Extended Data Fig. 3: TDP-43 modulation of miRNA target gene expression.
From: TDP-43 aggregation induced by oxidative stress causes global mitochondrial imbalance in ALS
a, Western blotting analysis of IPed Ago2 under mock-treated (left) and TDP-43 overexpression (right) conditions. GAPDH served as loading control. b, RT-qPCR analysis of the expression of targets for TDP-43 bound miRNAs (red) and for non-bound miRNA (gray) in response to TDP-43 depletion. c, Dual luciferase reporter assay on individual target reporters. In each, the MRE sequence is shown with the seed region base-paired with the miRNA. Three copies of each MRE was inserted in each reporter. Corresponding to each wt reporter is the mutant one in which the seed region was mutated. Each pair of reporters was assayed in response to specific miRNA mimics or inhibitors. d, and e, Cumulative distribution plots of siTDP-43 induced expression changes among up-regulated genes with or without predicted target sites for TDP-43 sequestrated miRNAs (d), and of siTDP-43 induced expression changes among down-regulated genes with or without predicted target sites for TDP-43 non-bound miRNAs (e). Different colored lines indicate non-targets (gray), all predicted targets (blue), genes that contain more than one miRNA target (orange), and genes that contain more than two predicted miRNA target sites (red). The significance for each shift from non-target genes is indicated by P value determined by Wilcoxon signed-rank test. f, Boxplots of TDP-43 depletion-induced expression changes for genes with (n = 9,217) or without (n = 2,140) TDP-43 binding peaks. g, Boxplots of expression for genes with (n = 3,974) or without (n = 2,140) TDP-43 binding peaks in 3′UTRs. The lower and upper hinges of the boxplots correspond to the first and third quartiles (the 25th and 75th percentiles). The upper whisker extends from the hinge to the largest value no further than 1.5 * IQR from the hinge (where IQR is the interquartile range, or distance between the first and third quartiles). The lower whisker extends from the hinge to the smallest value at most 1.5 * IQR of the hinge. Data beyond the end of the whiskers are outlying points that are plotted individually. Data in b and c are presented as mean ± s.d. (n = 3 independent experiments). For b, c, f, and g, the P values are based on a two-tailed unpaired t test: *p < 0.05, **p < 0.01, ***p < 0.001; N.S., non-significant. For a, a representative example of three independent experiments is shown. Data for graphs in b and c are available as source data online. Uncropped images for panels a are available as source data online.
