Journal:

Article Title: Thymidine phosphorylase induces angiogenesis in vivo and in vitro : an evaluation of possible mechanisms

doi: 10.1038/sj.bjp.0705216

Figure Lengend Snippet: The effect of thymidine phosphorylase and VEGF165 on HUVECs monolayer regeneration, following a mechanical injury. The top panel of phase contrast micrographs show: (a) the total denudation at the time of injury (T0); (b) basal regrowth in the presence of vehicle (5% FCS); (c) regeneration in the presence of VEGF (1 nM); (d) regeneration in the presence of thymidine phosphorylase (TP) (1 nM); (e) preincubation for 1 h and the continuous presence of TP inhibitor, reverts the TP-induced regeneration to basal level; (f) TP inhibitor has no effect on the VEGF165-induced recovery. Images were captured with a Nikon Diaphot inverted microscope at × 4 objective, coupled to a CCD (JVC), and grabbed using a Q500 Leica software. The calibration bar represents 400 μm. The graphs show the concentration–effect curve of TP and VEGF165, on (g) the recovery of a ‘wounded' area, and (h) the proliferation of endothelial cell. A synchronised monolayer of HUVECs was injured using a multichannel wounder, producing 11 linear lesions, and transferred to fresh media supplemented with 5% FCS, with appropriate treatments. They were incubated for a further 24 h, following which, they were either fixed and image analysed, or trypsinised and counted using a haemocytometer. Data expressed are mean±s.e.m. of at least four separate experiments with quadruplicate wells in each. In the wound recovery experiment, data are shown as percentage of T0 values. *P<0.05, **P<0.01, ***P<0.001 vs vehicle-treated controls, +P<0.05 vs corresponding TP-treated group.

Article Snippet: Human recombinant-thymidine phosphorylase or platelet-derived endothelial cell growth factor (rh PD-ECGF) and human recombinant vascular endothelial growth factor (VEGF) were procured from R&D Systems, USA.

Techniques: Inverted Microscopy, Software, Concentration Assay, Incubation

Journal:

Article Title: Thymidine phosphorylase induces angiogenesis in vivo and in vitro : an evaluation of possible mechanisms

doi: 10.1038/sj.bjp.0705216

Figure Lengend Snippet: Effect of 2-deoxy-D-ribose-1-phosphate (2-DDR-1-P) and thymine on tube formation by endothelial cells in a coculture system. Photomicrographs depict: (a) vehicle-treated cells, (b) VEGF-induced tubulogenesis, (c) suramin-induced inhibition of tubulogenesis, (d) tube formation induced by 2-DDR-1-P (10−6 M), (e) inhibition of tubulogenesis by thymine (10−4 M), and (f) reversal of thymine-inhibition by 2-DDR-1-P (10−4 M). A coculture of endothelial cells and fibroblasts (Angiokit) was used to study the tube formation by endothelial cells. The AngioKit was seeded with cells on day 0, and the optimised growth medium was changed on days 3, 5, 7, 10 and 12. It was then fixed, and stained for CD31, on day 14. Suramin (20 μM) and VEGF (2 ng ml−1) were used as negative and positive controls, respectively. Thymine and 2-DDR-1-P were added on day 4. The appropriate treatments were replenished with each medium change. Graph (g) shows the comparison of venule length following different treatments, as measured using a ‘AngioSys' image analysis system. Four images were grabbed per well, from the four quadrants. Experiments were run in quadruplicate, and data were expressed as the mean±s.e.m. *P<0.05, **P<0.01 vs vehicle control; #P<0.05 vs matched thymine concentration.

Article Snippet: Human recombinant-thymidine phosphorylase or platelet-derived endothelial cell growth factor (rh PD-ECGF) and human recombinant vascular endothelial growth factor (VEGF) were procured from R&D Systems, USA.

Techniques: Inhibition, Staining, Comparison, Control, Concentration Assay

Journal:

Article Title: Thymidine phosphorylase induces angiogenesis in vivo and in vitro : an evaluation of possible mechanisms

doi: 10.1038/sj.bjp.0705216

Figure Lengend Snippet: A proposed mechanism for the angiogenic effects of thymidine phosphorylase. Mechanical stress can lead to cell death (normal inside a solid tumour), and result in the release of thymidine in the microenvironment. Thymidine can enter live carcinoma cells, and is broken down by TP to 2-DDR-1-P, which can be dephosphorylated to 2-DDR. Inside a tumour cell (phase 1), reducing sugars can undergo rearrangement reactions, leading to the generation of free radicals. The concomitant upregulation of HO-1 has been implicated in increasing the expression of VEGF, MMPs and IL-8. These can then act on the host endothelial cells to induce an angiogenic effect in vivo. Furthermore, CO released would stabilise the neo-vessels through an antiapoptotic effect. Additionally, as seen in phase 2, the TP released from injured cells, or added exogenously, can act on thymidine and lead to the generation of 2-DDR, which is a chemotactic/chemokinetic factor, and promote angiogenesis. The 2-DDR that enters the cell can also be incorporated into the glycolytic machinery, and generate energy that can further serve towards a migratory phenotype. This model explains why both 2-DDR and 2-DLR could exert angiogenic effects in vivo, but only 2-DDR was active in vitro. Thin arrows indicate possible links elucidated in this study, while hashed arrows are putative links. The thicker arrows indicate pathways established by other studies (Brown et al., 2000).

Article Snippet: Human recombinant-thymidine phosphorylase or platelet-derived endothelial cell growth factor (rh PD-ECGF) and human recombinant vascular endothelial growth factor (VEGF) were procured from R&D Systems, USA.

Techniques: Expressing, In Vivo, In Vitro

Journal: FASEB journal : official publication of the Federation of American Societies for Experimental Biology

Article Title: Imaging NF-κB activity in a murine model of early stage diabetes

doi: 10.1096/fj.201801147R

Figure Lengend Snippet: A. Molecular model of the near-infrared ODN duplex probe (NIR-ODND) with a single NF-κB consensus binding site carrying a Cy5.5 fluorophore on one of the ODN strands. B-electrophoretic mobility (4–20% polyacrylamide gradient /TBE) of the NIR-ODND and single ODNs. ODND was labeled with either Cy5.5 (lane 3, red) or 800CW (lane 4, green). The migration of single strand NIR-fluorophore labeled ODNs serve as controls and are shown in lanes 1 and 2. C-electrophoretic mobility shift assay (EMSA, 10% PAGE): quantitative analysis of shifts using Cy5.5-labeled ODND (fluorescence emission – 700 nm) as a probe for activated NF-κB in pancreatic extracts collected on day 8. NIR-ODND was incubated in the presence of: lane 1- none; lane 2- HeLa nuclear extract (positive control); lane 3- islet nuclear extract-control; lane 4 - islet nuclear extract-LD-STZ; lane 5- islet cytoplasmic extract-control; lane 6- islet cytoplasmic extract-LD-STZ. The arrowhead points to the shifted ODN bands. D-quantification of fluorescence intensity of shifted bands due to NIR-ODND banding to the components of islet extracts. E-electrophoretic mobility shift assay for testing the specificity of NIR-ODND binding: lane 1- Cy5.5-labeled ODND, lane 2- HeLa nuclear extract; lane 3 – HeLa and excess of non-labeled ODND; lane 4: inactive HeLa extract; lane 5- LD-STZ treated islet extract; lane 6- LD-STZ treated islet extract and excess of non-labeled ODND; lane 7- inactive LD-STZ treated islet extract. For testing band supershift in the presence of anti- NF-κB p65 antibody Cy3-labeled ODND was analyzed on 7.5% PAGE gels: lane 8- ODND only, lane 9 - LD-STZ treated islet extract; lane 10 - LD-STZ treated islet extract and anti- NF-κB p65 antibody.

Article Snippet: Images were captured using Nikon TE2000-U inverted microscope equipped with a standard Cy5.5 set (XF141–2, Omega optical).

Techniques: Binding Assay, Labeling, Migration, Electrophoretic Mobility Shift Assay, Fluorescence, Incubation, Positive Control

Journal: Cell Reports

Article Title: Ets21c Governs Tissue Renewal, Stress Tolerance, and Aging in the Drosophila Intestine

doi: 10.1016/j.celrep.2019.05.025

Figure Lengend Snippet: Ets21c Functions Downstream of JNK Signaling in the Intestine (A) Setup and timeline of the TARGET expression system specific for the ISCs/EBs ( esg TS ) and ECs ( Myo1A TS ) of adult midguts. (B) qRT-PCR shows ets21c expression in 6-day-old midguts overexpressing hep WT and ets21c RNAi in ISCs/EBs ( esg TS ) (n = 4–5). (C–E) Representative confocal images of control 6-day-old posterior midguts (C) and those overexpressing hep WT only (D) or in combination with ets21c RNAi (E) in ISCs/EBs ( esg TS ) marked by GFP. Immunostaining labels cell membranes (Arm) and EEs (Pros). (F) Quantification of pH3 + cells per 6-day-old midgut overexpressing hep WT and ets21c RNAi in ISCs/EBs ( esg TS ) (n = 18–23). (G–I) Representative confocal images of control 6-day-old posterior midguts (G) and those overexpressing hep WT only (H) or in combination with ets21c RNAi (I) in ECs ( Myo1A TS ) marked by GFP. DAPI labels nuclei. (J) Quantification of pH3 + cells per 6-day-old midgut overexpressing hep WT and ets21c RNAi in ECs ( Myo1A TS ) (n = 16–36). (K) Setup and timeline of stress experiments using paraquat (PQ). (L–N) Representative confocal images of posterior midguts of control flies and those expressing ets21c RNAi in ISCs/EBs ( esg TS ) marked by GFP, fed for 24 h with mock solution (L) or 5 mM PQ (M and N). (O) Quantification of pH3 + cells per midgut of flies fed for 24 h with 5 mM PQ or mock solution and expressing ets21c RNAi in ISCs/EBs ( esg TS ) (n = 34–38). (P–R) Representative confocal images of posterior midguts of control flies and those expressing ets21c RNAi in ECs ( Myo1A TS ) marked by GFP, fed for 24 h with mock solution (P) or 5 mM PQ (Q and R). Immunostaining labels activated caspase Dcp1. DAPI labels nuclei. (S) Quantification of pH3 + cells per midgut of flies fed for 24 h with 5 mM PQ or mock solution and expressing ets21c RNAi in ECs ( Myo1A TS ) (n = 30–33). Data represent means (SDs); ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001, n.s. = non-significant. Scale bars: 50 μm. See also Figure S1 .

Article Snippet: Rabbit anti-phospho-Histone H3 (pH3) , Cell Signaling , Cat. #9701; RRID: AB_331535.

Techniques: Expressing, Quantitative RT-PCR, Control, Immunostaining

Journal: Cell Reports

Article Title: Ets21c Governs Tissue Renewal, Stress Tolerance, and Aging in the Drosophila Intestine

doi: 10.1016/j.celrep.2019.05.025

Figure Lengend Snippet: Ets21c Regulates Stem Cell Proliferation, Epithelial Turnover, and Tissue Aging (A) Quantification of pH3 + cells per midgut overexpressing ets21c WT and ets21c RNAi in ISCs/EBs ( esg TS ) (n = 30–41). (B–D) Representative confocal images of 6-day-old control posterior midguts (B) and those overexpressing ets21c WT (C) and ets21c RNAi (D) in ISCs/EBs ( esg TS ) marked by GFP. Immunostaining labels cell membranes (Arm), EEs (Pros), and ERK activation (dpERK). (E) Schematic representation of the esg TS -ReDDM tracing system. (F–H) Representative confocal images of 10-day-old control posterior midguts (F) and those overexpressing ets21c WT (G) and ets21c RNAi (H) in ISCs/EBs with the esg TS -ReDDM tracing system. ISCs/EBs are double positive for GFP and H2B-RFP. Newly generated diploid EEs (empty arrowhead) or polyploid ECs (filled arrowhead) are only labeled by H2B-RFP. DAPI labels nuclei. (I) qRT-PCR shows ets21c expression in 3-, 6-, and 20-day-old midguts of esg TS flies (n = 4). (J and K) Representative confocal images of 20-day-old control posterior midguts (J) and those expressing ets21c RNAi (K) in ISCs/EBs ( esg TS ) marked by GFP. Immunostaining labels cell membranes (Arm), EEs (Pros), and ERK activation (dpERK). Data represent means (SDs); ∗∗∗ p < 0.001. Scale bars: 50 μm.

Article Snippet: Rabbit anti-phospho-Histone H3 (pH3) , Cell Signaling , Cat. #9701; RRID: AB_331535.

Techniques: Control, Immunostaining, Activation Assay, Generated, Labeling, Quantitative RT-PCR, Expressing

Journal: Cell Reports

Article Title: Ets21c Governs Tissue Renewal, Stress Tolerance, and Aging in the Drosophila Intestine

doi: 10.1016/j.celrep.2019.05.025

Figure Lengend Snippet: Ets21c Drives Epithelial Renewal by Triggering EC Apoptosis (A–D) Representative confocal images of 6- and 20-day-old control posterior midguts (A and C) and those expressing ets21c RNAi (B and D) in ECs ( Myo1A TS ) marked by GFP. Immunostaining labels cell membranes (Arm). DAPI labels nuclei. (E) Diameter measurements of 10-day-old posterior midguts expressing ets21c WT and ets21c RNAi in ECs ( Myo1A TS ) (n = 18–22). (F–H) Representative confocal images of 10-day-old control posterior midguts (F) and those overexpressing ets21c RNAi (G) and ets21c WT (H) in ECs ( Myo1A TS ). Immunostaining labels activated caspase Dcp1, cell membranes (Arm), and EEs (Pros). (I–K) Representative confocal images of 6-day-old control posterior midguts (I) and those overexpressing ets21c RNAi (J) and ets21c WT (K) in ECs ( Myo1A TS ) marked by GFP. Images show single confocal sections of epithelial cross-sections with the gut lumen oriented to the bottom. Immunostaining labels cell membranes (Arm). DAPI labels nuclei. (L and M) Representative confocal images of 6-day-old posterior midguts overexpressing ets21c WT alone (L) or in combination with p35 (M) in ECs ( Myo1A TS ) marked by GFP. Immunostaining labels cell membranes (Arm). DAPI stains nuclei. (N) Quantification of pH3 + cells per midgut overexpressing ets21c WT and p35 in ECs ( Myo1A TS ) (n = 20–28). (O and P) Representative confocal images of recovery experiments showing posterior midguts of flies overexpressing ets21c WT in ECs ( Myo1A TS ) marked by GFP for 3 days at a permissive temperature (29°C) (O) compared to those shifted back to a restrictive temperature (18°C) for 3 additional days (P). Data represent means (SDs); ∗∗∗ p < 0.001; n.s. = non-significant. Scale bars: 50 μm. See also Figure S2 .

Article Snippet: Rabbit anti-phospho-Histone H3 (pH3) , Cell Signaling , Cat. #9701; RRID: AB_331535.

Techniques: Control, Expressing, Immunostaining

Journal: Cell Reports

Article Title: Ets21c Governs Tissue Renewal, Stress Tolerance, and Aging in the Drosophila Intestine

doi: 10.1016/j.celrep.2019.05.025

Figure Lengend Snippet: Cell-Type-Specific Sets of Target Genes Mediate the Cellular Responses to Ets21c (A–E) Representative confocal images of 6-day-old control posterior midguts (A) and those overexpressing ets21c WT alone (B) or in combination with RNAi-transgenes against pvf1 (C), upd3 (D), and imp (E) in ISCs/EBs ( esg TS ) marked by GFP. (F–J) Representative confocal images of 6-day-old control posterior midguts (F) and those overexpressing ets21c WT alone (G) or in combination with RNAi-transgenes against pvf1 (H), upd3 (I), and eip93F (J) in ECs ( Myo1A TS ) marked by GFP. (K–O) Representative confocal images of 10-day-old control posterior midguts (K) and those overexpressing ets21c WT alone (L) or in combination with RNAi-transgenes against pvf1 (M), upd3 (N), and eip93F (O) in ECs ( Myo1A TS ). Immunostaining labels activated caspase Dcp1. (P) Quantification of pH3 + cells per midgut overexpressing ets21c WT and RNAi-transgenes against pvf1 , upd3 , imp , and eip93F in ISCs/EBs ( esg TS ) or ECs ( Myo1A TS ) (n = 10–27). Data represent means (SDs); ∗∗∗ p < 0.001. (Q) Schematic representation of Ets21c functions in the Drosophila adult intestine. DAPI labels nuclei. Scale bars: 50 μm. See also Figure S4 .

Article Snippet: Rabbit anti-phospho-Histone H3 (pH3) , Cell Signaling , Cat. #9701; RRID: AB_331535.

Techniques: Control, Immunostaining

Journal: Cell Reports

Article Title: Ets21c Governs Tissue Renewal, Stress Tolerance, and Aging in the Drosophila Intestine

doi: 10.1016/j.celrep.2019.05.025

Figure Lengend Snippet:

Article Snippet: Rabbit anti-phospho-Histone H3 (pH3) , Cell Signaling , Cat. #9701; RRID: AB_331535.

Techniques: Recombinant, Reverse Transcription, In Situ, Software, Real-time Polymerase Chain Reaction, Microscopy, Inverted Microscopy

Journal: Ecotoxicology and environmental safety

Article Title: Role of ROS-mediated PERK/ATF4 signaling activation in extracorporeal tube formation injury of human umbilical vein endothelial cells induced by cooking oil fume PM 2.5 exposure.

doi: 10.1016/j.ecoenv.2023.115332

Figure Lengend Snippet: Fig. 3. COF-PM2.5 exposure activates the PERK/ATF4 signaling pathway in HUVECs. HUVECs were stimulated with COF-PM2.5 (100 μg/mL) for 0–12 h. (A) The representative western blotting images of p-PERK, p-eIF2α, as well as ATF4 expressions in HUVECs. (B-D) Quantification for p-PERK, p-eIF2α, and ATF4. All experimental data were expressed as mean ± SEM. (n = 3 per group). Compared with the untreated group, * P < 0.05, * * P < 0.01, * ** P < 0.001.

Article Snippet: Antibodies against PERK (3192 S), p-eIF2α (3398 S), eIF2α (2103 S), and β-Actin (4970) were from Cell Signaling Technology (Beverley, MA).

Techniques: Western Blot

Journal: Ecotoxicology and environmental safety

Article Title: Role of ROS-mediated PERK/ATF4 signaling activation in extracorporeal tube formation injury of human umbilical vein endothelial cells induced by cooking oil fume PM 2.5 exposure.

doi: 10.1016/j.ecoenv.2023.115332

Figure Lengend Snippet: Fig. 4. COF-PM2.5 exposure inhibits VEGFR2 expression via triggering PERK/ ATF4 signaling in HUVECs. (A-B) HUVECs tube formation was observed by inverted microscope after co-incubation with 100 nM GSK and 100 μg/mL COF-PM2.5 for 6 h. (A) The representative tube formation images of HUVECs in different groups. (B) Quantitative analysis of number of meshes in HUVECs. (C-H) HUVECs were pretreated with 100 nM GSK for 1 h prior to exposure to 100 µg/mL COF-PM2.5 for 6 h. (C) VEGFR2 protein expression in HUVECs was detected using western blotting. (D) Quantitative analysis for VEGFR2. (E) The representative western blotting images of p-PERK, p-eIF2α, as well as ATF4 expres sion in HUVECs. (F) Quantitative analysis for p-PERK. (G) Quantitative analysis for p- eIF2α. (H) Quantitative analysis for ATF4. All experimental data were expressed as mean ± SEM. (n = 3 per group). Compared with the control group, * * P < 0.01, * ** P < 0.001. Compared with COF-PM2.5 group, # P < 0.05, ### P < 0.001.

Article Snippet: Antibodies against PERK (3192 S), p-eIF2α (3398 S), eIF2α (2103 S), and β-Actin (4970) were from Cell Signaling Technology (Beverley, MA).

Techniques: Expressing, Inverted Microscopy, Incubation, Western Blot, Control

Journal: Ecotoxicology and environmental safety

Article Title: Role of ROS-mediated PERK/ATF4 signaling activation in extracorporeal tube formation injury of human umbilical vein endothelial cells induced by cooking oil fume PM 2.5 exposure.

doi: 10.1016/j.ecoenv.2023.115332

Figure Lengend Snippet: Fig. 6. NAC reverses COF-PM2.5-induced down-regulated expression of VEGFR2 via PERK/ATF4 signaling in HUVECs. (A-B) HUVECs tube formation was observed by inverted microscope after co-incubation with 200 μM NAC and 100 μg/mL COF- PM2.5 for 6 h. (A) The representative tube formation images of HUVECs in different groups. (B) Quantitative analysis of number of meshes in HUVECs. (C-H) HUVECs were pretreated with 200 μM NAC for 1 h prior to exposure to 100 µg/ mL COF-PM2.5 for 6 h. (C) VEGFR2 pro tein in HUVECs was detected using west ern blotting. (D) Quantitative analysis for VEGFR2. (E) The representative western blotting images of p-PERK, p-eIF2α, as well as ATF4 expression in HUVECs. (F) Quantitative analysis for p-PERK. (G) Quantitative analysis for p-eIF2α. (H) Quantitative analysis for ATF4. All experimental data were expressed as mean ± SEM. (n = 3 per group). Compared with the control group, * * P < 0.01, * ** P < 0.001. Compared with COF-PM2.5 group, # P < 0.05, ## P < 0.01.

Article Snippet: Antibodies against PERK (3192 S), p-eIF2α (3398 S), eIF2α (2103 S), and β-Actin (4970) were from Cell Signaling Technology (Beverley, MA).

Techniques: Expressing, Inverted Microscopy, Incubation, Western Blot, Control