Journal: Pharmaceutics

Article Title: Crotamine/siRNA Nanocomplexes for Functional Downregulation of Syndecan-1 in Renal Proximal Tubular Epithelial Cells.

doi: 10.3390/pharmaceutics15061576

Figure Lengend Snippet: Figure 4. Colocalization of syndecan-1 and crotamine/siRNA nanocomplexes on the cell sur- face of PTECs in vitro by multicolor confocal immunofluorescence microscopy. Human PTEC HK-2 cells were incubated with fluorescently labeled AF555-crotamine/AF488-siRNA complexes formed at a molar ratio of 100:1 with 50 nM of siRNA, at 4 ◦C for 30 min (A,B). Immunorecog- nition of syndecan-1 by mouse anti-human syndecan-1 (Alexa647) (C). Nuclei stained with DAPI (blue) (D,H,L). Differential interference contrast (DIC) image (E) and overlay of AF555-crotamine (red) and AF488-siRNA (green) (F,J). Overlay of AF555-crotamine (red) and AF647-syndecan-1 (cyan) (G,K), and image merge with all four channels including DIC (H,L). Magnified images of overlays are also shown (J–L). Colocalization was analyzed using the JacoP plugin for ImageJ soft- ware (1.44 version) based on Mander’s coefficient. Venn diagram values represent percentage positive pixels for (overlapping) stainings, measured in 20 cells per calculation (I). Bars = 25 µm. Images are representative of three independent assays. Objective 63× oil.

Article Snippet: The culture medium was removed and the cells were then incubated on ice with the primary antibody AF647 Alexa Fluor® 647 mouse anti-human syndecan-1 (CD138; Bio-Rad/AbD Serotec, Hercules, CA, USA—MCA2459A647) (diluted 1:100 in culture medium) for 30 min, followed by washing with medium and incubation with the secondary antibody donkey anti-mouse IgG conjugated with AF647 (Invitrogen, Waltham, MA, USA—A32787) (1:100 in culture medium), also for 30 min, on ice.

Techniques: In Vitro, Microscopy, Incubation, Labeling, Staining

Journal: Experimental & Molecular Medicine

Article Title: Upregulation of FcγRIIB by resveratrol via NF-κB activation reduces B-cell numbers and ameliorates lupus

doi: 10.1038/emm.2017.144

Figure Lengend Snippet: Resveratrol treatment decreases B cells in the bone marrow of MRL/ lpr mice. ( a ) The weight of spleens of MRL/ lpr mice from control and resveratrol-treated groups was measured and compared after the completion of treatment at 20 weeks of age ( P =0.0024). The percentages of ( b ) CD19 + B cells (control, n =6; resveratrol-treated, n =8) in the blood, spleen and bone marrow (BM) ( P =0.6634, 0.0479 and 0.0071, respectively) and ( c ) CD19 + GL7 + germinal center (GC) B cells in the spleen between control ( n =10) and resveratrol-treated ( n =9) groups were quantified by flow cytometry, compared and are presented as bar graphs ( P =0.0458). The percentages of ( d ) CD3 + T cells ( P =0.8675), ( e ) CD11b + myeloid cells and ( f ) CD11c + dendritic cells in the spleen were measured for comparison between control and resveratrol-treated groups. Data from control and resveratrol-treated mice are shown as the mean±s.e.m. * P <0.05 and ** P <0.01 denote statistical significance, whereas NS indicates no significance.

Article Snippet: Mouse IgG isotypes and mAbs specific to CD16/32 (clone 2.4G2), CD138-BV421 (clone 281-2), CD11b-PerCP-Cy5.5 (clone M1/70), CD11c-AlexaFluor 700 (clone HL3) and GL7-AlexaFluor 647 (clone GL7) were acquired from BD Biosciences (San Jose, CA, USA).

Techniques: Flow Cytometry

Journal: Experimental & Molecular Medicine

Article Title: Upregulation of FcγRIIB by resveratrol via NF-κB activation reduces B-cell numbers and ameliorates lupus

doi: 10.1038/emm.2017.144

Figure Lengend Snippet: Resveratrol treatment enhances the surface expression of FcγRIIB on B cells. Bar graphs of flow cytometry analyses on the expression levels of FcγRIIB of ( a ) total splenic CD19 + B cells ( P =0.0219) and ( b ) Germinal center (GC) B cells ( P =0.035) between control ( n =8) and resveratrol-treated ( n =7) groups. Bar graphs to compare the mean fluorescence intensity (MFI) of FcγRIIB expression on ( c ) CD19 + B cells ( P =0.0489) and ( d ) PCs ( P =0.0487) in the bone marrow between control ( n =9) and resveratrol-treated ( n =8) groups. Bar graphs of flow cytometric quantification to compare the MFI of FcγRIIB on splenic ( e ) CD11b + myeloid cells ( P =0.0457) and ( f ) CD11c + dendritic cells between control ( n =5) and resveratrol-treated ( n =9) groups. The MFI levels of FcγRIIB on cells were shown as the fold changes with respect to levels in the control. ( g ) Immunohistological examination and ( h ) quantification of the intensity (expression level) of FcγRIIB in B220 + B cells (circled regions) in follicles (>15 follicles per section) between control and resveratrol-treated mice. Scale bars denote 50 μm. Quantification results are illustrated in a bar graph. * P <0.05, ** P <0.01.

Article Snippet: Mouse IgG isotypes and mAbs specific to CD16/32 (clone 2.4G2), CD138-BV421 (clone 281-2), CD11b-PerCP-Cy5.5 (clone M1/70), CD11c-AlexaFluor 700 (clone HL3) and GL7-AlexaFluor 647 (clone GL7) were acquired from BD Biosciences (San Jose, CA, USA).

Techniques: Expressing, Flow Cytometry, Fluorescence