Additional file 1 of Loss of miR-101-3p in melanoma stabilizes genomic integrity, leading to cell death prevention
Creators: name: Lämmerhirt, Lisa
name: Kappelmann-Fenzl, Melanie
name: Fischer, Stefan
name: Meier, Paula
name: Staebler, Sebastian
name: Kuphal, Silke
name: Bosserhoff, Anja-Katrin
Publisher: name: figshare
Year: 2024
Subjects: subject: Medicine
subject: Cell Biology
subject: Genetics
subject: FOS: Biological sciences
subject: Molecular Biology
subject: Pharmacology
subject: Environmental Sciences not elsewhere classified
subject: Biological Sciences not elsewhere classified
subject: Developmental Biology
subject: Cancer
subject: Hematology

description: Additional file 1. Figure S1A. Quantitative real-time PCR analysis of miRNA expression of miR-101-3p in MELJUSO,SK-MEL-28 and MV3 after miR-101-3p mimic transfection compared to siCtrl (72 h) (Student’s t-test). Figure S1A. Quantitative real-time PCR analysis of miRNA expression of miR-101-3p in MELJUSO, SK-MEL-28 and MV3 after miR-101-3p mimic transfection compared to siCtrl (72 h) (Student’s t-test). Figure S1B. Proliferation Curves of real time cell analysis in dependency of impedance measurement of MEL-JUSO, SKMEL-28 and MV3 treated with miR-101-3p Mimic (72 h) and respective siCtrl (Student’s t-test). Figure S1C. Total protein lysate after RIPA of MEL-JUSO, SK-MEL-28 and MV3 transfected with miR-101-3p mimic and siCtrl (72 h) (Student’s t-test). Bars represent the means ± SEM (* = p ≤ 0.05, ns = not significant). Figure S2A. Clonogenic assay with NHEM treated with miR-101-3p mimic (18 h) and respective siCtrl (n=1). Representative images of NHEM stained with crystal violet, treated with miR-101-3p mimic and siCtrl. Scale bars equal 100 μm. Figure S2B. Proliferation Curves of real time cell analysis in dependency of impedance measurement of NHEM treated with miR-101-3p Mimic (18 h) and respective siCtrl (n=1,) (Student’s t-test). Figures S2C. Quantitative real-time PCR analysis of miRNA expression of miR-101-3p in NHEM after miR-101-3p mimic transfection compared to siCtrl (18 h, n=1). Figures S2D. Quantitative real-time PCR analysis of RNA expression of EZH2 and LMNB1 in NHEM after miR-101-3p mimic transfection compared to siCtrl (18 h, n=1). Figure S3. Volcano Plot of differentially expressed genes in miR-101-3p mimic transfected cells compared to siCtrl-transfected cells analyzed by RNA-Seq. Significantly (p-value &lt; 0.1) and strongly (log2 FoldChange &gt; 1.5 or &lt; −1.5 respectively) upregulated genes are marked in red and downregulated genes in blue. Figure S4A. EnrichR analysis of significant regulated genes after miR-101-3p transfection by using different bioinformatical databases: KEGG, WikiPathway, GO Biological Processes (genes with padj &lt; 0.1). Figure S4B. Distribution of log(norm counts) of the expressed genes in miR-101-3p mimic and siCtrl, determining that miR-101-3p target genes show lower expression overall in mimic transfected cells compared to siCtrl. Figure S4C. Enriched hallmark gene sets which cluster in 4 clusters based on the overrepresentation analysis of significantly downregulated miR-101-3p target genes via EnrichR against expressed genes of the RNA-Seq. Figure S5A,B,C. qRT-PCR analysis of potential target genes ATRX, CASP3 and PARP determined by EnrichR analysis using clustergram (Overlap of 40 enriched miR101-3p target genes within the top 20 overrepresented gene sets) (Student’s t-test). Bars represent the means ± SEM (* = p ≤ 0.05, ns = not significant). Figure S6A. Flow cytometry with the fluorescent dye propidium iodide for cell cycle staining of G1, S and G2 phase. MEL-JUSO and SK-MEL-28 transfected with miR-101-3p mimic (72 h) and siCtrl respectively (two-way ANOVA and subsequent Fisher’s LSD multiple comparison test). Figure S6B,C,D. Western blot analysis of full-length PARP and cleaved PARP following treatment 72 h with miR-101-3p mimic and siCtrl in MELJUSO, SK-MEL-28 and MV3 using β-actinashousekeeper. Representative images of PARP Western blots in MELJUSO, SK-MEL-28 and MV3 (Student’s t-test). Figure S6E. Representative Comet-Assay images from fluorescence microscopy at 10x magnification showing the fragmented DNA migration from the nucleoid body which forms a comet tail in MV3 transfected with miR-101-3p mimic (72h) and siCtrl. The cytostatic etoposide was used as positive control respectively. Bars represent the means ± SEM (* = p ≤ 0.05, ns = not significant).