Fig 1: Spatial interaction between CYB5R4 and RRM2.(a) The illustration shows APEXII-mediated proximal protein biotinylation. HAECs expressing CYB5R4-APEXII are incubated with biotin phenol and hydrogen peroxide (H2O2) to induce biotinylation. CYB5R4 is examined in the streptavidin pulldown and input samples using immunoblotting. (b) HAECs transfected with non-targeting (siNT) or CYB5R4-targeting (siCYB5R4) are used in the proximity ligation assay (PLA). Cells are either incubated with the negative isotype control or RRM2 and CYB5R4-specific antibodies. The RRM2 and CYB5R4 staining or PLA signal intensities are quantified in (c-e). N=3 for the control IgG or 6 for RRM2 and CYB5R4 antibody groups; * indicates p < 0.05 using one-way ANOVA with Tukey’s post-hoc test.
Fig 2: The effect of restoring hub genes in CYB5R4-silenced endothelial cells.(a) The illustration shows the strategy to screen the effect of hub genes on proliferation. HAECs are transduced with doxycycline (DOX)-inducible lentivirus (LV) expressing hub genes. Cells are pooled and transfected with CYB5R4 siRNA (siCYB5R4). After growing with or without DOX, genome DNA (gDNA)-incorporated transgenes are quantified using quantitative PCR (qPCR). (b) The qPCR results are summarized as the relative enrichment of transgenes in DOX-treated cells over the no DOX control. (c-e) The representative image and quantification of immunoblotting show CYB5R4 and RRM2 expression with transfection of non-targeting siRNA (NT), CYB5R4-targeting siRNA (KD), empty vector (EV), or RRM2-expressing vector (RRM2). N=6; * indicates p<0.05 using Student’s t-test. (f, g) Cell proliferation is examined with EdU incorporation under the same knockdown and overexpression conditions. N=5; * indicates p<0.05 using one-way ANOVA and Tukey’s post-hoc test.
Supplier Page from Sino Biological, Inc. for Human RRM2 Gene ORF cDNA clone in cloning vector