Fig 1: Effects of RANKL, OPG, and DMB on human immune cells.Flow cytometry analysis of (A) percent RANK+ cells in human PBMCs from healthy blood donors. (B) Percent intracellular RANKL-positive CD4 and CD8 T lymphocytes before (Basal) and after (Stim) activation of human PBMCs with plate-bound anti-CD3 (0.5 μg/ml) (n = 3). IFN-γ, TNF-α, and IL-17 cytokine production (C) in CD4 (left) and CD8 (right) T cells in PBMCs stimulated with plate-bound anti-CD3 (0.5 μg/ml) or (D) in CD45RA+ naïve CD4 T cells stimulated in the presence of CD14+ monocytes (MN) with plate-bound anti-CD3 (0.5 μg/ml) and soluble anti-CD28 (1.0 μg/ml) (n = 3 to 6). Cytokines were assessed in the absence (untreated) or presence of 500 ng/ml each of OPG (blue bar) or DMB (pink bar) for 72 hours. Intracellular cytokine production is shown on the y axis as the ratio (fold) of % cytokine+ cells treated with OPG or DMB versus untreated group. Dotted line (at 1.0) represents no difference in intracellular cytokine production between untreated and treated. *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001 versus untreated. (E) Levels (picograms per milliliter) of IL-1β and IL-6 in supernatants of CD14+ MNs incubated with RANKL (100 ng/ml) for 24 hours in the presence or absence of OPG (n = 6). ****P < 0.0001 versus basal; ##P < 0.05 versus RANKL. Individual symbols in the graphs represent independent experiments on different human immune cell donors, averaging triplicate samples for all experiments (A to E). All data represent means ± SEM. Statistical analysis was by t test (C and D) and by ANOVA with Tukey’s post hoc analysis (E).
Fig 2: Cytokine-induced rodent and human β cell death require RANKL/RANK.Adv-LacZ– or Adv-Cre–transduced mouse islet cells after 48 hours (A) were stained for insulin (green), Cre-recombinase (red), and DAPI (blue), as represented in the individual and merged images (white bar indicates the scale for the images); or (B) the ratio of Rank/actin mRNA was analyzed by real-time qPCR (n = 3). *P < 0.05 versus LacZ. (C) Percent TUNEL-positive β cells in mouse islet cells from WT or Rank-floxed (RANKFl) mice transduced with Adv-Cre for 48 hours and treated without (Ctrl) or with cytokines (CTK) for an additional 24 hours (n = 5 to 6). **P < 0.01 versus WT-Ctrl; #P < 0.05 versus WT-CTK. Percent of TUNEL-positive β cells in human islet cells treated without (Ctrl) or with CTK for 24 hours and (D) with Veh (V), 100 ng/ml of hOPG (O), or hRANKL (R) alone, or in combination at a 1:1, 1:5, or 5:1 ratio of OPG/RANKL (O + R), respectively; $P < 0.05, $$P < 0.01 versus all other groups without symbols, on duplicate samples of n = 9; or (E) with Veh or DMB (0.1 to 100 ng/ml), with Ctrl-Veh represented as 100% (Ctrl-Veh value 1.54 ± 0.42%; n = 4 to 8); ***P < 0.001 versus Ctrl-Veh; #P < 0.05, ##P < 0.01 versus CTK-Veh. Experiments were done in duplicate with 5 to 10 fields and 1646 ± 124 β cells per sample analyzed for mouse islets (C), and 5 to 10 fields and 1144 ± 167 β cells per sample analyzed for human islets (D and E). Individual symbols in the graphs represent independent experiments on individual mouse or human islet preps, averaging duplicate samples for all experiments (A to E). All data represent means ± SEM. All statistical analysis was by ANOVA with Tukey’s post hoc analysis.
Supplier Page from Enzo Life Sciences, Inc. for RANKL (soluble) (human), (recombinant)