Journal: Oncotarget

Article Title: Regulation of tumor growth by circulating full-length chromogranin A

doi: 10.18632/oncotarget.12237

Figure Lengend Snippet: A. Induction of protease nexin-1 mRNA in endothelial cells . HUVECs were treated with various doses of recombinant CgA for 20 h. The presence of PN-1 transcripts in cells was analyzed by real time PCR. PN-1 mRNA expression is reported as fold change compared to untreated cells. The cumulative results of 3 experiments are shown: in each experiment each sample was analyzed 5-6 times by real-time PCR (each in triplicate). Box-plots with median, interquartiles and 5-95 percentile values are shown (dotted line, mean value of controls). ***, P < 0.001, by t test (2-tailed). B. Effect of PN1 gene silencing on the activity of CgA in the endothelial spheroid sprouting assay . HUVEC cells spheroids were transfected with PN-1 siRNA mixture (siPN1) or with control siRNA (siCtrl) as described in Methods. After transfection, spheroids were treated with or without and 0.2 nM recombinant CgA 1-439 for 16 hours. The number of sprouts was from each spheroid was then counted. Cumulative results of two independent experiments (8-16 spheroids/experiment). The number of spheroids used is indicated in each panel ( n ). *, P < 0.05; **, P < 0.01 by t test. C. - D. Effect of anti-PN-1 antibodies on the activity of CgA in the endothelial spheroid sprouting assay . Cumulative results of two independent experiments (8 spheroids/experiment, total n = 16). Spheroids were incubated with or without recombinant CgA (0.2 nM) in the absence or the presence of anti-PN-1 polyclonal immunoglobulins (goat IgGs anti-human Serpin E2/PN1), or control immunoglobulins (normal goat IgGs) as indicated. Bars: mean ± SE. ****, P < 0.0001, ***, P < 0.001, by t test (2-tailed). Representative microphotographs of spheroids are shown in ( C ) (arrows: endothelial cell sprouts). E. Effect of anti-PN-1 antibodies on the anti-tumor activity of CgA . WEHI-164- or TS/A-tumor-bearing mice were treated with recombinant CgA (i.p.) in combination with anti-PN-1, or with control immunoglobulins (normal goat IgGs) at the indicated doses. Antibodies were given 2.5 h before CgA. Arrows indicate the day of treatment. Tumor volume (mean ± SE). The area under the curve for each mouse was calculated using the GraphPad Prism Software. Statistical analysis was performed by Mann-Whitney test on the calculated areas (6 animals per group; **, P < 0.01; *, P < 0.05).

Article Snippet: Goat IgGs anti-mouse serpin E2/PN1, goat IgGs anti-human serpin E2/PN1 and normal goat immunoglobulins were from R&D System.

Techniques: Recombinant, Real-time Polymerase Chain Reaction, Expressing, Activity Assay, Transfection, Incubation, Software, MANN-WHITNEY

Journal: Ecotoxicology and environmental safety

Article Title: The influence of phenolic environmental estrogen on the transcriptome of uterine leiomyoma cells: A whole transcriptome profiling-based analysis.

doi: 10.1016/j.ecoenv.2021.111945

Figure Lengend Snippet: Fig. 4. The effects of 10.0 μmol/L BPA, 32.0 μmol/L NP on genes and proteins expression in uterine leiomyoma cells. (a) Hierarchically clustered heatmap of differentially expressed genes associated with cell cycle after BPA and NP exposure. (b) E2F1, CCND1, CCNE2, MCM2, MCM3, MCM4, MCM5, MCM6 genes expression by q-PCR presented as relative changes. There were increased E2F1, CCND1, CCNE2, MCM2, MCM3, MCM4, MCM5, MCM6 genes expression in uterine leiomyoma cells treated with10.0 μmol/L BPA, 32.0 μmol/L NP for 48 h compared to control group (*P < 0.05). (c) Representative western blots and (d) band intensity bar graphs of proteins. There were significantly (*P < 0.05) higher expression levels of E2F1, CCND1, CCNE2, MCM2, MCM3, MCM4, MCM5, MCM6 in the cells treated with BPA and NP for 48 h compared to controls. The western band images shown are representative of three independent experiments and the data were expressed as mean ± SE done in three independent experiments.

Article Snippet: The following primary antibodies were used for the western blotting: E2F1 Monoclonal Antibody (66515-1-Ig; Proteintech; diluted 1:1000); Cyclin D1 Monoclonal Antibody (60186-1-Ig; Proteintech; diluted 1:5000); Cyclin E2 Polyclonal Antibody (11935-1-AP; Proteintech; diluted 1:500); MCM2 Polyclonal Antibody (10513-1-AP; Proteintech; diluted 1:1500); MCM3 Polyclonal Antibody (15597-1-AP; Proteintech; diluted 1:1000); MCM4 Polyclonal Antibody (13043-1-AP; Proteintech; diluted 1:600); MCM5 Polyclonal Antibody (11703-1-AP; Proteintech; diluted 1:1000); MCM6 Polyclonal Antibody (13347-2-AP; Proteintech; diluted 1:8000); phospho-Rb monoclonal (ab184796; abcam; diluted 1:1000); phospho-PI3K polyclonal (ab182651; abcam; diluted 1:500); phospho- AKT polyclonal (ab38449; abcam; diluted 1:500).

Techniques: Expressing, Control, Western Blot

Journal: Infectious agents and cancer

Article Title: Silencing of UBE2D1 inhibited cell migration in gastric cancer, decreasing ubiquitination of SMAD4.

doi: 10.1186/s13027-021-00402-2

Figure Lengend Snippet: Fig. 1 UBE2D1 was elevated in GC tissues. A The mRNA level of UBE2D1 was analyzed from TCGA-STAD data. B The mRNA level of UBE2D1 was detected in 25 pairs of tumors and paracancerous samples. ***, P < 0.001. C The protein level of UBE2D1 in tissue array (32 pairs) was determined by IHC staining. D Impact of UBE2D1 expression on OS in GC patients in TCGA

Article Snippet: Primary antibodies were listed as following: UBE2D1 (1:800, Proteintech, Rosemont, IL, USA), SMAD4 (1:1000, Abcam, St. Louis, MO, USA), MMP2 (1:1000, Abcam), MMP9 (1:800, Abcam), and β-actin (1:2000, Abcam).

Techniques: Immunohistochemistry, Expressing

Journal: Infectious agents and cancer

Article Title: Silencing of UBE2D1 inhibited cell migration in gastric cancer, decreasing ubiquitination of SMAD4.

doi: 10.1186/s13027-021-00402-2

Figure Lengend Snippet: Fig. 2 Expression level of UBE2D1 was measured by real-time PCR western blot assay. A UBE2D1 expression level in different cell lines, including GSE-1, AGS, BGC-823, MGC-803, MKN45, and SGC-7901. B Knock-down efficiency was evaluated after transduction with shUBE2D1 lentiviruses in AGS and MKN45 cells. ***, P < 0.001 versus shNC. C Overexpression efficiency was evaluated after transduction with UBE2D1 overexpression lentivirus in MGC-803 cells. ***, P < 0.001 versus vector

Article Snippet: Primary antibodies were listed as following: UBE2D1 (1:800, Proteintech, Rosemont, IL, USA), SMAD4 (1:1000, Abcam, St. Louis, MO, USA), MMP2 (1:1000, Abcam), MMP9 (1:800, Abcam), and β-actin (1:2000, Abcam).

Techniques: Expressing, Real-time Polymerase Chain Reaction, Western Blot, Knockdown, Transduction, Over Expression, Plasmid Preparation

Journal: Infectious agents and cancer

Article Title: Silencing of UBE2D1 inhibited cell migration in gastric cancer, decreasing ubiquitination of SMAD4.

doi: 10.1186/s13027-021-00402-2

Figure Lengend Snippet: Fig. 4 Overexpression of UBE2D1 promoted cell migration in vitro. A–C Transwell (A and B) and wound healing (C) assays were performed to analyze cell migration after transduction with UBE2D1 overexpression lentivirus in MGC-803 cells. ***, P < 0.001 versus vector. D A western blot assay was performed to measure the protein levels of UBE2D1, MMP2, and MMP9

Article Snippet: Primary antibodies were listed as following: UBE2D1 (1:800, Proteintech, Rosemont, IL, USA), SMAD4 (1:1000, Abcam, St. Louis, MO, USA), MMP2 (1:1000, Abcam), MMP9 (1:800, Abcam), and β-actin (1:2000, Abcam).

Techniques: Over Expression, Migration, In Vitro, Transduction, Plasmid Preparation, Western Blot

Journal: Advanced science (Weinheim, Baden-Wurttemberg, Germany)

Article Title: Graphene Quantum Dot Sensitized Heterojunctions Induce Tumor-Specific Cuproptosis to Boost Sonodynamic and Chemodynamic Enhanced Cancer Immunotherapy.

doi: 10.1002/advs.202410606

Figure Lengend Snippet: Figure 6. a) CRT levels in 4T1 cells were measured following various treatments. b) Following different treatments, Western blot analysis was used to examine the presence of DLAT and DLAT oligomers in 4T1 cells. c,d) By utilizing flow cytometry, we determined the expression of CD80 and CD86 in DCs. e) WB analysis of FDX1 and LIAS in 4T1 cells after different treatments. f) Bio-TEM images of 4T1 cells before and after GQD/Cu2O + US treatment. Statistical significance between the experimental group and the control group is calculated with a two-tailed Student’s t-test. Data are presented as the mean ± SD. (n = 3). *p < 0.05 and ***p < 0.001.

Article Snippet: After incubation with antibodies to DLAT (1:20 000, proteintech), LIAS (1:1000, Abcam), FDX1 (1:1000, Abcam), γ-H2AX (Abcam), or α- tubulin (1:1000, Abcam), the cell lysates were analyzed and detected with a chemiluminescent imaging system.

Techniques: Western Blot, Cytometry, Expressing, Control, Two Tailed Test

Journal: International Journal of Molecular Sciences

Article Title: Externalization of Mitochondrial PDCE2 on Irradiated Endothelium as a Target for Radiation-Guided Drug Delivery and Precision Thrombosis of Pathological Vasculature

doi: 10.3390/ijms23168908

Figure Lengend Snippet: Anti-PDCE2 antibodies bind to the surface of irradiated endothelial cells under optimized shear flow in a parallel-plate flow system. ( A ) Confocal images demonstrate binding of fluorescently tagged PDCE2 antibody (AF-647, green) at two different concentrations (1.25–2.5 µg/mL) to the irradiated cell surface at day 2 and day 4 post-irradiation (0–25 Gy) after 15 min of circulation in growth medium in the parallel-plate flow system. Nuclei were stained with DAPI (blue). Magnification 200× (Bar = 50 µm). ( B ) Confocal images from three independent experiments were analyzed using NIH Image J. Data (integrated density) are shown as mean ± SEM and were analyzed using two-way ANOVA followed by Tukey’s post-hoc analysis. * p < 0.05, *** p < 0.001, **** p < 0.0001, compared to corresponding non-irradiated cells. #### p < 0.0001, comparison between timepoints. ns, not significant.

Article Snippet: Click-&-GoTM Lys-to-Lys Protein-Protein Conjugation Kit (Click Chemistry Tools, Scottsdale, USA) was used to conjugate purified anti-PDCE2 antibody (166899, Santa Cruz, CA, USA) and human thrombin (Jomar Life Research, Scoresby, VIC, Australia).

Techniques: Irradiation, Shear, Binding Assay, Staining, Comparison

Journal: International Journal of Molecular Sciences

Article Title: Externalization of Mitochondrial PDCE2 on Irradiated Endothelium as a Target for Radiation-Guided Drug Delivery and Precision Thrombosis of Pathological Vasculature

doi: 10.3390/ijms23168908

Figure Lengend Snippet: PDCE2-targeting coaguligand induces selective fibrin clot formation on irradiated cells in a parallel-plate flow system. Representative confocal images showing FITC-tagged fibrinogen (red) forming fibrin clots on confluent endothelial monolayers at day 2 post-irradiation (0, 5, 15 and 25 Gy) in the parallel-plate flow system containing whole human blood. Anti-PDCE2 antibody–thrombin coaguligands were injected into the circulation at concentrations of 2.5 and 5 µg/mL for 15 min. Nuclei were stained with Hoechst (blue). Cells were imaged with phase contrast microscopy. All images were captured at a magnification of 200× (Bar = 50 µm). It should be noted that in response to radiation, cell layers retain full confluence; however, as they undergo significant hypertrophy (cell and nuclear enlargement), fewer cells are observed per field of view.

Article Snippet: Click-&-GoTM Lys-to-Lys Protein-Protein Conjugation Kit (Click Chemistry Tools, Scottsdale, USA) was used to conjugate purified anti-PDCE2 antibody (166899, Santa Cruz, CA, USA) and human thrombin (Jomar Life Research, Scoresby, VIC, Australia).

Techniques: Irradiation, Injection, Staining, Microscopy

Journal: International Journal of Molecular Sciences

Article Title: Externalization of Mitochondrial PDCE2 on Irradiated Endothelium as a Target for Radiation-Guided Drug Delivery and Precision Thrombosis of Pathological Vasculature

doi: 10.3390/ijms23168908

Figure Lengend Snippet: Fibrin clot formation is dependent on radiation dose and coaguligand concentration. Total ( A ) and average ( B ) fibrin volume per field of view were quantified by IMARIS after 3D volume rendering of fibrin clots. Total fibrin volume represents all fluorescence captured per field of view; average fibrin volume represents the sum of the fluorescence of each individual fibrin clot divided by the total number of clots per field of view. All thrombosis assays in the parallel-plate flow system were performed at day 2 after radiation exposure (0, 5, 15, 25 Gy) in the presence of coaguligand (2.5 or 5 µg/mL), or thrombin (2.5 µg/mL, equivalent activity), with or without excess non-specific IgG (12.5 µg/mL). Data are shown as mean ± SEM (at least 3 independent experiments) and were analyzed by two-way ANOVA with Tukey’s post-hoc analysis. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001, comparison to corresponding non-irradiated cells. # p < 0.5, ## p < 0.01, ### p < 0.001, #### p < 0.0001, comparison within and between radiation groups. ns, not significant. ( C ) Representative confocal images showing fibrin clot formation of FITC-tagged fibrinogen (red) with and without excess non-specific IgG antibody in the presence of anti-PDCE2-thrombin coaguligand (2.5 µg/mL). Experiments were performed only with the highest coaguligand and radiation dose at day 2. Nuclei were stained with Hoechst (blue). Magnification = 200× (Bar = 50 µm). ( D ) Representative images of fibrin clots (red) visualized on endothelial layers using confocal microscopy after 3D reconstruction of Z-stacks (volume rendering) using IMARIS.

Article Snippet: Click-&-GoTM Lys-to-Lys Protein-Protein Conjugation Kit (Click Chemistry Tools, Scottsdale, USA) was used to conjugate purified anti-PDCE2 antibody (166899, Santa Cruz, CA, USA) and human thrombin (Jomar Life Research, Scoresby, VIC, Australia).

Techniques: Concentration Assay, Fluorescence, Activity Assay, Comparison, Irradiation, Staining, Confocal Microscopy

Journal: International Journal of Molecular Sciences

Article Title: Externalization of Mitochondrial PDCE2 on Irradiated Endothelium as a Target for Radiation-Guided Drug Delivery and Precision Thrombosis of Pathological Vasculature

doi: 10.3390/ijms23168908

Figure Lengend Snippet: Fibrin clot formation is dependent on coaguligand concentration and radiation dose under static conditions (static Petri dish model). ( A ) Representative confocal images of FITC-tagged fibrinogen (red) forming fibrin clots on irradiated cells (0, 5, 15, 25 Gy) at day 2. Anti-PDCE2 antibody–thrombin coaguligands (2.5 or 5 µg/mL) or free thrombin (1.25 µg/mL, equivalent activity) were added to confluent cell layers in 2 mL of whole human blood for 10 min before washing, confocal imaging and 3D rendering. Nuclei were stained with Hoechst (blue); all images were taken at magnification 200× (Bar = 50 µM). ( B ) Total fibrin volume per field of view was quantified by IMARIS. Data are shown as mean ± SEM (3 independent experiments) and were analyzed by two-way ANOVA with Tukey’s post-hoc analysis. * p < 0.05, comparison to corresponding non-irradiated cells. # p < 0.05, ### p < 0.001, comparison within radiation groups.

Article Snippet: Click-&-GoTM Lys-to-Lys Protein-Protein Conjugation Kit (Click Chemistry Tools, Scottsdale, USA) was used to conjugate purified anti-PDCE2 antibody (166899, Santa Cruz, CA, USA) and human thrombin (Jomar Life Research, Scoresby, VIC, Australia).

Techniques: Concentration Assay, Irradiation, Activity Assay, Imaging, Staining, Comparison

Journal: International Journal of Molecular Sciences

Article Title: Externalization of Mitochondrial PDCE2 on Irradiated Endothelium as a Target for Radiation-Guided Drug Delivery and Precision Thrombosis of Pathological Vasculature

doi: 10.3390/ijms23168908

Figure Lengend Snippet: High throughout, static approaches for assessment of fibrin deposition demonstrate lower sensitivity (static multi-well model). ( A ) Representative fluorescent images of a 96-well microplate demonstrating extent of fibrin clot deposition (FITC, green) 2 days after irradiation (25 Gy only) in the presence of anti-PDCE2-thrombin coaguligand (0–10 µg/mL) and 200 µL whole human blood for 10 min. Images were captured using an EVOS Imaging System. Magnification, 100× (Bar = 400 µm). ( B ) FITC fluorescence (integrated density) in fluorescent images was analyzed using NIH Image J. ( C ) FITC-labelled fibrinogen binding was assessed in the same plates using a fluorescent microplate reader. For both analyses, average value of triplicate wells per individual treatment group is shown ± SEM (one independent experiment). Data were analyzed using two-way ANOVA with Tukey’s post-hoc analysis. * p < 0.5, comparison relative to non-irradiated, saline-treated cells (no coaguligand). # p < 0.05, ## p < 0.01, comparison within radiation group.

Article Snippet: Click-&-GoTM Lys-to-Lys Protein-Protein Conjugation Kit (Click Chemistry Tools, Scottsdale, USA) was used to conjugate purified anti-PDCE2 antibody (166899, Santa Cruz, CA, USA) and human thrombin (Jomar Life Research, Scoresby, VIC, Australia).

Techniques: Irradiation, Imaging, Fluorescence, Binding Assay, Comparison, Saline