https://orcid.org/0000-0003-4954-3498
Figures & data
Figure 1 Effect of passive adsorption of BSA on aggregation and surface potential of FDP-NV functionalized with carboxyl groups and suspended in water, culture medium and biological buffers, respectively.
Notes: Particles were suspended in the various dispersants, applied into capillary cuvettes, and positioned into a Zetasizer instrument (Malvern Inc.) for measuring Z-average, diameter size (A) and ζ-potential (B). Data are presented as means ± SD from three measurements of independent samples. (*) P<0.001 and (**) P<0.01 for the difference between FDP-NV-BSA and native FDP-NV, in a particular dispersant, calculated using one-way ANOVA.
Abbreviations: FDP-NV, fluorescent diamond particles with NV active centers; HUVEC, human umbilical vein endothelial cells; BSA, bovine serum albumin; PBS, phosphate-buffered saline; SD, standard deviation.
Figure 2 Effect of FDP-NV on HepG-2 cell proliferation determined by direct evaluation of cell numbers after 24 hours.
Notes: (A) Graphic presentation of the percentage increase in HepG-2 cell numbers after 24 hours incubation with or without FDP-NV-BSA, or vincristine. The data represent means ± SD from 5 independent wells, and evaluation of 7 observation fields for each well. (*) P<0.001 between control and treated group, calculated by one-way ANOVA. (B) Representative images of observation fields of cells applied for the determination of cell numbers using ImageJ software. Images were captured in a fluorescence microscope (Olympus IX81) using a10x objective and DAPI (blue) and TRITC (red) filters. White arrows indicate internalized particles into the flanking cells of colonies.
Abbreviations: FDP-NV, fluorescent diamond particles with NV active centers; HepG-2, human liver hepatocellular carcinoma; standard deviation; DAPI, 4ʹ,6-diamidino-2-phenylindole; TRITC, tetramethylrhodamine.
Figure 3 Effect of FDP-NV on HUVEC proliferation determined by direct evaluation of cell numbers after 24, 48 and 72 hours.
Notes: (A) Graphic presentation of the percentage increase in HUVEC number after incubation for the indicated times with or without FDP-NV-BSA, or vincristine. The data represent means ± SD from 5 independent wells, and evaluation of 7 observation fields for each well. (*) P<0.001, (**) P = 0.02 between control and treated groups, calculated by one-way ANOVA. (B) Representative images of observation fields of cells applied for the determination of cell numbers using ImageJ software after 24 hours incubation. Images were captured in a fluorescence microscope (Olympus IX81) using a 10x objective and DAPI (blue) and TRITC (red) filters.
Abbreviations: FDP-NV, fluorescent diamond particles with NV active centers; HUVEC, human umbilical vein endothelial cells; SD standard deviation; DAPI, 4ʹ,6-diamidino-2-phenylindole; TRITC, tetramethylrhodamine.
Figure 4 Effect of FDP-(NV) on HepG-2 (A) and HUVEC (B) NADPH-dependent oxidoreductase activity tested in the MTT assay.
Notes: Values “% of control” were calculated based on absorption at 562 nm for control samples (no FDP-NV) measured on the same plate. Data are presented as means ± SD for three independent experiments, calculated by One-way ANOVA between control and treated groups (*) P<0.001; (**) P<0.01
Abbreviations: FDP-NV, fluorescent diamond particles with NV active centers; HepG-2, liver hepatocellular carcinoma; HUVEC, human umbilical vein endothelial cells; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; NADPH, nicotinamide adenine dinucleotide phosphate; SD, standard deviation.
Figure 5 Effect of FDP-(NV) on HepG-2 (A) and HUVEC (B) cytoplasmic esterase activity monitored using the calcein AM assay.
Notes: Values “% of control” were calculated based on the fluorescence of control samples (no FDP-NV) as measured on the same plate. Data are presented as means ± SD for three independent experiments, calculated by One-way ANOVA between control and treated groups (*) P<0.001; (**) P<0.01
Abbreviations: FDP-NV, fluorescent diamond particles with NV active centers; HepG-2, liver hepatocellular carcinoma; HUVEC, human umbilical vein endothelial cells; AM, acetoxymethyl ester; SD, standard deviation.
Figure 6 Effect of FDP-NV on the migration of HUVEC stimulated by 2% FBS in a scratch assay.
Notes: “Scratch closure” stimulated by 2% FBS in the presence or absence of FDP-NV is presented in (A). Non-stimulated cells (negative control) were treated with medium containing 0.1% FBS. Data are presented as means ± SD for three independent experiments, calculated by One-way ANOVA between control and treated groups (*) P<0.001 for comparison with control (2% FBS treated) in One-way ANOVA. Shown in (B) are representative images of scratches as visualized in a fluorescence microscope (Olympus IX81) at 20x magnification and using DAPI (blue) and TRITC (red) filters.
Abbreviations: FDP-NV, fluorescent diamond particles with NV active centers; HUVEC, human umbilical vein endothelial cells; FBS, fetal bovine serum; SD, standard deviation; DAPI, 4ʹ,6-diamidino-2-phenylindole; TRITC, tetramethylrhodamine.
Figure 7 Effect of FDP-NV on the phosphorylation of MAPK Erk1/2 induced by FBS.
Notes: HepG-2 cells (A) or HUVEC (B) serum-starved for 24 hrs, were stimulated with 2% FBS for 10 and 20 minutes. After stripping, total MAPK Erk1/2 was re-probed in PVDF membrane with an anti-phospho antibody. Bar graphs on the right show the ratios of the intensities of the total protein bands to the phosphorylated protein bands. Green bars represent ratios for control (non-treated cells), whereas red bars for FDP-NV treated cells. Left panes show representative Western blot images for each cell type. Data are presented as means ± SD for three independent experiments. (*) P<0.01 for comparison between FBS-treated or non-treated cells, calculated by One-way ANOVA.
Abbreviations: FDP-NV, fluorescent diamond particles with NV active centers; HepG-2, liver hepatocellular carcinoma; HUVEC, human umbilical vein endothelial cells; FBS, fetal bovine serum; MAPK Erk1/2, mitogen-activated protein kinase extracellular-signal regulated; SD, standard deviation.
Figure 8 Identification of phospho- and total-MAPK Erk1/2 in the cytoplasm and nuclei of HepG-2 cells and HUVEC in the presence and absence of FDP-NV and TPA.
Notes: HepG-2 cells (A) or HUVEC (B) were treated or not with FDP-NV-BSA (0.1 mg/mL), and after 24 hours serum-starvation, stimulated or not with TPA. Cells were lysed and fractionated into cytoplasmic and nuclear fractions. Fractions were probed by WB using indicated antibodies. Mek-1 was used as a marker for the cytoplasmic fraction, whereas HDAC1 as a marker for the nuclear fraction. HepG-2 cells (C) or HUVEC (D) were grown on chamber slides, serum-starved for 24 hours, and then exposed to FDP-NV-BSA. After treatment or not with TPA, cells were immunostained with anti-phospho-MAPK Erk 1/2, followed by staining with FITC-conjugated goat anti-rabbit. Slides were analyzed in a fluorescence microscope (Olympus IX81) at 400x magnification using an oil objective and FITC (green) and TRITC (red) filters. Overlapping areas of green and red are seen in yellow. White arrows indicate the high accumulation of particles in TPA-treated cells; blue arrows indicate nuclei of cell non-treated with TPA; red arrows indicate nuclei of cell treated with TPA.
Abbreviations: FDP-NV, fluorescent diamond particles with NV active centers; HepG-2, human liver hepatocellular carcinoma; HUVEC, human umbilical vein endothelial cells; MAPK Erk1/2, mitogen-activated protein kinase extracellular-signal regulated; Mek-1, MAP or Erk kinase, HDAC1, histone deacetylase 1, TPA, tetradecanoyl phorbol acetate; FITC, fluorescein isothiocyanate; TRITC, tetramethylrhodamine., WB, Western blotting.
Figure 9 Effect of FDP-NV on the induction of apoptosis and ER stress in HepG-2 cells and HUVEC.
Notes: (A) Western blot analysis of cleavage of caspase 3 in the presence or absence of FDP-NV (0.1 mg/mL) in HepG-2 and HUVEC. Vincristine was used as a positive control for apoptosis. The localization of molecular weight markers is indicated by arrows on the left side of images. (B) Western blot analysis of expression of chaperons in ER in the presence or absence of FDP-NV (0.1 mg/mL) in HepG-2 cells and HUVEC. Tunicamycin was used as a positive control for ER-stress.
Abbreviations: FDP-NV, fluorescent diamond particles with NV active centers; ER, endoplasmic reticulum; HepG-2, liver hepatocellular carcinoma; HUVEC, human umbilical vein endothelial cells; kDa, kilodaltons; CHOP, C/EBP (emopamil-binding protein) homologous protein; BiP, binding immunoglobulin protein.
