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. 2021 Mar 17;29(7):2281–2293. doi: 10.1016/j.ymthe.2021.03.014

Figure 3.

Figure 3

Anti-miR-33a/b ASO treatment increased miR-33 target gene expression levels and ABCA1 protein localization in NHP RPE cell layer

(A) Expression levels of ABCA1, PRKAA1, CPT1A, CROT, SIRT6, and SIK1 were measured by quantitative RT-PCR in RPE cells isolated from NHPs injected with anti-miR-33 ASO or vehicle for 6 weeks (n ≤ 4). mRNA expression levels were normalized to PPIH or HPRT1. (B) Protein blot showing the ABCA1 protein expression in RPE/choroid lysate extracted from vehicle or anti-miR-33 injected NHP (n = 3) and the bar graph showing the relative fold change. (C) Retinal cryosections prepared from NHPs that were treated with vehicle or anti-miR-33 ASO by subcutaneous injections were immunostained for ABCA1 and DAPI nuclear stain (n = 3). Scale bar: 10 μm. (D) Free and esterified cholesterol levels were measured in the lipid extracts from RPE/choroid of NHPs that were injected with anti-miR-33 ASO or vehicle for 6 weeks (n = 3). (E) NHP retinal sections showing four highlighted regions (R1–4) from the fovea to the periphery were chosen to quantify filipin III staining in the RPE cell layer of vehicle- or anti-miR-33 ASO-treated NHP retinal sections. Arrow points to fovea; scale bar: 1 mm. (F) Retinal sections of NHPs that were injected with anti-miR-33a/b ASO or vehicle for 6 weeks were stained with filipin III to label unesterified cholesterol. Four regions (R1–4) from the fovea to the periphery shown in (E) chosen to quantify filipin III staining in the RPE cell layer of vehicle- or anti-miR-33 ASO-treated NHP retinal sections (n = 9). Scale bar: 50 μm. (G) Retinal sections of NHPs that were injected with anti-miR-33 ASO or vehicle for 6 weeks were pretreated with cholesterol esterase and then stained with filipin III to label esterified cholesterol. Four regions (R1–4) from the fovea to the periphery shown in (E) chosen to quantify filipin III staining in the RPE cell layer of vehicle- or anti-miR-33 ASO-treated NHP retinal sections (n = 4). Scale bar: 50 μm. All error bars represent ± SEM. Statistical differences between vehicle- and anti-miR-33 ASO-injected NHPs were calculated by unpaired t test. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001.