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. 2023 Jun 9;4(6):101072. doi: 10.1016/j.xcrm.2023.101072

Figure 5.

Figure 5

FAM3D causes eNOS uncoupling through FPR1/2-mediated oxidative stress

(A) Representative western blot and quantification of monomer/dimer eNOS ratios in HUVECs treated with FAM3D (10 nmol/L) for 6 h after preincubation with CsH (1 μmol/L) and/or WRW4 (1 μmol/L) for 2 h. n = 4. Data are represented as mean ± SEM. Two-way ANOVA followed by Tukey’s multiple comparisons test, ∗p < 0.05.

(B) Quantification of intracellular NO by DAF-FM DA staining in human umbilical vein ECs (HUVECs) treated with FAM3D (10 nmol/L) for 6 h after preincubation with CsH (1 μmol/L) and/or WRW4 (1 μmol/L) for 2 h. n = 4. Data are represented as mean ± SEM. Two-way ANOVA followed by Tukey’s multiple comparisons test, ∗p < 0.05.

(C) Quantification of ROS by DCFH-DA staining in HUVECs treated with FAM3D (10 nmol/L) for various times. n = 3. Data are represented as mean ± SEM. One-way ANOVA followed by Dunnett’s multiple comparisons test, ∗p < 0.05.

(D) Quantification of ROS by DCFH-DA staining in HUVECs treated with FAM3D (10 nmol/L) for 60 min after preincubation with CsH (1 μmol/L) and/or WRW4 (1 μmol/L) for 2 h. n = 4. Data are represented as mean ± SEM. Two-way ANOVA followed by Tukey’s multiple comparisons test, ∗p < 0.05.

(E) Quantification of O2− generation by DHE staining in HUVECs treated with FAM3D (10 nmol/L) in the presence or absence of L-NAME (200 μmol/L) for 60 min. n = 3. Data are represented as mean ± SEM. Two-way ANOVA followed by Tukey’s multiple comparisons test, ∗p < 0.05.

(F) Quantification of intracellular NO by DAF-FM DA staining in HUVECs treated with FAM3D (10 nmol/L) for 6 h after preincubation with NAC (5 mmol/L) or apocynin (100 μmol/L) for 4 h. n = 4. Data are represented as mean ± SEM. Two-way ANOVA followed by Sidak’s multiple comparisons test, ∗p < 0.05.

(G) Representative western blot and quantification of monomer/dimer eNOS radios in HUVECs treated with FAM3D (10 nmol/L) for 6 h after preincubation with NAC (5 mmol/L) or apocynin (100 μmol/L) for 4 h. n = 3. Data are represented as mean ± SEM. Two-way ANOVA followed by Sidak’s multiple comparisons test, ∗p < 0.05.