Fig. 8: ZRR complexes regulate complement-induced vascular injury in vivo.
HUVECs stably transduced with an NF-κB luciferase reporter were treated with PRA and luciferase activity were assessed following treatment with PRA for 6 h (a). Quantitative epifluorescence analysis of nuclear translocation of the P65 NF-κB subunit in HUVECs transfected with siRNA and treated with PRA for 4 h (b). qRT-PCR of siRNA-transfected HUVECs exposed to PRA for 4 h (c). EC:T cell cocultures were performed using HUVECs transfected with various siRNA as indicated, and CFSE-labeled T cells were harvested and analyzed by FACS after 10–14 days in coculture (d). Male WT or Rubicon-/-mice received 200 μL tail vein injection of MOPC or anti-H-2b Ab (750 μg) and 24 h later, male skin grafts were harvested and analyzed by I.F. implanted onto female SCID/bg hosts that had passively received 1 × 106 female WT C57/Bl6 splenocytes. Three weeks post-implantation, skin grafts were analyzed (e). Following MOPC or anti-H-2b Ab injection i.v. (750 μg), kidney tissues from age-matched WT or Rubicon-/- mice were harvested 24 h later and analyzed for glomerular VCAM-1 staining on CD31+ECs prior to host implantation (f, n = 8 mice per group). Three weeks post-implantation, male skin grafts treated with MOPC or anti-H-2b Ab as above were analyzed by I.F. (g, n = 8 mice per group) and H&E sections (h, n = 8 mice per group). Schema of the proposed mechanism for ZRR complexes (i). For Fig. 8a–d, experiments repeated 3 times with different HUVEC donors. For Fig. 8a–d, f–h two-way ANOVA followed by Tukey’s pairwise comparison was used for statistical analysis where p < 0.05 is considered statistically significant. Figures 8e and 8i were created with BioRender.com. All scale bars, 400 μm. p-values are indicated in the figures.
