Fig 1: Disturbed shear stress reduces the expression of Aff3ir-ORF2 in vivo and in vitro.(A) RT-PCR analysis of the mRNA levels of Aff3ir-ORF2 and AF4/FMR2 family member 3 (Aff3) in the intima of thoracic aorta (TA) and aortic arch (AA) of C57BL/6 mice. Data are presented as mean ± SEM (n=6 mice per group). *p<0.05, unpaired two-tailed t-test. (B, C) Western blot analysis of the expression of the indicated proteins in the intima of TA and AA of C57BL/6 mice. Protein levels were normalized to those of Gapdh, and the relative expression values were compared to those of the TA group. Data are presented as mean ± SEM (n=6 mice per group). *p<0.05, unpaired two-tailed t-test. (D, E) En-face immunofluorescence staining of Aff3ir-ORF2, VE-cadherin, and DAPI, and quantification of Aff3ir-ORF2 expression in inner curvature of the AA (AA inner), outer curvature of the AA (AA outer), and TA of C57BL/6 mice. Scale bar, 20 μm. The immunofluorescence intensity of Aff3ir-ORF2 was normalized to that of DAPI, and the relative expression values were compared to that of the TA group. Data are presented as mean ± SEM (n=6 mice per group). *p<0.05, one-way ANOVA with Tukey post-test. (F) Representative immunofluorescent staining for von Willebrand factor (vWF), Aff3ir-ORF2, and Aff3 in longitudinal aortic sections of C57BL/6 mice. n=6 mice per group. Scale bar, 25 μm. Inner, inner curvature of the AA; outer, outer curvature of the AA; BIF, Bifurcation. (G, H) Mouse embryonic fibroblasts (MEFs) isolated from the embryo of C57BL/6 mice were subjected to static (ST) or oscillatory shear stress (OSS, 0.5±4 dyn/cm2, 1 Hz) for indicated time. Western blot analysis of the indicated proteins. Protein levels were normalized to Gapdh and the relative expression values were compared to that of the ST group. Data are mean ± SEM (n=6 independent experiments). *p<0.05, one-way ANOVA with Tukey post-test. (I) MEFs were subjected to ST or oscillatory shear stress (OSS) treatment for 6 hr. RT-PCR analysis of the mRNA levels of Aff3ir, Aff3, intercellular adhesion molecule 1 (Icam1), and vascular cell adhesion molecule 1 (Vcam1) in MEFs. Data are mean ± SEM (n=6 independent experiments). *p<0.05, unpaired two-tailed t-test. Figure 1—source data 1.The table summarizes the data in the statistical graph for Figure 1A, C, E, H and I. Figure 1—source data 2.The original file of the full raw uncropped, unedited polyacrylamide gels for Figure 1B. Figure 1—source data 3.Figures with the uncropped polyacrylamide gels with the relevant bands clearly labeled for Figure 1B. Figure 1—source data 4.The original file of the full raw uncropped, unedited polyacrylamide gels for Figure 1G. Figure 1—source data 5.Figures with the uncropped polyacrylamide gels with the relevant bands clearly labeled for Figure 1G.
Fig 2: Disturbed shear stress reduces the expression of Aff3ir-ORF2 in vivo and in vitro.(A) RT-PCR analysis of the mRNA levels of Aff3ir-ORF2 and AF4/FMR2 family member 3 (Aff3) in the intima of thoracic aorta (TA) and aortic arch (AA) of C57BL/6 mice. Data are presented as mean ± SEM (n=6 mice per group). *p<0.05, unpaired two-tailed t-test. (B, C) Western blot analysis of the expression of the indicated proteins in the intima of TA and AA of C57BL/6 mice. Protein levels were normalized to those of Gapdh, and the relative expression values were compared to those of the TA group. Data are presented as mean ± SEM (n=6 mice per group). *p<0.05, unpaired two-tailed t-test. (D, E) En-face immunofluorescence staining of Aff3ir-ORF2, VE-cadherin, and DAPI, and quantification of Aff3ir-ORF2 expression in inner curvature of the AA (AA inner), outer curvature of the AA (AA outer), and TA of C57BL/6 mice. Scale bar, 20 μm. The immunofluorescence intensity of Aff3ir-ORF2 was normalized to that of DAPI, and the relative expression values were compared to that of the TA group. Data are presented as mean ± SEM (n=6 mice per group). *p<0.05, one-way ANOVA with Tukey post-test. (F) Representative immunofluorescent staining for von Willebrand factor (vWF), Aff3ir-ORF2, and Aff3 in longitudinal aortic sections of C57BL/6 mice. n=6 mice per group. Scale bar, 25 μm. Inner, inner curvature of the AA; outer, outer curvature of the AA; BIF, Bifurcation. (G, H) Mouse embryonic fibroblasts (MEFs) isolated from the embryo of C57BL/6 mice were subjected to static (ST) or oscillatory shear stress (OSS, 0.5±4 dyn/cm2, 1 Hz) for indicated time. Western blot analysis of the indicated proteins. Protein levels were normalized to Gapdh and the relative expression values were compared to that of the ST group. Data are mean ± SEM (n=6 independent experiments). *p<0.05, one-way ANOVA with Tukey post-test. (I) MEFs were subjected to ST or oscillatory shear stress (OSS) treatment for 6 hr. RT-PCR analysis of the mRNA levels of Aff3ir, Aff3, intercellular adhesion molecule 1 (Icam1), and vascular cell adhesion molecule 1 (Vcam1) in MEFs. Data are mean ± SEM (n=6 independent experiments). *p<0.05, unpaired two-tailed t-test. Figure 1—source data 1.The table summarizes the data in the statistical graph for Figure 1A, C, E, H and I. Figure 1—source data 2.The original file of the full raw uncropped, unedited polyacrylamide gels for Figure 1B. Figure 1—source data 3.Figures with the uncropped polyacrylamide gels with the relevant bands clearly labeled for Figure 1B. Figure 1—source data 4.The original file of the full raw uncropped, unedited polyacrylamide gels for Figure 1G. Figure 1—source data 5.Figures with the uncropped polyacrylamide gels with the relevant bands clearly labeled for Figure 1G.
Fig 3: Aff3ir-ORF2 overexpression alleviates disturbed flow-induced inflammation in endothelial cells (ECs).(A) Mouse embryonic fibroblasts (MEFs) isolated from C57BL/6 mice were infected with indicated adenoviruses (Ad-Scramble or Ad-Aff3ir-ORF2) for 48 hr and then exposed to static (ST) or oscillatory shear stress (OSS, 0.5±4 dyn/cm2, 1 Hz) for another 6 hr. RT-PCR analysis of mRNA levels of Aff3, Vcam1, Icam1, interleukin-6 (Il6), and interleukin-1 beta (Il1b). The relative expression values were compared to MEFs infected with Ad-Scramble and treated with ST. Data are presented as mean ± SEM (n=6 independent experiments). *p<0.05, two-way ANOVA with Tukey post-test. (B) Human umbilical vein endothelial cells (HUVECs) were infected with Ad-Scramble or Ad-Aff3ir-ORF2 for 48 hr and then exposed to ST or OSS for another 6 hr. RT-PCR analysis of mRNA levels of Aff3, Vcam1, Icam1, Il6, and Il1b. The relative expression values were compared to HUVECs infected with Ad-Scramble and treated with ST. Data are presented as mean ± SEM (n=6 independent experiments). *p<0.05, two-way ANOVA with Tukey post-test. (C–D) Enface immunofluorescence staining of Aff3ir-ORF2, VE-cadherin, and DAPI and quantification of Aff3ir-ORF2 expression. Scale bar, 20 μm. The immunofluorescence intensity of Aff3ir-ORF2 was normalized to DAPI and the relative expression values were compared to that of the Ad-Scramble group. Data are presented as mean ± SEM (n=6 mice per group). *p<0.05, unpaired two-tailed t-test. (E), Eight-wk-old male Apoe−/− mice were subjected to partial ligation of the carotid artery along with 10 μL of adenovirus suspension at 1×108 transducing units (TU)/mL was instilled into the left carotid arteries. The mice were then fed high-fat diet for 4 wk. Right carotid arteries were sectioned for hematoxylin and eosin staining. Figure 2—figure supplement 1—source data 1.The table summarizes the data in the statistical graph for Figure 2—figure supplement 1A, B and D.
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