Journal: Frontiers in Cellular Neuroscience

Article Title: MiR-29a-deficiency causes thickening of the basilar membrane and age-related hearing loss by upregulating collagen IV and laminin

doi: 10.3389/fncel.2023.1191740

Figure Lengend Snippet: MiR-29a regulation of target gene expression in cell lines. (A–G) The Renilla/firefly luciferase activity measured in HEK293T cells of the wild-type (WT) psiCHECK2-Col4a1:3′-UTR binding site mouse and mutant psiCHECK2-Col4a1:3′-UTR binding site mouse cotransfected without (control) or with mmu-miR-29a mimics (A) . (B) WT or mutant psiCHECK2-Col4a2:3′-UTR binding site mouse and without or with mmu-miR-29a mimics. (C) WT or mutant psiCHECK2-Col4a3:3′-UTR binding site mouse and without or with mmu-miR-29a mimics. (D) WT or mutant psiCHECK2-Col4a4:3′-UTR binding site mouse and without or with mmu-miR-29a mimics. (E) WT or mutant psiCHECK2-Col4a5:3′-UTR binding site mouse and without or with mmu-miR-29a mimics. (F) WT or mutant psiCHECK2-Lamb2:3′-UTR binding site mouse and without or with mmu-miR-29a mimics. (G) WT or mutant psiCHECK2-Lamc1:3′-UTR binding site mouse and without or with mmu-miR-29a mimics. (H) Relative mRNA expression levels in HEI-OC1 cells transfected with mmu-miR-29a inhibitors. All data are shown as the mean ± SD, n = 4 biological replicates, statistical analysis was performed by paired t -test (A–G) and independent t -test (H) , where P < 0.05 was considered significant (* P < 0.05, ** P < 0.01, and *** P < 0.001).

Article Snippet: The primary antibodies used in this study were COL4A1 (IF: 1:10, Novus, NBP1-26549) and LAMB2 (IF:1:40, Santa Cruz, sc-377379).

Techniques: Targeted Gene Expression, Luciferase, Activity Assay, Binding Assay, Mutagenesis, Control, Expressing, Transfection

Journal: Frontiers in Cellular Neuroscience

Article Title: MiR-29a-deficiency causes thickening of the basilar membrane and age-related hearing loss by upregulating collagen IV and laminin

doi: 10.3389/fncel.2023.1191740

Figure Lengend Snippet: MiR-29a regulation of target gene expression in cochlea from miR-29a +/+ and miR-29a –/– mice. (A–G) With β-actin as a control, COL4A1, COL4A2, COL4A3, COL4A4, COL4A5, LAMB2, and LAMC1 protein levels in cochlea were analyzed by Western blot. The independent t -test was used to compare data between two groups [COL4A1, t (4) = 9.920, P < 0.001; COL4A2, t (4) = 3.847, P = 0.018; COL4A3, t (4) = 3.389, P = 0.028; COL4A4, t (4) = 2.842, P = 0.047; COL4A5, t (4) = 3.782, P = 0.019; LAMB2, t (4) = 3.915, P = 0.017; LAMC1, t (4) = 3.088, P = 0.037]. (H) The mRNA expression levels of target genes as measured by qRT-PCR [independent t -test, Col4a1, t (4) = 6.050, P = 0.004; Col4a2, t (4) = 13.145, P < 0.001; Col4a3, t (4) = 3.735, P = 0.02; Col4a4, t (4) = 7.238, P = 0.002; Col4a5, t (4) = 13.141, P < 0.001; Lamb2, t (4) = 8.529, P = 0.001; Lamc1, t (4) = 3.126, P = 0.035]. All data are shown as the mean ± SD, n = 3 biological replicates, * P < 0.05, ** P < 0.01, and *** P < 0.001 between two groups.

Article Snippet: The primary antibodies used in this study were COL4A1 (IF: 1:10, Novus, NBP1-26549) and LAMB2 (IF:1:40, Santa Cruz, sc-377379).

Techniques: Targeted Gene Expression, Control, Western Blot, Expressing, Quantitative RT-PCR

Journal: Frontiers in Cellular Neuroscience

Article Title: MiR-29a-deficiency causes thickening of the basilar membrane and age-related hearing loss by upregulating collagen IV and laminin

doi: 10.3389/fncel.2023.1191740

Figure Lengend Snippet: Specific expression of COL4A1 and LAMB2 in the basilar membrane (BM) area. (A) Double immunostaining for COL4A1 (red) and LAMB2 (green) in cochlear frozen sections from miR-29a +/+ and miR-29a –/– mice at 4 months. Nuclei were visualized by DAPI (blue). Scale bar = 100 μm for global images, scale bar = 50 μm for higher magnification. (B,C) Quantification of COLA1 and LAMB2 immunofluorescence in the BM area [independent t -test, COL4A1, t (4) = 5.867, P = 0.004; Lamb2, t (4) = 4.143, P = 0.014]. Data are shown as the mean ± SD, n = 3 biological replicates, * P < 0.05 and ** P < 0.01 between two groups.

Article Snippet: The primary antibodies used in this study were COL4A1 (IF: 1:10, Novus, NBP1-26549) and LAMB2 (IF:1:40, Santa Cruz, sc-377379).

Techniques: Expressing, Membrane, Double Immunostaining, Immunofluorescence