Additional file 1 of Systematic characterization of seed overlap microRNA cotargeting associated with lupus pathogenesis
Creators: name: Kitai, Hiroki
name: Kato, Noritoshi
name: Ogami, Koichi
name: Komatsu, Shintaro
name: Watanabe, Yu
name: Yoshino, Seiko
name: Koshi, Eri
name: Tsubota, Shoma
name: Funahashi, Yoshio
name: Maeda, Takahiro
name: Furuhashi, Kazuhiro
name: Ishimoto, Takuji
name: Kosugi, Tomoki
name: Maruyama, Shoichi
name: Kadomatsu, Kenji
name: Suzuki, Hiroshi I.
Publisher: name: figshare
Year: 2022
Subjects: subject: Biophysics
subject: Biochemistry
subject: Genetics
subject: FOS: Biological sciences
subject: Molecular Biology
subject: 59999 Environmental Sciences not elsewhere classified
subject: FOS: Earth and related environmental sciences
subject: Ecology
subject: Immunology
subject: FOS: Clinical medicine
subject: 69999 Biological Sciences not elsewhere classified
subject: Hematology

description: Additional file 1: Fig. S1. Additional results of qRT-PCR analysis. Additional validation of downregulated miRNAs by qRT-PCR. Data are means ± SD (N = 8-12). Fig. S2. Characterization of the human pDC-like cell line CAL-1. (A-C) FACS analysis of the cell-surface markers CD11c (A), CD304 (B), and CD123 (C) in CAL-1 cells. Isotype controls are shown in blue. Fig. S3. Overexpression of miR-128 and miR-148a in CAL-1 and HeLa cells. (A) Overexpression of miR-128-3p and miR-148a-3p in CAL-1 cells. Transfection with miR-control, miR-128-3p and miR-control, miR-148a-3p and miR-control, or miR-128-3p and miR-148a-3p, followed by qRT-PCR analysis (N = 3 per group, ***P &lt; 0.001, ****P &lt; 0.0001, one-way ANOVA and post hoc Tukey test). (B) Overexpression of miR-128-3p and miR-148a-3p in HeLa cells. HeLa cells were transfected with pri-miRNA empty or overexpression plasmids. At 48 hours after transfection, qRT-PCR analyses were performed (N = 3 per group, **P &lt; 0.01, ***P &lt; 0.001, one-way ANOVA and post hoc Tukey test). Fig. S4. Transcriptomic effects of miR-128 and miR-148a overexpression in HeLa cells. (A) Principal component analysis (left) and K-means clustering analysis (right) of the 1,000 most variable genes in RNA-seq datasets. The bottom panel shows enrichment of miR-128-3p and mIR-148a-3p target genes in each DEG group, classified by K-means clustering analysis. (B, C) Cumulative distributions of fold changes (FCs) of genes with single 8mer, 7mer-m8, 7mer-A1, and 6mer sites of miR-128-3p (B) and miR-148a-3p (C) upon single or double miRNA transfection. P values for downregulation (vs. genes with no sites) were calculated by one-tailed Wilcoxon rank sum test. Fig. S5. Deep conservation of KLF4 seed overlap cotarget sites between human and Coelacanth. TargetScan sequence alignments around the seed overlap cotarget site within KLF4 3′ UTRs are shown. Fig. S6. Conservation trends of miRNA target sites based on TargetScan and MirGeneDB classification. (A) Density and contour plots showing the distribution of BLS values and number of species in which the sites are conserved. Results for target sites of major miRNA groups in TargetScan (top) and MirGeneDB (bottom) classification are shown. (B) Conservation patterns of non-conserved sites, conserved sites, two classes of conserved sites for major miRNA groups in TargetScan (top) and MirGeneDB (bottom) classification across 84 vertebrate species (according to the species number threshold (n = 62)). Fig. S7. Additional seed overlap analysis of miRNA genes. (A) Summary showing relative occurrence (dashed lines) and statistics (-log10(P value), solid lines) of the maximum overlap for each seed type (8mer, 7mer-m8, 7mer-A1, and 6mer) among all pairs of (1) broadly conserved miRNAs, (2) broadly conserved and conserved miRNAs, and (3) broadly conserved, conserved, and poorly conserved miRNAs. Note that the results were similar when different miRNA groups were combined. (B) Frequency of extensive seed overlap in real data (vertical lines) and GC content-matched random seed sequences. Results of 1,000 randomizations are shown as density distributions. Fig. S8. Analysis of evolutionary trends in “seed overlap” miRNA cotargets and probability of haplo-insufficient genes. (A) Summary of differences and statistics of the number of species in which the sites are conserved. Target sites with “conserved overlap” and other target sites were compared. The results for the 50 miRNA pairs shown in Figure 7 are shown by site type. P values were calculated by one-tailed Wilcoxon rank sum test for either direction. (B) Summary of differences and statistics of the probability of haplo-insufficient genes between target genes with “conserved overlap” or other target sites. Results for 50 miRNA pairs shown in Figure 7 are shown. P values were calculated by one-tailed Wilcoxon rank sum test for either direction. Fig. S9. Additional seed overlap analysis of miRNA genes. Density and contour plots showing the distribution of BLS values and number of species in which the sites are conserved. Results for sites with “seed overlap” cotargeting (left), “neighborhood” cotargeting for the same miRNA (middle), and “neighborhood” cotargeting for all broadly conserved miRNAs (right) are shown. Vertical and horizontal dashed lines indicate BLC cutoffs (1.8 for 8mer, 2.8 for 7mer-m8, and 3.6 for 7mer-A1) and the species number threshold (n = 62). The trends do not markedly differ between groups. Fig. S10. Images of the full-size original blots. Uncropped images for Fig. 2 are shown.