Journal: Oxidative Medicine and Cellular Longevity

Article Title: MicroRNA-93 Regulates Hypoxia-Induced Autophagy by Targeting ULK1

doi: 10.1155/2017/2709053

Figure Lengend Snippet: miR-93 targets ULK1 and regulates its expression. (a) Predicted binding sites of mmu-miR-93 to the 3′ UTR of ULK1. The short vertical lines indicate complementary paired bases of miR-93 and ULK1. The 302–309 nucleotides of ULK1 3′ UTR present the miRNA regulatory element (MRE). The underlined bases were mutant MRE. (b) Luciferase reporter assay by the interaction between miR-93 and the predicted MRE in MEFs. Each luciferase construct was cotransfected with scramble NC (NC), scramble IN-NC (IN-NC), miR-93 mimics (93), or miR-93 inhibitor (IN-93). At approximately 24 h after transfection, the luciferase activity was detected. The firefly luciferase activity was normalized to Renilla . Data were shown as the mean ± S.D. from three independent experiments. ∗∗ P < 0.01. (c) miR-93 represses the endogenous ULK1 expression in MEFs. NC, 93, IN-NC, and IN-93 were transfected into MEFs. At 24 h after transfection, qPCR was performed to detect the expression of miR-93 and ULK1. Data were from three independent experiments. ∗ P < 0.05; ∗∗ P < 0.01; ∗∗∗ P < 0.001. (d) miR-93 represses the endogenous ULK1 expression in CHO cells. NC, 93, IN-NC, and IN-93 were transfected into CHO cells. At 24 h after transfection, qPCR was performed to detect the expression of miR-93 and ULK1. Data were from three independent experiments. ∗∗ P < 0.01; ∗∗∗ P < 0.001. (e) Cell lysates in (c) were prepared and subjected to Western blot analysis by using anti-ULK1 and anti- β -actin antibody. The densitometric ratios of ULK1/actin from the samples were quantified by using ImageJ. Data were from three independent experiments. Representative data are shown. (f) Cell lysates in (d) were prepared and subjected to Western blot analysis by using anti-ULK1 and anti- β -actin antibody. The densitometric ratios of ULK1/actin from the samples were quantified by using ImageJ. Data were from three independent experiments. Representative data are shown. (g, h) MEFs and CHO cells were transfected with miR-93 (93) and miR-93 inhibitor (IN-93) for 24 h, 48 h, and 72 h, individually, setting group NC as a control. Cell lysates were prepared and subjected to Western blot analysis by using anti-ULK1, anti-ATG5, anti-ATG7, anti-Beclin1, anti-Tom20, and anti-Gapdh. Data were from three independent experiments. Representative data are shown.

Article Snippet: The following antibodies were used: anti-ULK1 (Sigma-Aldrich, A7481), anti-P62 (MBL, PM045), anti-P62 (Abcam, ab56416), anti-LC3B (Sigma, L7543), anti-c-Myc monoclonal antibody (Sigma, C3956), anti- β -actin (Sino Biological, 100162-RP02-100), anti-Gapdh (Transgen, HC301), anti-ULK1 (Sigma, A7481), anti-ATG5 (Sigma, A0731), anti-ATG7 (Sigma, A2856), anti-Beclin1 (CST, 3738), anti-Tom20 (BD Biosciences, 612278), DAPI (CST, 4083), HRP Affinipure goat anti-mouse IgG (Earthox, E030110), and HRP Affinipure goat anti-rabbit IgG (Earthox, E030120).

Techniques: Expressing, Binding Assay, Mutagenesis, Luciferase, Reporter Assay, Construct, Transfection, Activity Assay, Western Blot, Control

Journal: Oxidative Medicine and Cellular Longevity

Article Title: MicroRNA-93 Regulates Hypoxia-Induced Autophagy by Targeting ULK1

doi: 10.1155/2017/2709053

Figure Lengend Snippet: miR-93 inhibits hypoxia-induced autophagy. (a) The expression of ULK1 is induced by hypoxia. MEFs are subjected to hypoxia conditions for 0 h, 6 h, 12 h, and 24 h. Endogenous ULK1, P62, LC3, and β -actin were blotted by corresponding antibodies. Densitometric ratios of LC3-II/I from the samples were quantified by using ImageJ. (b) Expression levels of endogenous miR-93 and ULK1 in MEFs under hypoxia condition for 0 h, 6 h, 12 h, or 24 h by qPCR. (c) MEFs were transfected with scramble NC (NC), miR-93 mimics (93), scramble IN-NC (IN-NC), or miR-93 inhibitor (IN-93) for 12 h; afterward, MEFs were sealed in a hypoxic or normoxic condition for another 24 h with or without Bafilomycin A1 (BAF1). Samples were blotted with anti-ULK1, anti-P62, anti-LC3, and anti- β -actin antibodies. (d) MEFs were treated as the same as (c). Cells were fixed and immunostained with anti-ULK1 (green) and anti-P62 (red) antibodies. Scale bar, 20 μ m.

Article Snippet: The following antibodies were used: anti-ULK1 (Sigma-Aldrich, A7481), anti-P62 (MBL, PM045), anti-P62 (Abcam, ab56416), anti-LC3B (Sigma, L7543), anti-c-Myc monoclonal antibody (Sigma, C3956), anti- β -actin (Sino Biological, 100162-RP02-100), anti-Gapdh (Transgen, HC301), anti-ULK1 (Sigma, A7481), anti-ATG5 (Sigma, A0731), anti-ATG7 (Sigma, A2856), anti-Beclin1 (CST, 3738), anti-Tom20 (BD Biosciences, 612278), DAPI (CST, 4083), HRP Affinipure goat anti-mouse IgG (Earthox, E030110), and HRP Affinipure goat anti-rabbit IgG (Earthox, E030120).

Techniques: Expressing, Transfection