Additional file 1 of EphrinA5 regulates cell motility by modulating Snhg15/DNA triplex-dependent targeting of DNMT1 to the Ncam1 promoter
Creators: name: Yildiz, Can Bora
name: Kundu, Tathagata
name: Gehrmann, Julia
name: Koesling, Jannis
name: Ravaei, Amin
name: Wolff, Philip
name: Kraft, Florian
name: Maié, Tiago
name: Jakovcevski, Mira
name: Pensold, Daniel
name: Zimmermann, Olav
name: Rossetti, Giulia
name: Costa, Ivan G.
name: Zimmer-Bensch, Geraldine
Publisher: name: figshare
Year: 2024
Subjects: subject: Genetics
subject: FOS: Biological sciences

description: Additional file 1: Figure S1. Western blots confirming the specificity of antibodies against DNMT1 and EZH2. (a) Immunoprecipitations from N2a whole cell lysate with normal rabbit IgG (#12–370, Merck) and rabbit anti-DNMT1 (#70–201, BioAcademia) antibodies. Detection was performed with the mouse anti-DNMT1 antibody (1:250, # ab13537, abcam). (b) The EZH2 antibody (1:1000, #5246, Cell Signaling Technology) was used for detection on whole cell lysate from the murine medial ganglionic eminence (MGE). Protein sizes are indicated in kDa, identified through SERVA Protein standard III. Figure S2. CLIP revealed no interaction of Snhg15 and EZH2 in CB cells. RNA recovery for IgG and anti-EZH2 antibody CLIP samples in CB cells (N = 5 biological replicates). For all investigated amplicons, the recovery for EZH2 could not be statistically differentiated from the IgG-based pulldown. Whiskers of the box plots extend 1.5 times the interquartile range from the 25th and 75th percentiles (Tukey style) while outliers are represented by hollow dots. Significances were determined with two-tailed Student’s t-test. ctrl-Fc: control Fc, efnA5-Fc: ephrinA5-Fc, CB: cerebellar granule, CLIP: UV cross-linking and immunoprecipitation. Figure S3. Expression of NCAM1/Ncam1 is implicated in low-grade glioma as well as the migration of CB cells, where it can be downregulated via RNA silencing. (a) High expression levels of NCAM1 are associated with increased patient survival in low-grade glioma. Survival analysis is based on clinical data and gene expression counts from tumor samples of lower grade glioma patients downloaded from BioPortal ( http://www.cbioportal.org/study/clinicalData?id=lgg_tcga ) and The Cancer Genome Atlas (TCGA), respectively. (b) Knockdown efficiency of the applied Ncam1 siRNA (N = 3 biological replicates). (c) Quantitative analysis of migration distance of CB cells (n = 557 for ctrl siR + ctrl-Fc, n = 455 for ctrl siR + efnA5-Fc, n = 481 for Ncam1 siR + ctrl-Fc, n = 495 for Ncam1 siR + efnA5-Fc, N = 4 biological replicates). Significances were determined with log-rank (a), Wilcoxon-Mann–Whitney test (b) and one-way ANOVA (c). Significance levels: p value &lt; 0.05 *; p value &lt; 0.01 **; p value &lt; 0.001 ***. ctrl: control. efnA5: ephrinA5. LGG: low-grade glioma. siR: siRNA. Figure S4. Migratory analysis of CB cells upon stimulation with ephrinA5-Fc and downregulation of EphA2. The motility of CB cells was reduced upon stimulation with ephrinA5-Fc. (a) Temporal color-coded migratory distance over 20 h of imaging. The starting point of migration for each cell is shown in dark blue and the end point in white. (b) Quantitative analysis of average migratory speed (n = 268 for ctrl siR + ctrl-Fc, n = 244 for ctrl siR + efnA5-Fc, n = 273 for EphA2 siR + ctrl-Fc, n = 238 for EphA2 siR + efnA5-Fc, N = 3 biological replicates). (c) Knockdown efficiency of the applied EphA2-siRNA (N = 3 biological replicates). Significances were determined with one-way ANOVA (b) and Wilcoxon-Mann–Whitney test (c). Significance levels: p value &lt; 0.05 *; p value &lt; 0.01 **; p value &lt; 0.001 ***. Scale bar: 100 μm. ctrl: control. efnA5: ephrinA5. siR: siRNA. Figure S5. The murine Ncam1 locus shows bivalent regulation in primary cerebellar tissue and CB cells. (a) The peaks depict publicly available ChIP-seq data for H3K27me3 and H3K4me3 in the murine Ncam1 locus in cerebellar tissue from various developmental stages. The corresponding GEO accession numbers for the datasets from top to bottom are as follows: GSM1000143, GSE29184, GSM1000090, GSM769027. Genomic regions targeted by the primers are shown in turquoise. The promoter is shown in dark blue, candidate cis-regulatory elements in red, CpG islands in green, and putative Snhg15-binding sites in pink. (b-d) Native ChIP using anti-DNMT1 and anti-H3K27me3 with qPCR analysis targeting the Ncam1 promoter region normalized against the input material and IgG (N =