P < 0.0001. (C) Expression profile of Ackr1 in vascular endothelial subtypes of various organs from a mouse single-cell transcriptome atlas (accession code: E-MTAB-8077 ). Data were generated with EC Atlas web-based visualization.

Article Snippet: We custom made a microarray-based autoantibody detection kit (RayBiotech) where a glass slide surface was spotted with purified human recombinant ACKR1 protein (Cat #TP304680; OriGene) that could be recognized by ACKR1 autoantibodies present in human plasma.

Techniques: Flow Cytometry, Expressing, Generated

Journal: Life Science Alliance

Article Title: Inflammatory risk contributes to post-COVID endothelial dysfunction through anti-ACKR1 autoantibody

doi: 10.26508/lsa.202402598

Figure Lengend Snippet: (A) Scatterplot of the plasma levels of anti-ACKR1 autoantibodies in COVID-19 survivors and non-infected controls (mean ± s.e.m., Mann–Whitney test). Anti-ACKR1 concentrations were calculated based on normalized ratio over positive control of human immunoglobulin G (50 μg/ml) and presented as fold change to non-infected controls. (B) Heatmap depicting plasma concentrations of anti-ACKR1 autoantibodies, cytokines (quantified by Luminex multiplex assay), and enumeration of circulating endothelial cells in COVID-19 survivors and non-infected controls. Spearman’s correlation analysis revealed positive associations between anti-ACKR1 levels and the quantity of circulating endothelial cells, as well as each cytokine listed, with significant P -values indicated. Heatmap was generated using the MORPHEUS visualization software. (C) Spearman’s correlation analysis between anti-ACKR1 levels and reactive hyperemia index in individuals (n = 10) with endothelial dysfunctions (characterized by natural log-transformed reactive hyperemia index < 0.51). Spearman’s correlation coefficient r and P -values (two-tailed test) are indicated. (D) Kaplan-Meier plot shows the probability of primary vascular disease outcomes in patients without prior established cardiovascular diseases in a median follow-up period of 6.7 yr. Hazard ratio and 95% confidence intervals (CI) compare the time of blood collection to the first occurrence of vascular outcomes according to the presence of anti-ACKR1 autoantibodies (n = 16 undetectable anti-ACKR1, n = 10 anti-ACKR1 > 0 μg/ml).

Article Snippet: We custom made a microarray-based autoantibody detection kit (RayBiotech) where a glass slide surface was spotted with purified human recombinant ACKR1 protein (Cat #TP304680; OriGene) that could be recognized by ACKR1 autoantibodies present in human plasma.

Techniques: Infection, MANN-WHITNEY, Positive Control, Luminex, Multiplex Assay, Generated, Software, Transformation Assay, Two Tailed Test

Journal: Life Science Alliance

Article Title: Inflammatory risk contributes to post-COVID endothelial dysfunction through anti-ACKR1 autoantibody

doi: 10.26508/lsa.202402598

Figure Lengend Snippet: (A) Scatterplot of the plasma levels of anti-ACKR1 autoantibodies in COVID-19 survivors and non-infected controls (mean ± s.e.m., Mann–Whitney test). Anti-ACKR1 levels were determined based on flow cytometry detection of bound anti-ACKR1 to K562 cells, a human erythroleukemic cell line ectopically overexpressing ACKR1. (B) ACKR1 (DARC) polymorphism, G125A (rs12075) was genotyped to identify individuals carrying FYA (G125) and FYB (125A) alleles, indicated under “Observed Frequencies.” “Expected frequencies” were extracted from literature as a comparison. (C) Spearman’s correlation analysis to assess the associations between the circulating levels of anti-ACKR1 autoantibodies and red blood cell count (n = 46).

Article Snippet: We custom made a microarray-based autoantibody detection kit (RayBiotech) where a glass slide surface was spotted with purified human recombinant ACKR1 protein (Cat #TP304680; OriGene) that could be recognized by ACKR1 autoantibodies present in human plasma.

Techniques: Infection, MANN-WHITNEY, Flow Cytometry, Comparison, Cell Counting

Journal: Life Science Alliance

Article Title: Inflammatory risk contributes to post-COVID endothelial dysfunction through anti-ACKR1 autoantibody

doi: 10.26508/lsa.202402598

Figure Lengend Snippet: (A) Experimental workflow of intervening autoantibody-antigen interactions to investigate the functional impact of anti-ACKR1 autoantibodies on human endothelial cells. The three-dimensional structure of ACKR1 was produced using I-TASSER server. (B) Flow cytometry analysis of the percentage of necrotic and late apoptotic endothelial cells after 24 h of experimental treatments. (C) Transendothelial electrical resistance assay to evaluate endothelial barrier permeability after experimental treatments. (D) Number of transmigrated PBMCs in an endothelial-immune cell co-culture transwell assay. (E) Degree of cytotoxicity was determined by quantifying lactate dehydrogenase activity in cell-free supernatants obtained from endothelial cells cultured with purified immunoglobulin G (IgG) and/or PBMCs. (F) Degree of antibody-dependent cellular cytotoxicity was determined from endothelial cells exposed to purified IgG and PBMCs. Purifie IgG was pre-incubated with and without blocking peptide or liposome ACKR1. For (B, C, D, E, F), data represent mean ± s.d.; one-way ANOVA; * P < 0.05, ** P < 0.01; *** P < 0.001, **** P < 0.0001; ns, non-significant. Data points represent biological replicates.

Article Snippet: We custom made a microarray-based autoantibody detection kit (RayBiotech) where a glass slide surface was spotted with purified human recombinant ACKR1 protein (Cat #TP304680; OriGene) that could be recognized by ACKR1 autoantibodies present in human plasma.

Techniques: Functional Assay, Produced, Flow Cytometry, Permeability, Co-Culture Assay, Transwell Assay, Activity Assay, Cell Culture, Purification, Incubation, Blocking Assay

Journal: Arteriosclerosis, Thrombosis, and Vascular Biology

Article Title: CD34 Hybrid Cells Promote Endothelial Colony-Forming Cell Bioactivity and Therapeutic Potential for Ischemic Diseases

doi: 10.1161/atvbaha.112.301052

Figure Lengend Snippet: Figure 1. The role of CD34− accessory cells in CD34+ stem cell–derived endothelial progenitor cell (EPC) commitment. A, A schematic diagram of the human EPC colony-forming assay (CFA) used to evaluate the effect of functional CD34− cells on EPC colony-forming units (CFUs). B, Differentiation of 2 types of EPC colony clusters, small EPC-CFUs and large EPC-CFUs, from 3 kinds of cell populations. Small EPC-CFUs were round-shaped, and large EPC-CFUs were spindle-shaped (magnification, ×40). C, Standard quantification of EPC-CFUs was performed by counting the number of small, large, and total EPC-CFUs. Results are shown as the mean±SEM (*P<0.05 and **P<0.01 vs CD34+ cells). D, Schematic diagram of the insert culture assay used to assess the effect of functional CD34− cells on CD34+ cell- mediated endothelial cell (EC)-lineage commitment. E, Standard quantification of EPC-CFUs was performed by calculating the number of expanded EC progenitor colonies using CD34+ cells (lower chamber) cocultured with CD34− cells, CD11b+/CD34− cells (macrophage), or CD11b−/CD34− cells (upper chamber). The results were shown as the mean±SEM (*P<0.05 and **P<0.01 vs CD34+ cells). F, Expression of angiogenic cytokines in tumor necrosis factor (TNF)α-treated or untreated CD34− cell–derived macrophages. G, Effect of stromal cell– derived factor (SDF)-1α and vascular endothelial growth factor (VEGF) on human EPC-CFUs. In response to stimulation with SDF-1α and VEGF, the frequency of large EPC-CFUs was significantly increased (*P<0.05 and **P<0.01 vs basal control cytokines: stem cell factor, VEGF, interleukin-3, basic fibroblast growth factor, epidermal growth factor, insulin-like growth factor-1). H, Expression of endothelial lin- eage markers for kinase insert domain receptor, C-X-C chemokine receptor 4, and Tie2 on transwell-cultured cells with or without CD34− cells. HUCB indicates human umbilical cord blood; and MNC, mononuclear cell.

Article Snippet: SDF-1α and VEGF levels were determined by Quantikine ELISA Human SDF-1α Immunoassay and Quantikine ELISA Human VEGF Immunoassay (R&D Systems, Minneapolis, MN, USA) following the manufacturer’s instructions.

Techniques: Derivative Assay, Functional Assay, Expressing, Control, Cell Culture

Journal: Arteriosclerosis, Thrombosis, and Vascular Biology

Article Title: CD34 Hybrid Cells Promote Endothelial Colony-Forming Cell Bioactivity and Therapeutic Potential for Ischemic Diseases

doi: 10.1161/atvbaha.112.301052

Figure Lengend Snippet: Figure 2. Characterization and functional capacity of CD34−/CD34+ cell–derived endothelial colony-forming cells (hybrid-dECFCs) and CD34+ cell–derived ECFCs (stem-dECFCs). A, Hybrid-dECFCs and stem-dECFCs are spindle-shaped cells (similar to outgrowth endothelial cells [OECs]; magnification, ×40). B, Human ECFCs were characterized by the endothelial cell (EC) markers CD31, vascular endothelial growth factor (VEGFR)2 (KDR), and von Willebrand factor (vWF), as well as pivotal markers of functional ECFCs, including phosphor-protein kinase B, endothelial nitric oxide synthase (eNOS), and p-eNOS. C, Expression of surface markers for CD34, CD133, kinase insert domain receptor (KDR), C-X-C chemokine receptor 4 (CXCR4), and c-Kit on 2 types of ECFCs measured by flow cytometry. Fluorescence-activated cell sorter (FACS) analysis indicated the ratios of CD34/CD133, KDR, CXCR4, or c-Kit positive cells. D, Hybrid- dEPC (passages 6–20) and stem-dEPC (passages 6–16) lysates containing equal amounts of total protein were analyzed by Western blot- ting using anti-cyclin-dependent kinase (Cdk)-2, anticyclin E, anti-Cdk4, and anticyclin D1 antibodies. E, Both types of ECFCs (passages 6–16) were treated with VEGF (100 ng/mL), and cell proliferation was examined via the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetra- zolium bromide assay after 24 hours. F, Senescence-associated β-galactosidase (SA-β-gal) activity of the 2 types of ECFCs (passages 6–16). The green cells were counted as positive for senescence (magnification, ×40). G, Hybrid-dECFC (passages 6–20) and stem-dECFC (passages 6–16) lysates containing equal amounts of total protein were analyzed by Western blotting using senescence markers with anti-senescence marker protein (SMP)-30 and antip21 antibodies. Results are shown as the mean±SEM (*P<0.05 and **P<0.01 vs hybrid- dECFCs). DAPI indicates 4',6-diamidino-2-phenylindole; EBM, endothelial basal medium; and SMP, senescence marker protein.

Article Snippet: SDF-1α and VEGF levels were determined by Quantikine ELISA Human SDF-1α Immunoassay and Quantikine ELISA Human VEGF Immunoassay (R&D Systems, Minneapolis, MN, USA) following the manufacturer’s instructions.

Techniques: Functional Assay, Derivative Assay, Expressing, Flow Cytometry, Fluorescence, Western Blot, Activity Assay, Marker

Journal: Arteriosclerosis, Thrombosis, and Vascular Biology

Article Title: CD34 Hybrid Cells Promote Endothelial Colony-Forming Cell Bioactivity and Therapeutic Potential for Ischemic Diseases

doi: 10.1161/atvbaha.112.301052

Figure Lengend Snippet: Figure 5. Proliferation, survival, and secretion of angiogenic growth factors of endothelial colony-forming cells (ECFCs) in hind-limb isch- emia. At day 3 after surgery, samples harvested from hind-limb ischemic tissues were stained to determine the proliferation, survival, and secretion of angiogenic growth factors of the transplanted ECFCs (passage 6). A, Proliferative cells in ischemic injury sites visualized by immunofluorescent staining for proliferating cell nuclear antigen (PCNA; red). B, Standard quantification of proliferating cells represented as the number of PCNA/4',6-diamidino-2-phenylindole (DAPI) double-positive cells per high-power field. Results are shown as the mean±SEM (*P<0.05 and **P<0.01 vs sham, ##P<0.05 vs CD34+ cell–derived ECFCs [stem-dECFCs]). C, Proliferative transplanted cells at the ischemic injury sites indicated by human nuclear antigen (HNA; red), Ki-67 (green), and DAPI (blue) triple-positive cells. White color indicates triple-positive cells in the merged images. D, Apoptotic transplanted cells in ischemic injury shown as HNA (red), caspase-3 (green), and DAPI (blue) triple-positive cells. E, Standard quantification of proliferating transplanted cells represented as the number of HNA/Ki-67/DAPI triple-positive cells (white) per high-power field. F, Standard quantification of apoptotic cells represented as the num- ber of HNA/caspase-3/DAPI triple-positive cells (white) per high-power field. Results are shown as the mean±SEM (**P<0.01 vs CD34−/ CD34+ cell–derived ECFCs [hybrid-dEPC]). G–K, Secretion of angiogenic growth factor from transplanted ECFCs in injury sites visualized by fibroblast growth factor (FGF-2; G), hepatocyte growth factor (HGF; H), stromal cell–derived factor (SDF)-1α (I), vascular endothelial growth factor (VEGF; J), and interleukin (IL)-8 (K) staining (green) and HNA staining (red). L At day 3 after surgery, samples harvested from hind-limb ischemic tissues were analyzed to confirm the secretion of angiogenic growth factor at the injury sites by Western blot- ting. Western blots of ischemic tissue homogenates indicated secretion of FGF-2, HGF, SDF-1α, VEGF, and IL-8. Hvf indicates high visual field.

Article Snippet: SDF-1α and VEGF levels were determined by Quantikine ELISA Human SDF-1α Immunoassay and Quantikine ELISA Human VEGF Immunoassay (R&D Systems, Minneapolis, MN, USA) following the manufacturer’s instructions.

Techniques: Staining, Derivative Assay, Western Blot

Journal: bioRxiv

Article Title: Syndecan-4 is required for VE-Cadherin trafficking during pathological angiogenesis

doi: 10.1101/736553

Figure Lengend Snippet: (A) Papilloma formation is reduced in Sdc4-/- mice in the DBMA/TPA model. Micrographs of animals (left) and sections of skin (right, H&E stained) from WT and Sdc4-/- animals at week 19 (scale bar = 2 mm). (B) Number of large tumors (≥2 mm/mouse) over time and (C) size of papillomas at end of the experiment (n=7 mice/group). (D) Time-course of papilloma incidence in WT and Sdc4-/- mice. The percentage of tumor-free animals at each time point is shown. Papillomas were observed in WT and Sdc4-/- mice at 9 weeks and after week 12 none of the animals was tumor-free. Because the proportional hazards assumption appeared correct, a survival plot was generated and analyzed via log-rank (Mantel-Cox) test. (E) Tumor sections from WT and Sdc4-/- animals were immuno-stained for the EC marker CD31and vessel width was measured. (H) Vessels from Sdc4-/- papillomas were narrower. (G) Micrographs of B16F1 melanomas from WT and Sdc4-/- animals showing reduced tumor volume as quantified in (H) (n=5-6 mice/group). (I) Tumor vessels (arrowheads) appear in WT sections but are not obvious in B16F1 melanomas from Sdc4-/- mice (Ki-67, blue; CD31 red, scale bar = 100 µM), (J) quantification of tumor vessel coverage (n=5/group, 3 images/animal). *P < 0.05. Error bars indicate SEM. Levels of NK cells are equal in WT and Sdc4-/- animals in both spleen (K) and B16F1 tumor immune infiltrates (L) (n=3 mice/group).

Article Snippet: The following dyes and reagents were used in this work; lectin GS-II Alexa594 (Thermo Fisher Cat#L21416), DAPI (Sigma-Aldrich, Cat#D9542), Draq5 (Biostatus limited Cat#DR05500), recombinant mouse VEGFA 164 (R&D Systems Cat#493-MV-025m), Bradykinin (Sigma Cat#B3259), recombinant syndecan-4 (R&D Systems Cat# 6267-SD-050) and recombinant syndecan-2 (R&D Systems Cat#6585-SD-050), proteome profiler human phospho-kinase array kit (R&D Systems Cat#ARY003B).

Techniques: Staining, Generated, Marker

Journal: bioRxiv

Article Title: Syndecan-4 is required for VE-Cadherin trafficking during pathological angiogenesis

doi: 10.1101/736553

Figure Lengend Snippet: (A) Micrographs showing the pre-retinal neovascularization response (stained with BS1-isolectin) to OIR in P17 WT neonates is greater than in equivalent Sdc4-/- animals (Scale bar = 500 µM). (B) Quantification of pre-retinal neovascularization (∼40 eyes/group). (C) Micrographs showing Sdc4-/- animals exhibit less angiogenesis in the laser induced CNV model as evident from reduced lesion area (D) (n=6-8 animals/group, Scale bar = 2.4 mm). (E) Staining of CNV lesions with lectin GS-II followed by 3D confocal reconstruction. (F) CNV lesion volume was calculated using Imaris Bitplane software on the basis of lectin GS-II staining. n=5-6 animals for each group. Centre line, median. Plus sign, mean. Error bars indicate min and max values. *P < 0.05; **P < 0.01; ***P<0.001. (G) Syndecan gene expression profile during early stages of murine retinal angiogenesis. Eyes were enucleated at postnatal day 0, 4 and 7, mRNA extracted and quantified by qPCR. (H) Syndecan gene expression in neonates subjected to OIR (P12 vaso-obliteration phase, P17 angiogenic phase) and in untreated controls. Centre line, median. Plus sign, mean. N=4-6 animals for each group. Error bars indicate min and max values. *P < 0.05; **P < 0.01.

Article Snippet: The following dyes and reagents were used in this work; lectin GS-II Alexa594 (Thermo Fisher Cat#L21416), DAPI (Sigma-Aldrich, Cat#D9542), Draq5 (Biostatus limited Cat#DR05500), recombinant mouse VEGFA 164 (R&D Systems Cat#493-MV-025m), Bradykinin (Sigma Cat#B3259), recombinant syndecan-4 (R&D Systems Cat# 6267-SD-050) and recombinant syndecan-2 (R&D Systems Cat#6585-SD-050), proteome profiler human phospho-kinase array kit (R&D Systems Cat#ARY003B).

Techniques: Staining, Software, Expressing

Journal: bioRxiv

Article Title: Syndecan-4 is required for VE-Cadherin trafficking during pathological angiogenesis

doi: 10.1101/736553

Figure Lengend Snippet: (A and B) Consecutive sections of human proliferative diabetic retinopathy membrane stained for CD31 marker of blood vessels), VEGFR2 (marker of new immature vessels), and SDC4. Scale bar, 200 μm. Images are representative of n=6 patients. Correlation between SDC4, CD31 and VEGFR2 expression in neovascular membranes from diabetic patients (C) . Multiple linear regression analysis of correlation between SDC4 and CD31 + or VEGFR2 + area (%) (Correlation R=0.751, p=0.036, coefficients CD31=0.963, VEGFR2=0.032). n=6 patients (2-3 sections per patient), individual ROIs (n=11) were used for the analysis. (D) Matrigel supplemented with PBS or VEGFA (50 ng) was injected subcutaneously in the flank of WT or Sdc4-/- mice. Images are representative of plugs extracted 7 days post-injection. Scale bar, 1 cm. (E) Plug vascularity was expressed as the amount of hemoglobin released from the plugs per ml of Matrigel and normalized for the plug weight. n=5-6 animal for each group (2 plugs per animal). (F) Fragments (1 mm2) of choroid were dissected from adult WT, Sdc4 +/- and Sdc4-/- littermates and cultured as explants in VEGFA-containing medium. Explants were stained with lectin GS-II (red) and anti-αSMA (green) after 7 days in culture. Arrows show angiogenic sprouts. Scale bar, 10 µM. (G) Sprouting was quantified by manually counting the number of sprouts per explant. n=5-6 animal for each group, 10 explants per animal. (H) Quantification of sprout formation from aortic ring explants (n=5-6 animal for each group, 15-20 rings per animal).

Article Snippet: The following dyes and reagents were used in this work; lectin GS-II Alexa594 (Thermo Fisher Cat#L21416), DAPI (Sigma-Aldrich, Cat#D9542), Draq5 (Biostatus limited Cat#DR05500), recombinant mouse VEGFA 164 (R&D Systems Cat#493-MV-025m), Bradykinin (Sigma Cat#B3259), recombinant syndecan-4 (R&D Systems Cat# 6267-SD-050) and recombinant syndecan-2 (R&D Systems Cat#6585-SD-050), proteome profiler human phospho-kinase array kit (R&D Systems Cat#ARY003B).

Techniques: Membrane, Staining, Marker, Expressing, Injection, Cell Culture

Journal: bioRxiv

Article Title: Syndecan-4 is required for VE-Cadherin trafficking during pathological angiogenesis

doi: 10.1101/736553

Figure Lengend Snippet: (A) MLECs were scratched and incubated in serum-free (SF) medium or with VEGFA (100 ng/ml) or FBS (10%) containing medium for 16 hours. Phase contrast images showing final scratch area (white lines). Scale bar, 200 µm. (B) Migrated area was calculated by subtracting the final scratch area to the initial scratch area using ImageJ. n=3. (C) Evans blue was injected into the tail vein of WT and Sdc4-/- mice, followed by subcutaneous injections of PBS, VEGFA (100 ng) or Bradykinin (BK, 100 μg). Images show the local extravasation of the dye from underneath the skin 90 minutes post-injection, white circles indicate the approximate injection area. (D) Skin punches corresponding to the injection sites were collected and Evans blue extracted in formamide overnight. Permeability was quantified by measuring the optical density at 620 nm of the skin extracts and values were normalized on the basis of tissue weight. n=6-8 animals per condition. Data are mean ± s.e.m. *P < 0.05; **P < 0.01; ****P<0.0001. Statistical comparisons were made between PBS and treatments within the same genotype unless otherwise indicated. (E) MLECs from WT and Sdc4-/- mice were washed with PBS and incubated with anti-VE-Cadherin antibody at 4 °C for 1 hour. Unbound antibody was washed away and cells stimulated with VEGFA (30 ng/ml) for 10 min at 37 °C to promote VE-Cadherin internalization. Cells were then subjected to an acid wash to remove cell surface antibody. VE-Cadherin (red), DAPI (blue). Scale bar, 20 μm. (F) Images of MLECs treated with VEGFA and acid-washed were analyzed on Imaris software and VE-cadherin+ vesicles were counted and divided by the number of nuclei in the field of view. n=3. Images are representative of one experiment where 9 images per condition were analyzed. (G) Two representative images of WT and Sdc4-/- OIR retinas paraffin-embedded, sectioned and stained with isolectin GS-IB4 (red), VE-Cadherin (green) and DAPI (blue). Arrows indicate VE-Cadherin+ vesicles. GCL: ganglion cell layer. Blood vessels are pre-retinal tufts. Scale bar, 20 μm. VEGFR2 signaling is unaffected in Sdc4-/- primary MLECS. (H) Western blots of either WT or Sdc4-/- MLEC lysates harvested at different time points of showing a VEGFA stimulation. Levels of phoshpo-VEGR2, Erk, Src and VE-Cadherin were assayed. (I) Cell surface expression of VEGR2 is the same on WT and Sdc4-/- MLECs as measured by flow cytometry.

Article Snippet: The following dyes and reagents were used in this work; lectin GS-II Alexa594 (Thermo Fisher Cat#L21416), DAPI (Sigma-Aldrich, Cat#D9542), Draq5 (Biostatus limited Cat#DR05500), recombinant mouse VEGFA 164 (R&D Systems Cat#493-MV-025m), Bradykinin (Sigma Cat#B3259), recombinant syndecan-4 (R&D Systems Cat# 6267-SD-050) and recombinant syndecan-2 (R&D Systems Cat#6585-SD-050), proteome profiler human phospho-kinase array kit (R&D Systems Cat#ARY003B).

Techniques: Incubation, Injection, Permeability, Software, Staining, Western Blot, Expressing, Flow Cytometry

Journal: bioRxiv

Article Title: Syndecan-4 is required for VE-Cadherin trafficking during pathological angiogenesis

doi: 10.1101/736553

Figure Lengend Snippet: (A) Immunofluorescence staining for endogenous SDC4 (green) shows co-localisation with VE-Cadherin (red) and VEGFR2 (purple) at EC junctions. Scale bar, 10 µm. (B) The complete coding sequence of eGFP was inserted into murine SDC4 between I 32 and D 33 and cloned into lentiviral expression vectors (diagram). Phase contrast and fluorescent images at x20 and x40 of transfected HUVECS are shown on the right and these confirm Sdc4-eGFP localizes to EC junctions. Scale bar, 100 µm. (C) Cell surface VEGFR2 expression in HUVECs is unaffected by overexpression of Sdc4-eGFP and Sdc4(Y/A)-eGFP as measured by flow cytometry. (D) Western blots showing that VEGR2 phosphorylation (Y 1175 ) in response to VEGFA is not affected in HUVECs expressing Sdc4-eGFP and Sdc4(Y/A)-eGFP. (E) Confocal images of FRAP of HUVECs expressing Sdc4-eGFP and Sdc4(Y/A)-eGFP at the time points indicated in the presence or absence of VEGFA stimulation. (F) Quantification of fluorescence recovery of Sdc4-eGFP and Sdc4(Y/A)-eGFP and (G) plots of the half-life of recovery for each of the treatments.

Article Snippet: The following dyes and reagents were used in this work; lectin GS-II Alexa594 (Thermo Fisher Cat#L21416), DAPI (Sigma-Aldrich, Cat#D9542), Draq5 (Biostatus limited Cat#DR05500), recombinant mouse VEGFA 164 (R&D Systems Cat#493-MV-025m), Bradykinin (Sigma Cat#B3259), recombinant syndecan-4 (R&D Systems Cat# 6267-SD-050) and recombinant syndecan-2 (R&D Systems Cat#6585-SD-050), proteome profiler human phospho-kinase array kit (R&D Systems Cat#ARY003B).

Techniques: Immunofluorescence, Staining, Sequencing, Clone Assay, Expressing, Transfection, Over Expression, Flow Cytometry, Western Blot, Fluorescence

Journal: bioRxiv

Article Title: Syndecan-4 is required for VE-Cadherin trafficking during pathological angiogenesis

doi: 10.1101/736553

Figure Lengend Snippet: (A) Confocal micrographs of HUVECs showing Proximity ligation on the cell surface (red dots) between HA tagged SDC4 and VE-Cadherin (nuclei, blue; VE-Cad, green). An example of a junctional dots (yellow arrow) and a non-junctional dots (white arrow) is shown on the right hand micrograph. Scale bar, 20 µm. (B) Quantification of SDC4HA/VE-Cadherin PLA puncta on the cell surface over time after VEGFA stimulation (10ng/ml). (C) Quantification of SDC4HA/VE-Cadherin PLA puncta at EC junctions over time after VEGFA stimulation (10ng/ml). (D) Cell surface VE-Cadherin is reduced after VEGFA stimulation in Sdc4-eGFP transfected cells but not in HUVECs transfected with Sdc4(Y/A)-eGFP. Cell surface biotinylation was performed on transfected HUVECs and biotin labelled proteins were ‘pulled-down’ using streptavidin beads. Lysates were analyzed by western blotting for VE-Cadherin. (E) Phosphorylated SDC4 is required for VE-Cadherin internalization. VE-Cadherin vesicles are evident in cells (white arrows) after VEGA stimulation in Sdc4-eGFP cells but not in Sdc4(Y/A)-eGFP treated cells, where VE-Cadherin remains at EC junctions. (F) Quantification of VE-Cadherin positive vesicles before and after VEGFA stimulation in HUVECs transfected with either Sdc4-eGFP or Sdc4(Y/A)-eGFP. (G) Quantification of eGFP+ vesicles (n=20 cells/condition).

Article Snippet: The following dyes and reagents were used in this work; lectin GS-II Alexa594 (Thermo Fisher Cat#L21416), DAPI (Sigma-Aldrich, Cat#D9542), Draq5 (Biostatus limited Cat#DR05500), recombinant mouse VEGFA 164 (R&D Systems Cat#493-MV-025m), Bradykinin (Sigma Cat#B3259), recombinant syndecan-4 (R&D Systems Cat# 6267-SD-050) and recombinant syndecan-2 (R&D Systems Cat#6585-SD-050), proteome profiler human phospho-kinase array kit (R&D Systems Cat#ARY003B).

Techniques: Ligation, Transfection, Western Blot

Journal: bioRxiv

Article Title: Syndecan-4 is required for VE-Cadherin trafficking during pathological angiogenesis

doi: 10.1101/736553

Figure Lengend Snippet: (A) SolS4 disrupts PLA between SDC4 and VE-Cadherin at EC junctions. (B) Confocal images of VE-Cadherin antibody ‘washout’ experiments. WT MLECs were washed with PBS and incubated with anti-VE-Cadherin antibody at 4 °C for 1 hour. Unbound antibody was washed away and cells stimulated with VEGFA (30 ng/ml) for 10 min with or without SolS4 (3.5 nM) at 37 °C to promote VE-Cadherin internalization. Cells were then subjected to an acid wash to remove cell surface antibody. VE-Cadherin (white). Scale bar, 20 μm. (C) Images of MLECs treated with VEGFA and acid-washed were analyzed on Imaris software and VE-cadherin+ vesicles were counted and divided by the number of nuclei in the field of view. Images are representative of one experiment were 9 images per condition were analyzed. n=3 Scale bar, 20 μm. (D) HUVECs were scratched and incubated in serum-free media with or without VEGFA (20 ng/ml) and with or without SolS4 or solS2 (3.5 nM) for 16 hours. Migrated area was calculated by subtracting the final scratch area to the initial scratch area using ImageJ. n=3. (E) Confluent HUVECs were serum-starved for 2 hours and treated with soluble SDC4 (solS4, 3.5 nM), VEGFA (20 ng/ml) or both for 10 min. Lysates were then analyzed using a human phospho-kinase membrane-based sandwich immunoassay. Phosphorylated kinases were quantified using ImageJ, signals normalized using the reference spots. Phosphorylation sites were as follows, pERK1/2 (T202/Y204, T185/Y187), pGSK-3a,b (S21/S9), pAkt1/2/3 (S473), pCREB (S133), pSrc (Y419), pPRAS40 (T246). (F and G) Rings (1 mm in width) of aorta were dissected from adult WT rats, embedded in Collagen I and cultured as explants in VEGFA-containing medium with or without solS4 (3.5 nM) for 7 days and manually quantified. (n=4, 5-15 rings/condition). Scale bar, 10 μm. (H) Fundus fluorescein angiograms of WT animals at day 7 post laser induced CNV. Intravitreal injection of 1 µl of either PBS, Aflibercept (10 µg) or solS4 (100 ng) were performed at day 0 post-laser. Scale bar, 2.4 mm. (I) The CNV lesion areas were quantified using ImageJ, each dot represents the average of 3 lesions per eye. n=7-9 animals per condition. Data are means and error bars indicate SEM in B, C and G and min and max values in E. *P < 0.05; **P < 0.01; ***P<0.001.

Article Snippet: The following dyes and reagents were used in this work; lectin GS-II Alexa594 (Thermo Fisher Cat#L21416), DAPI (Sigma-Aldrich, Cat#D9542), Draq5 (Biostatus limited Cat#DR05500), recombinant mouse VEGFA 164 (R&D Systems Cat#493-MV-025m), Bradykinin (Sigma Cat#B3259), recombinant syndecan-4 (R&D Systems Cat# 6267-SD-050) and recombinant syndecan-2 (R&D Systems Cat#6585-SD-050), proteome profiler human phospho-kinase array kit (R&D Systems Cat#ARY003B).

Techniques: Incubation, Software, Membrane, Cell Culture, Injection

Journal: Molecular oncology

Article Title: The miR-200 family differentially regulates sensitivity to paclitaxel and carboplatin in human ovarian carcinoma OVCAR-3 and MES-OV cells.

doi: 10.1016/j.molonc.2015.04.015

Figure Lengend Snippet: Figure 1 e Characterization of OVCAR-3 GFP and OVCAR-3/TP GFP cell lines. (A) The cells were treated with different concentrations of paclitaxel (left panel) and carboplatin (right panel) during 72 h. Cells survival was measured by SRB assay. All data are expressed as the average percentage of survival values relative to an untreated control ± SD with significance determined between the indicated cell lines per paclitaxel or carboplatin concentration tested (*, P < 0.05; **, P < 0.01; ***, P < 0.001). (B) The constitutive expression of miR-200 family members was determined by real time PCR 48 h after cell seeding, using the RNU6 gene as an internal loading control, and calculating the ratio of parental to resistant cells. (C) The constitutive expression of CDH1, FN1 and VIM was measured in cells by using real time PCR 48 h after seeding. All data are expressed as the average of at least three measurements. Significance was determined between the OVCAR-3/TP GFP compared to the OVCAR-3 GFP cell line (*, P < 0.05; **, P < 0.01; ***, P < 0.001), (B and C). (D) EMT marker proteins were measured in cells 48 h after seeding using flow cytometry, and representative histograms of 10,000 events per cell line for each channel (E-cadherin-PE, Vimentin-Brilliant Violet 421, and Fibronectin-APC) are shown. Results for OVCAR-3 GFP cells are shown in orange and OVCAR-3/TP-GFP cells in blue.

Article Snippet: For intracellular staining, cells were exposed to Flow Cytometry Fixation Buffer (R & D Systems) for 20 min at 4 C in the dark, followed by the addition of Flow Cytometry Permeabilization buffer for 5 min at room temperature (R & D Systems).

Techniques: Sulforhodamine B Assay, Control, Concentration Assay, Expressing, Real-time Polymerase Chain Reaction, Marker, Cytometry