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stem1603-sup-0001-suppfig01.tif4092KSupplemental Figure S1 miR-26b accelerates human adipocyte differentiation. (A-C) hMADS-3 cells were transfected with miR-26b or control (miR-C) and subsequently adipocyte differentiation was induced. (A) 48 h after transfection, RNA was prepared and subjected to qPCR. miR-26b abundance was normalized to 5S rRNA and is presented relative to control transfected cells. ***P<0.001 (n=3) (B-C) At early (ew) and late (lw) stages of adipocyte differentiation, cells were analyzed by (B) Oil Red O staining (representative pictures, image magnification: 40x) and (C) quantification of triglyceride accumulation. *P<0.05 (n=5, paired t-test).
stem1603-sup-0002-suppfig02.tif3185KSupplemental Figure S2 Global effects of miR-26a during human adipocyte differentiation. hMADS-2 and hMADS-3 cells (2 biological replicates each) were transfected with miR-26a or miR-C and subsequently adipocyte differentiation was induced. RNA from cells at day 9 of differentiation was used to assess global changes in mRNA expression. (A) Cumulative distributions of mRNA fold changes between miR-26a- and miR-C-transfected cells. Significant difference between the distribution of all mRNAs (black) and the subset of mRNAs harboring at least 1 miR-26 7mer seed match (red) was assessed by a one-sided Kolmogorov-Smirnov test. (B) Heat map of transcripts most affected by miR-26a. Total data was filtered for mRNAs that on average were at least 2-fold up- or downregulated by miR-26a. Transcripts are specified by NCBI RefSeq ID and official symbol. Grey fields: missing values due to raw data post-processing. BR1-4: four individual biological replicates.
stem1603-sup-0003-suppfig03.tif3572KSupporting Information Figure 3. Supplemental Figure S3 miR-26b promotes brown adipocyte characteristics during human adipocyte differentiation. (A-C) hMADS-3 cells were transfected with miR-26b or control (miR-C) and subsequently adipocyte differentiation was induced. At the late brite (lb) stage of adipocyte differentiation, cells were analyzed by (A) Oil Red O staining (representative pictures, image magnification: 40x) and (B) quantification of triglyceride accumulation. (C) Analysis of UCP1 and ß-tubulin (TUB ß) protein levels at early white (ew), late white (lw), and lb stages of adipocyte differentiation were analyzed by Western blot.
stem1603-sup-0004-suppfig04.tiff6849KSupplemental Figure S4 Combined transcriptomic and in silico approaches to identify potential direct miR-26 targets. (A) hMADS-2 and hMADS-3 cells (2 biological replicates each) were transfected at confluence with miR-26a or control (miR-C). Two days later, total RNA was isolated to assess global changes in mRNA expression using microarrays. The graph depicts cumulative distributions of mRNA fold changes between miR-26a- and miR-C-transfected cells. Significant difference between the distribution of all mRNAs (black) and the subset of mRNAs harboring at least 1 miR-26 7mer seed match (red) was assessed by a one-sided Kolmogorov-Smirnov test. (B) Different types of seed/ seed match pairing for miR-26a/b. Upper part depicts sequences of miR-26a and -26b; differences are marked by (*). Lower part depicts sequences of canonical and marginal seed matches. Bold red letters denote the seed region of miRNAs and corresponding seed match sequences of potential direct target mRNAs. (C) Heat map of the 80 transcripts most downregulated by miR-26a (z score ≤ -2.17), including prediction of direct miR-26 targeting. Columns 1 to 5 depict the color-coded expression levels for the individual biological replicates (BR1-4) and the mean expression level. Columns 6-10 summarize two distinct approaches for identification of predicted direct miR-26 targets: (i) the fraction of miRNA-target prediction algorithms (microrna.org, microCosm, PITA, PicTar 4-way, PicTar 5-way, TargetScan-conserved, TargetScan-nonconserved, DIANA microT, ElMMo, rna22) predicting a direct interaction (mtpt / 10), and (ii) the presence of different miRNA seed matches (as shown in (B)) within the 3'UTR of the respective transcript. For intuitive visualization, the number of identified miRNA target sites was divided by 4. Depicted transcripts are specified by NCBI RefSeq ID and official symbol. Grey fields indicate missing values due to raw data post-processing (columns 1-4), no prediction for direct miR-26 targeting (column 6), or lack of the respective seed match within the transcript 3'UTR (columns 7-10). Black arrows denote the transcripts which w subsequently selected for the RNAi screen.ere
stem1603-sup-0005-suppfig05.tif22KSupplemental Figure S5 Evolutionary conservation of miR-26 binding site in ADAM17 3'UTR. Genomic sequences for human ADAM17 as well as orthologous genes in mouse, rat, dog, and chicken were obtained from Ensembl via BioMart and multiple sequence alignment was performed with muscle (www.ebi.ac.uk/Tools/msa/muscle). Bold underlined letters highlight the conserved miR-26 seed match.
stem1603-sup-0006-supptab01.pdf8KSupplemental Table S1: Oligonucleotides used for transfection
stem1603-sup-0007-supptab02.pdf45KSupplemental Table S2: RT-qPCR primer sequences
stem1603-sup-0008-supptab03.pdf7KSupplemental Table S3: Primer sequences for generation of Luciferase reporter vectors containing wt or mutated ADAM17 3'UTR
stem1603-sup-0009-supptab04.pdf25KSupplemental Table S4. Gene Set Enrichment Analysis for transcripts upregulated by miR-26a at early white stage. Gene Set Enrichment Analysis (GSEA) was performed to identify gene sets which are significantly (false discovery rate (FDR) q-value < 0.1) overrepresented among mRNAs upregulated by miR-26a in the distinct KEGG pathways and GO classes. Adipogenesis-related gene sets are highlighted in bold and italic.
stem1603-sup-0010-supptab05.pdf12KSupplemental Table S5. Gene Set Enrichment Analysis for transcripts downregulated by miR-26a at early white stage. Gene Set Enrichment Analysis (GSEA) was performed to identify gene sets which are significantly (false discovery rate (FDR) q-value < 0.1) overrepresented among mRNAs downregulated by miR-26a in the distinct KEGG pathways and GO classes. Adipogenesis-related gene sets are highlighted in bold and italic.
stem1603-sup-0011-supptab06.pdf7KSupplemental Table S6. Data on Liposuction Material.
stem1603-sup-0012-suppinfo01.pdf100KSupplemental Information
stem1603-sup-0013-suppinfo02.pdf94KSupplemental Information

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