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Figure 8. miR-423-5p protects endothelial cells from apoptosis and promotes endothelial migration and angiogenesis. (A) Immunoblot and densitometric analysis of cleaved PARP1 in endothelial cells transfected with scrambled miR mimic control (Ctrl) or miR-423-5p mimics (10 nM) and exposed to normal medium or serum starvation for 4 hours. <t>TUBA1B</t> was used as a loading control; n = 2–3. Scale bar: 200 µm (B) Quantification of caspase-3 activity in endothelial cells transfected with Ctrl or miR-423-5p mimics (10 nM) exposed to N or SS for 4 hours; n = 3. Scale bar: 100 µm (C) Endothelial cells transfected with scrambled miR mimic control (Ctrl) or miR-423-5p mimics (10 nM) were mechanically injured, and wound closure was followed over a 6-hour period. The wound healing assay results are expressed as the percentage of wound closure ± SEM; n = 4 for each condition. Representative pictures at 6 hours after injury are presented. (D) Endothelial cells were transfected with scrambled miR mimic control (Ctrl) or miR-423-5p mimics (10 nM), and capillary-like structures were quantified after 6 hours on Matrigel. Angiogenic activity was assessed by quantifying the number of segments per field ± SEM; n = 4 for each condition. Representative images of tubule formation are presented in the right panel. P values were obtained by 1-way ANOVA and the Bonferroni post hoc test (*P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001).
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Figure 8. miR-423-5p protects endothelial cells from apoptosis and promotes endothelial migration and angiogenesis. (A) Immunoblot and densitometric analysis of cleaved PARP1 in endothelial cells transfected with scrambled miR mimic control (Ctrl) or miR-423-5p mimics (10 nM) and exposed to normal medium or serum starvation for 4 hours. <t>TUBA1B</t> was used as a loading control; n = 2–3. Scale bar: 200 µm (B) Quantification of caspase-3 activity in endothelial cells transfected with Ctrl or miR-423-5p mimics (10 nM) exposed to N or SS for 4 hours; n = 3. Scale bar: 100 µm (C) Endothelial cells transfected with scrambled miR mimic control (Ctrl) or miR-423-5p mimics (10 nM) were mechanically injured, and wound closure was followed over a 6-hour period. The wound healing assay results are expressed as the percentage of wound closure ± SEM; n = 4 for each condition. Representative pictures at 6 hours after injury are presented. (D) Endothelial cells were transfected with scrambled miR mimic control (Ctrl) or miR-423-5p mimics (10 nM), and capillary-like structures were quantified after 6 hours on Matrigel. Angiogenic activity was assessed by quantifying the number of segments per field ± SEM; n = 4 for each condition. Representative images of tubule formation are presented in the right panel. P values were obtained by 1-way ANOVA and the Bonferroni post hoc test (*P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001).
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Figure 8. miR-423-5p protects endothelial cells from apoptosis and promotes endothelial migration and angiogenesis. (A) Immunoblot and densitometric analysis of cleaved PARP1 in endothelial cells transfected with scrambled miR mimic control (Ctrl) or miR-423-5p mimics (10 nM) and exposed to normal medium or serum starvation for 4 hours. <t>TUBA1B</t> was used as a loading control; n = 2–3. Scale bar: 200 µm (B) Quantification of caspase-3 activity in endothelial cells transfected with Ctrl or miR-423-5p mimics (10 nM) exposed to N or SS for 4 hours; n = 3. Scale bar: 100 µm (C) Endothelial cells transfected with scrambled miR mimic control (Ctrl) or miR-423-5p mimics (10 nM) were mechanically injured, and wound closure was followed over a 6-hour period. The wound healing assay results are expressed as the percentage of wound closure ± SEM; n = 4 for each condition. Representative pictures at 6 hours after injury are presented. (D) Endothelial cells were transfected with scrambled miR mimic control (Ctrl) or miR-423-5p mimics (10 nM), and capillary-like structures were quantified after 6 hours on Matrigel. Angiogenic activity was assessed by quantifying the number of segments per field ± SEM; n = 4 for each condition. Representative images of tubule formation are presented in the right panel. P values were obtained by 1-way ANOVA and the Bonferroni post hoc test (*P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001).
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Figure 8. miR-423-5p protects endothelial cells from apoptosis and promotes endothelial migration and angiogenesis. (A) Immunoblot and densitometric analysis of cleaved PARP1 in endothelial cells transfected with scrambled miR mimic control (Ctrl) or miR-423-5p mimics (10 nM) and exposed to normal medium or serum starvation for 4 hours. <t>TUBA1B</t> was used as a loading control; n = 2–3. Scale bar: 200 µm (B) Quantification of caspase-3 activity in endothelial cells transfected with Ctrl or miR-423-5p mimics (10 nM) exposed to N or SS for 4 hours; n = 3. Scale bar: 100 µm (C) Endothelial cells transfected with scrambled miR mimic control (Ctrl) or miR-423-5p mimics (10 nM) were mechanically injured, and wound closure was followed over a 6-hour period. The wound healing assay results are expressed as the percentage of wound closure ± SEM; n = 4 for each condition. Representative pictures at 6 hours after injury are presented. (D) Endothelial cells were transfected with scrambled miR mimic control (Ctrl) or miR-423-5p mimics (10 nM), and capillary-like structures were quantified after 6 hours on Matrigel. Angiogenic activity was assessed by quantifying the number of segments per field ± SEM; n = 4 for each condition. Representative images of tubule formation are presented in the right panel. P values were obtained by 1-way ANOVA and the Bonferroni post hoc test (*P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001).
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Figure 8. miR-423-5p protects endothelial cells from apoptosis and promotes endothelial migration and angiogenesis. (A) Immunoblot and densitometric analysis of cleaved PARP1 in endothelial cells transfected with scrambled miR mimic control (Ctrl) or miR-423-5p mimics (10 nM) and exposed to normal medium or serum starvation for 4 hours. <t>TUBA1B</t> was used as a loading control; n = 2–3. Scale bar: 200 µm (B) Quantification of caspase-3 activity in endothelial cells transfected with Ctrl or miR-423-5p mimics (10 nM) exposed to N or SS for 4 hours; n = 3. Scale bar: 100 µm (C) Endothelial cells transfected with scrambled miR mimic control (Ctrl) or miR-423-5p mimics (10 nM) were mechanically injured, and wound closure was followed over a 6-hour period. The wound healing assay results are expressed as the percentage of wound closure ± SEM; n = 4 for each condition. Representative pictures at 6 hours after injury are presented. (D) Endothelial cells were transfected with scrambled miR mimic control (Ctrl) or miR-423-5p mimics (10 nM), and capillary-like structures were quantified after 6 hours on Matrigel. Angiogenic activity was assessed by quantifying the number of segments per field ± SEM; n = 4 for each condition. Representative images of tubule formation are presented in the right panel. P values were obtained by 1-way ANOVA and the Bonferroni post hoc test (*P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001).
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Figure 8. miR-423-5p protects endothelial cells from apoptosis and promotes endothelial migration and angiogenesis. (A) Immunoblot and densitometric analysis of cleaved PARP1 in endothelial cells transfected with scrambled miR mimic control (Ctrl) or miR-423-5p mimics (10 nM) and exposed to normal medium or serum starvation for 4 hours. TUBA1B was used as a loading control; n = 2–3. Scale bar: 200 µm (B) Quantification of caspase-3 activity in endothelial cells transfected with Ctrl or miR-423-5p mimics (10 nM) exposed to N or SS for 4 hours; n = 3. Scale bar: 100 µm (C) Endothelial cells transfected with scrambled miR mimic control (Ctrl) or miR-423-5p mimics (10 nM) were mechanically injured, and wound closure was followed over a 6-hour period. The wound healing assay results are expressed as the percentage of wound closure ± SEM; n = 4 for each condition. Representative pictures at 6 hours after injury are presented. (D) Endothelial cells were transfected with scrambled miR mimic control (Ctrl) or miR-423-5p mimics (10 nM), and capillary-like structures were quantified after 6 hours on Matrigel. Angiogenic activity was assessed by quantifying the number of segments per field ± SEM; n = 4 for each condition. Representative images of tubule formation are presented in the right panel. P values were obtained by 1-way ANOVA and the Bonferroni post hoc test (*P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001).

Journal: JCI insight

Article Title: Endothelial extracellular vesicle miR-423-5p regulates microvascular homeostasis and renal function after ischemia-reperfusion injury.

doi: 10.1172/jci.insight.181937

Figure Lengend Snippet: Figure 8. miR-423-5p protects endothelial cells from apoptosis and promotes endothelial migration and angiogenesis. (A) Immunoblot and densitometric analysis of cleaved PARP1 in endothelial cells transfected with scrambled miR mimic control (Ctrl) or miR-423-5p mimics (10 nM) and exposed to normal medium or serum starvation for 4 hours. TUBA1B was used as a loading control; n = 2–3. Scale bar: 200 µm (B) Quantification of caspase-3 activity in endothelial cells transfected with Ctrl or miR-423-5p mimics (10 nM) exposed to N or SS for 4 hours; n = 3. Scale bar: 100 µm (C) Endothelial cells transfected with scrambled miR mimic control (Ctrl) or miR-423-5p mimics (10 nM) were mechanically injured, and wound closure was followed over a 6-hour period. The wound healing assay results are expressed as the percentage of wound closure ± SEM; n = 4 for each condition. Representative pictures at 6 hours after injury are presented. (D) Endothelial cells were transfected with scrambled miR mimic control (Ctrl) or miR-423-5p mimics (10 nM), and capillary-like structures were quantified after 6 hours on Matrigel. Angiogenic activity was assessed by quantifying the number of segments per field ± SEM; n = 4 for each condition. Representative images of tubule formation are presented in the right panel. P values were obtained by 1-way ANOVA and the Bonferroni post hoc test (*P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001).

Article Snippet: Antibodies against: PARP1 (9542; Cell Signaling Technology), TUBA1B (11224-1-AP; Proteintech), PLVAP (NB100-77668; Novus Biological), ACTB (a5441; Sigma-Aldrich), PECAM1 (AF3628-SP; R&D Systems), CD82 (ab66400; Abcam), CD81 (66866-1-IG; Thermo Fisher Scientific), SDCBP (SC-515538; Santa Cruz Biotechnology), PSMA3 (SC-67340, Santa Cruz Biotechnology; 11887-1-AP, Proteintech), HSPG2/LG3 (AF2364; R&D Systems or polyclonal antibody against recombinant LG3; MediMabs), and histone H3C1 (9715S; Cell Signaling Technology).

Techniques: Migration, Western Blot, Transfection, Control, Activity Assay, Wound Healing Assay