Journal:

Article Title: The neurotrophin receptor p75 NTR triggers endothelial cell apoptosis and inhibits angiogenesis: implications for diabetes-induced impairment of reparative neovascularization

doi: 10.1161/CIRCRESAHA.108.177386

Figure Lengend Snippet: (a) HUVEC were infected with 100, 250 and 500 M.O.I. of Ad.Null or Ad.p75NTR, as indicated. After 48h, cell lysates were collected and subjected to western blotting with antibodies to p75NTR, cleaved caspase-3, and GAPDH (used as loading control). (b) FACS analyses for p75NTR show abundant receptor expression (95.8±3%) at 12h from gene transfer with Ad.p75NTR (250 M.O.I.), while at 12h from Null gene transfer, p75NTR-expressing HUVEC are only 6.7±0.7%. (c) p75NTR-expressing HUVEC were gated and studied at 12h, 24h, and 48h for co-expression with Annexin-V and propidium iodide (PI) to detect apoptosis. This analysis revealed that at 12h from Ad.p75NTR, less than 9% of p75NTR-carrying cells presented Annexin-V on the external plasma membrane. Early apoptosis (cells positive for Annexin-V and negative for PI, lower right squares) peaked at 24h (39.1±3% of p75NTR-expressing HUVEC), followed by late apoptosis (cells positive for both Annexin-V and PI, upper right squares) at 48h from Ad.p75NTR (17.05±2% of p75NTR-expressing HUVEC). (d) HUVEC were treated as described in (a) and apoptotic nuclei were detected by TUNEL assay. Fluorescent images are representative of apoptosis rate in Null-HUVEC and p75NTR-HUVEC. Bar graphs quantify apoptosis, which is expressed as percentage of TUNEL-positive nuclei (green fluorescence) to total nuclei (stained in blue fluorescence by DAPI). Data are presented as means±SEM. *P<0.05 and **P<0.001 vs. Ad.Null. (e,f) Caspase-3 activity assay was performed on HUVEC (e) or MVEC (f). Cells (5000 cells/well) were infected with 100, 250 or 500 M.O.I. of Ad.Null or Ad.p75NTR or left uninfected (PBS). After 48h, Caspase-Glo 3/7 was incubated for 1h before recording luminescence. The apoptosis inducer staurosporin (stauro, 1μM) was used as reference in (e). Values are means±SEM. *P<0.05 and **P<0.001 vs. Ad.Null. (g) Upper panel: Concentration of endothelial apoptotic microparticles (EMP) released by Null-HUVEC or p75NTR-HUVEC under no other stimulation or following incubation with the apoptosis inducer staurosporin (100 nM, 16h). Values are means±SEM. **P<0.01 vs. Ad.Null. Lower panel: p75NTR is present in EMP released by HUVEC. The shadow peak corresponds to fluorescence background obtained with the isotypic control of p75NTR antibody. The second peak represents the specific labelling of EMPs with fluorescent p75NTR antibody. (h) Caspase-3 activity assay performed on HUVEC infected with Ad.Null or Ad. p75NTR (each at 100 and 250 M.O.I.) before being treated with PBS, proNGF (5ng/mL), NGF (100ng/mL), or BDNF (100ng/mL) for 24h. Values are means±SEM. *P<0.01 vs. PBS.

Article Snippet: HUVEC and MVEC were purchased from Cambrex (Belgium) and grown in EGM-2 medium (EBM-2 supplemented with EGM-2 SingleQuots, Cambrex) containing 5% FBS (Cambrex).

Techniques: Infection, Western Blot, Control, Expressing, Clinical Proteomics, Membrane, TUNEL Assay, Fluorescence, Staining, Caspase-3 Activity Assay, Incubation, Concentration Assay

Journal: Journal of Extracellular Biology

Article Title: Chemo‐small extracellular vesicles released in cisplatin‐resistance ovarian cancer cells are regulated by the lysosomal function

doi: 10.1002/jex2.157

Figure Lengend Snippet: A2780cis CDDP‐resistant cells have more capacity to produce and secrete exosomes. (a) Representative confocal microscopy images of A2780 and A2780cis PFA‐fixed cells immunofluorescent stained with anti‐CD63. Scale Bar 10 μm. (b) Analysis of the number of structures and (c) area of structures of CD63 per cell from images as those shown in (a). Bars indicated the mean with SEM; *** p < 0.001; one‐tiled unpaired parametric t ‐test ( n > 50 cells from three independent experiments). (d) Representative TEM micrograph shows MVEs of A2780 and A2780cis cells. Scale Bar 0.5 μm. (e) Semiquantitative analysis of the area of MVEs and (f) ILVs per MVEs of A2780 and A2780cis cells, from images as those shown in (d). Bars indicated the mean with SEM; * p < 0.05, *** p < 0.001; one‐tiled, unpaired, parametric t ‐test ( n > 50 cells from three independent experiments). (g) Analysis of detergent‐soluble protein extracts obtained from A2780 and A2780cis cells by Western blot with anti‐HRS, anti‐ALIX, anti‐TSG101, anti‐LAMP2A and anti‐β‐actin. The image is representative of three independent experiments. (h) Densitometric quantification of the signal of HRS, ALIX, TSG101 and LAMP2A from images as those shown in (g). The signal was normalized with β ‐actin signal. Bars indicated the mean with SEM; * p < 0.05, ** p < 0.01, one‐tiled, paired, non‐parametric Mann‐Whitney test ( n = 3). (i) Analysis of detergent‐soluble protein extracts obtained from A2780 and A2780cis cells by Western blot with anti‐RAB11A, anti‐RAB22A, anti‐RAB27A, anti‐RAB35 and anti‐β‐actin. The image is representative of three independent experiments. (j) Densitometric quantification of the signal of RAB11A, RAB22A, RAB27A and RAB35 from images as those shown in (i). The signal was normalized with β ‐actin signal. Bars indicated the mean with SEM; ** p < 0.01, NS: not significant; one‐tiled, paired non‐parametric Mann‐Whitney test ( n = 3).

Article Snippet: In general, the type and quantity of EVs are controlled intracellularly through its biogenesis at the level of MVEs and ILVs and/or with the trafficking and fusion of MVEs with the PM (Dixson et al., ; Steinbichler et al., ; van Niel et al., ).

Techniques: Confocal Microscopy, Staining, Western Blot, MANN-WHITNEY

Journal: PLoS ONE

Article Title: Comparison of polypeptides that bind the transferrin receptor for targeting gold nanocarriers

doi: 10.1371/journal.pone.0252341

Figure Lengend Snippet: Endocytosis of peptides by hCMEC/D3, MVEC and bEnd.3 cells analysed by FACS. Data shown is the mean ±SEM of the median fluorescence of 3 biological replicates. Binding of each peptide on different cell types was compared using an unpaired t-test. (*** = p<0.001, ** = p<0.01 and * = p<0.05).

Article Snippet: hCMEC/D3, bEnd.3 or MVEC (Lonza, CC-2527) cells were grown to confluence in a 12-well or 24-well plate and washed 2x with HBSS at 37°C before being treated with stated concentrations of each peptide (equal fluorescence intensity).

Techniques: Fluorescence, Binding Assay