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. 2021 Oct 9;10(10):2697. doi: 10.3390/cells10102697

Figure 4.

Figure 4

miR-1273g-3p impairs mitochondrial function in H4-APPswe and SH-SY5Y cells. (A–C) Representative images of MitoTracker Red CMX Ros (MitoTracker Red) staining for H4-APPswe cells transfected with miR-1273g-3p mimic or negative control. Magnified views of the boxed area are presented in the second row. Scale bar: first row, 20 μm; second row, 10 μm (A). Intensity of MitoTracker Red is shown in (B) (n = 3, 26-65 cells were analyzed per experiment) and analysis of mitochondrial morphological changes is shown in (C) (n = 3, 43-135 cells per experiment). (D–G) Relative concentration of H2O2 (D) (n = 3), oxygen consumption rate (OCR) analysis (E) (n = 4), quantification of Bcl-2, Bax, and Bak mRNAs by qPCR (F) (n = 4), and percentage of cell viability assessed by WST-1 (G) (n = 3) in H4-APPswe cells transfected with miR-1273g-3p mimic or negative control. (H–J) Representative images of MitoTracker Red staining for SH-SY5Y cells transfected with miR-1273g-3p mimic or negative control. Magnified views of the boxed area are presented in the second row. Scale bar: first row, 20 μm; second row, 10 μm (H). Intensity of MitoTracker Red is shown in (I) (n = 3, 42-137 cells per experiment) and analysis of mitochondrial morphological changes is shown in (J) (n = 3, 57-131 cells per experiment). (K–M) Measurements of OCR (K) (n = 4), neurite outgrowth analysis by immunostaining of MAP2 (L) (n = 3; Scale bar: 100 μm), and Western blotting for BACE1 (M) (n = 3) in SH-SY5Y cells transfected with miR-1273g-3p mimic or negative control. Mitochondrial morphology was defined as fragmented, mainly round shape; tubulated, mainly long shape; and intermediate, mixture of round and tubulated shape. Real-time OCR data are presented as a percentage relative to the first negative control measurement; bar graph indicates the percentage of OCR relative to the basal value of the negative control. All data are presented as mean ± SEM. * p < 0.05; ** p < 0.01; *** p < 0.001; n.s., non-significant (Student’s t-test). NC, mimic negative control; 1273, miR-1273g-3p mimic.