Journal: bioRxiv
Article Title: CYB5R4 sustains endothelial proliferation and ischemia-induced angiogenesis by maintaining RRM2-dependent nucleotide balance
doi: 10.1101/2025.09.17.676962
Figure Lengend Snippet: HAECs were transfected with non-targeting (siNT), CYB5R4 -targeting (siCYB5R4), or SCD -targeting (siSCD) siRNA. (a, c) Gene silencing efficiency was verified using immunoblotting. N = 4; * indicates p < 0.05 using Student’s t-tests. (b, d) After siRNA transfection, cells were allowed to grow to confluence for 48 hours and used in a scratch wound healing assay. The representative images show the wound edges (highlighted in red) at the beginning and the end of the assay. The recovered wound areas are quantified in the right panels. N = 4; * indicates p < 0.05 using Student’s t-tests. (e) HAECs were treated with deuterium (D2)-labeled stearic acid (18:0) to measure the conversion rate of stearic acid to oleic acid (18:1). N = 4; * indicates p < 0.05 between linked groups using two-way ANOVA and Tukey’s post hoc test. (f) The representative density plot and quantification of cell cycle analysis using siRNA-transfected HAECs. N = 5; * indicates p < 0.05 between linked groups using one-way ANOVA and Tukey’s post-hoc test. (g) The representative dot density plot showing the gating strategy and the quantification of 5-ethynyl-2’-deoxyuridine (EdU) using siRNA-transfected HAECs. N = 3; * indicates p < 0.05 between linked groups using one-way ANOVA and Tukey’s post-hoc test. (h) After siRNA transfection, HAECs were treated with palmitic (PA), stearic (SA), palmitoleic (POA), or oleic acids (OA) and used in the EdU incorporation assay. N = 3; * indicates p < 0.05 between linked groups using one-way ANOVA and Tukey’s post-hoc test.
Article Snippet: Membranes were blocked for 1 h at room temperature in PBS with 3% BSA and 0.05% Tween-20, then incubated overnight at 4 °C with primary antibodies to CYB5R4 (Santa Cruz, sc-390569, 1:1,000), SCD (Cell Signaling, 2438, 1:1,000), RRM2 (Proteintech, 11661-1-AP, 1:1,000), and α-tubulin (Sigma, T6074, 1:10,000).
Techniques: Transfection, Western Blot, Wound Healing Assay, Labeling, Cell Cycle Assay
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