Fig 2: FBXO38 and Nox1 Interact and Co-localize. (a) Use immunofluorescence staining to analyze the co-localization of FBXO38 and Nox1 proteins in the cytoplasm of HUVECs. Stain the cell nuclei with DAPI. Magnification: ×240, scale bar = 10 μm. (b) Use immunocoprecipitation to evaluate the interaction between FBXO38 and Nox1.

Fig 3: FBXO38 overexpression attenuates LOSS-induced oxidative stress and apoptosis. HUVECs with over-expressed FBXO38 (OE group) and negative control group (NC group) are established, respectively, for subsequent experiments. (a) qPCR analysis of FBXO38 mRNA in HUVECs of NC group and OE group (⁣∗∗p < 0.01; n = 3). (d, g) Western blot detection and quantification of FBXO38 protein levels in the abovementioned two groups of HUVECs (⁣∗∗∗p < 0.001; n = 3). (b, c) qPCR analysis of FBXO38, Nox1, Bax, Bcl-2, and Caspase3 mRNA in two groups of HUVECs exposed to LOSS for 0 and 24 h (ns, p > 0.05; ⁣∗p < 0.05; ⁣∗∗p < 0.01; n = 3). (e, f, h, i) Western blot detection and quantification of FBXO38, Nox1, Bax, Bcl-2, and C-caspase3 protein levels under the same conditions (ns, p > 0.05; ⁣∗∗p < 0.01; ⁣∗∗∗p < 0.001; n = 3). (j) Representative immunofluorescence images showing FBXO38 (red) and Nox1 (green) in two groups of HUVECs at 0 and 24 h after LOSS (magnification: ×20, scale bar = 1000 μm). (k, l) Fluorescence intensity quantification of FBXO38 and Nox1, respectively (ns, p > 0.05; ⁣∗p < 0.05; ⁣∗∗p < 0.01; ⁣∗∗∗p < 0.001; n = 3). (m) Representative images of ROS staining (two groups of HUVECs treated for 0 and 24 h under LOSS; magnification: ×20, scale bar = 1000 μm). (n) Quantification of ROS fluorescence intensity (⁣∗p < 0.05; ⁣∗∗p < 0.01; ⁣∗∗∗p < 0.001; n = 3). (o, p) Flow cytometry analysis the apoptosis of HUVECs under the same conditions (ns, p > 0.05; ⁣∗p < 0.05; ⁣∗∗∗p < 0.001; n = 3). Data are presented as mean ± SEM from three independent experiments (n = 3). Significance is determined by one-way ANOVA with Tukey's post-hoc test (unless otherwise specified). “ns” indicates no statistical significance.

Fig 4: Knockdown of Nox1 reverses elevated ROS and apoptosis in FBXO38-deficient HUVECs. (a) qPCR analysis of Nox1 mRNA in HUVECs after transfecting sgRNA of Nox1 (⁣∗∗∗p < 0.001; n = 3). (d, g) Western blot detection and quantification of Nox1 protein levels in HUVECs after transfecting sgRNA of Nox1 (⁣∗∗∗p < 0.001; n = 3). Set up the siRNA FBXO38 group, the siRNA FBXO38 + sgRNA Nox1 group, and the blank control group (Mock group) for subsequent experiments. (b, c) qPCR analysis of FBXO38, Nox1, Bax, Bcl-2, and Caspase3 mRNA in HUVECs of the above three groups.(⁣∗p < 0.05; ⁣∗∗p < 0.01; n = 3). (e, f, h, i) Western blot detection and quantification of FBXO38, Nox1, Bax, Bcl-2, and C-caspase3 protein levels under the same conditions (⁣∗p < 0.05; ⁣∗∗p < 0.01; ⁣∗∗∗p < 0.001; n = 3). (j) Representative immunofluorescence images showing FBXO38 (red) and Nox1 (green) under the same conditions (magnification: ×20, scale bar = 1000 μm). (k, l) Fluorescence intensity quantification of FBXO38 and Nox1, respectively (⁣∗p < 0.05; ⁣∗∗p < 0.01; ⁣∗∗∗p < 0.001; n = 3). (m) Representative images of ROS staining (three groups of HUVECs under the same conditions as above; magnification: ×20, scale bar = 1000 μm). (n) Quantification of ROS fluorescence intensity (⁣∗p < 0.05; ⁣∗∗∗p < 0.001; n = 3). (o, p) Flow cytometry analysis the apoptosis of HUVECs under the same conditions as above (⁣∗p < 0.05; ⁣∗∗p < 0.01; n = 3). Data are presented as mean ± SEM from three independent experiments (n = 3). Significance is determined by one-way ANOVA with Tukey's post hoc test (unless otherwise specified).

Fig 5: FBXO38 knockdown exacerbates LOSS-induced oxidative stress and apoptosis. (a) qPCR analysis of FBXO38 mRNA in HUVECs after transfecting different siRNA of FBXO38 (⁣∗p < 0.05; ⁣∗∗p < 0.01; n = 3). (d, g) Western blot detection and quantification of FBXO38 protein levels in HUVECs after transfecting different siRNA of FBXO38 (⁣∗p < 0.05; ⁣∗∗p < 0.01; n = 3). After verification, HUVECs transfected with SiRNA-3 are selected as the knockdown group (Si) and used with the negative control group (NC) for subsequent experiments. (b, c) qPCR analysis of FBXO38, Nox1, Bax, Bcl-2, and Caspase3 mRNA in two groups of HUVECs exposed to LOSS for 0 and 24 h (⁣∗p < 0.05; ⁣∗∗p < 0.01; ⁣∗∗∗p < 0.001; n = 3). (e, f, h, i) Western blot detection and quantification of FBXO38, Nox1, Bax, Bcl-2, and C-caspase3 protein levels under the same conditions (ns, p > 0.05; ⁣∗p < 0.05; ⁣∗∗p < 0.01; ⁣∗∗∗p < 0.001; n = 3). (j) Representative immunofluorescence images showing FBXO38 (red) and Nox1 (green) in two groups of HUVECs at 0 and 24 h after LOSS (magnification: ×20, scale bar = 1000 μm). (k, l) Fluorescence intensity quantification of FBXO38 and Nox1, respectively (⁣∗p < 0.05; ⁣∗∗p < 0.01; ⁣∗∗∗p < 0.001; n = 3). (m) Representative images of ROS staining (two groups of HUVECs treated for 0 and 24 h under LOSS; magnification: ×20, scale bar = 1000 μm). (n) Quantification of ROS fluorescence intensity (⁣∗p < 0.05; ⁣∗∗p < 0.01; n = 3). (o, p) Flow cytometry analysis the apoptosis of HUVECs under the same conditions (⁣∗p < 0.05; ⁣∗∗p < 0.01; n = 3). Data are presented as mean ± SEM from three independent experiments (n = 3). Significance is determined by one-way ANOVA with Tukey's post hoc test (unless otherwise specified). “ns” indicates no statistical significance.