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. 2021 Feb 8;6(3):e142299. doi: 10.1172/jci.insight.142299

Figure 2. NGBR knockdown inhibits the proliferation of HemSCs in vitro.

Figure 2

(A) NGBR knockdown was characterized by Western blot and RT-PCR. *P < 0.05 vs. control (siControl) cells (n = 3). (B) Cell proliferation modulated by NGBR deficiency was evaluated by cell proliferation assay after treatment with EGF, FGF2, and VEGF in HemSCs. The results were expressed as fold change relative to the initial cell number. *P < 0.05 vs. control (siControl) cells. #P < 0.05 vs. control (siControl) cells treated with EGF, FGF2, and VEGF (n = 4), 3 repeats. (C) NGBR knockdown inhibited BrdU incorporation in HemSCs treated with growth factors. Cells treated with EGF, FGF2, and VEGF displayed more BrdU-positive cells than control groups. NGBR knockdown abolished EGF-induced, FGF2-induced, and VEGF-induced BrdU incorporation. Quantitative analysis of BrdU-positive cells was determined by ImageJ software. *P < 0.05 vs. control (siControl) cells. #P < 0.05 vs. control (siControl) cells treated with EGF, FGF2, and VEGF (n = 4), 3 repeats. (D) HemSCs expressing FUCCI cell cycle markers were transiently transfected with siControl or siNGBR. After 24 hours, the cells were fixed, and the images were taken with a confocal microscope. Quantitative analysis of G1 phase cells was determined by ImageJ software. *P < 0.05 vs. control (siControl) cells (n = 3), 3 repeats. (E) HemSCs transfected with siNGBR underwent G1 phase arrest. The cell cycle distribution was analyzed by flow cytometry using PI staining. The percentage of different cell cycle phases presented in histogram form has been summarized in the bar graph (right). *P < 0.05 vs. control (siControl) cells (n = 3), 3 repeats. (F) Protein levels of NGBR, p21, p53, cyclin D1, RB1, and phosphorylated RB1 in HemSCs treated with siControl and siNGBR were determined by Western blot. *P < 0.05 vs. control (siControl) cells (n = 3). Statistical analyses: 2-tailed unpaired Student’s t test (A, D, E, and F) and 1-way ANOVA with Bonferroni’s post hoc test (B and C); data are expressed as mean ± SEM. HemSCs, hemangioma stem cells; NGBR, NOGOB receptor; siControl, control siRNA; siNGBR, NGBR siRNA; FUCCI, fluorescence ubiquitination cell cycle indicator; RB1, retinoblastoma; PI, propidium iodide.