Page 68 - Haematologica May 2022

G. Purgatorio et al.
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Figure 2. Impaired GATA2 activity and expression alter eNOS mRNA and protein expression. (A) Real time polymerase chain reaction (PCR) of the endothelial nitric oxide synthase gene (eNOS) mRNA expression in blood outgrowth endothelial cells (BOEC) from healthy controls, the unaffected family member (I1) and the GATA2-deficient family members. eNOS mRNA expression is reported as fold change versus healthy control BOEC normalized to a housekeeping mRNA (GAPDH). Values represent mean ± standard error of the mean (SEM) of 8 repeated measures from 6 controls and 3 different preparations from the family members (**P<0.001 vs. controls; one-way ANOVA followed by Dunnett’s multiple comparison test). (B) Western blotting of eNOS protein in BOEC from healthy controls, the unaffected family member (I1) and the GATA2 deficiency family members. Actin was used as loading control. Optical densitometric analysis was performed using ImageJ software and results are expressed in arbitrary units. Values represent mean ± SEM of 6 repeated measures from 6 controls and 3 different preparations from the patients (one-way ANOVA followed by Dunnett’s multiple comparison test, *P<0.005, **P<0.001 vs. controls). (C) Real time PCR of GATA2-coding mRNA extracted from BOEC after incubation with three dif- ferent small interfering RNA (siRNA) directed against GATA2 (25 nM) and their combinations. GATA2 expression was normalized to that of GAPDH. Values represent mean ± SEM of 4 repeated measures (*P<0.05 vs. controls, one-way ANOVA followed by Bonferroni’s multiple comparison test). (D) Western blotting of GATA2 protein extracted from BOEC after incubation with siRNA directed against GATA2 (25 nM) for 24-48 hours (h). HPRT was used as a loading control. Optical densitometric analysis was performed using ImageJ software and results are expressed in arbitrary units. Values represent mean ± SEM of 4 repeated measures (**P<0.001 vs. untreated BOEC, two-way ANOVA followed by Dunnett’s multiple comparison test). (E) Expression of eNOS mRNA after 24 h and 36 h of incubation of control BOEC with siRNA direct- ed against GATA2 (25 nM) measured by real time PCR. The expression of eNOS is reported as fold change versus untreated BOEC and normalized to a housekeeping mRNA (GAPDH). Values are means ± SEM of 6 different transfection experiments (**P<0.005 vs. control BOEC, one way-ANOVA followed by Bonferroni’s multiple com- parison test). (F) Western blotting of eNOS protein expression in BOEC treated with siRNA directed against GATA2 (25 nM) for 24, 36 and 48 h. HPRT was used as loading control. Optical densitometric analysis was performed using ImageJ software and results are expressed in arbitrary units. Values are mean ± SEM of 6 different trans- fection experiments (**P<0.001 vs. control BOEC, two-way ANOVA followed by Dunnett’s multiple comparison test).
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