Fig 1: EWS-WT1 loss leads to large-scale gene expression changes in DSRCT cells.a Hierarchical clustering of gene expression changes following silencing of EWS-WT1 in JN-DSRCT-1 cells (JNDSRCT1) or BER cells (BER) relative to a non-targeting control siRNA in the same cells (JN_control or BER_control). Data represent 1879 gene expression changes that are either increased (red) or decreased (blue) by a log FC of >2. b Volcano plots showing the magnitude of gene expression change as a function of q value (false discovery rate-adjusted P value) with silencing. Gene expression changes are both common (red) across the two cell lines and unique to the cell line (blue). More genes are repressed by EWS-WT1 than are induced. Libraries were prepared from 500 ng of total RNA using the KAPA-stranded mRNAseq Kit (v5.17). RNA was sheared to 300–400 bp. Prior to PCR amplification, cDNA fragments were ligated to Bio Scientific NEXTflex dual adapters. Quality and quantity were determined using a combination of Agilent DNA High Sensitivity chip, QuantiFluor® dsDNA System, and Kapa Illumina Library Quantification qPCR assays. Individually indexed libraries were pooled, and 75-bp, paired-end sequencing was performed on an Illumina NextSeq 500 sequencer using a 75-bp HO sequencing kit (v2). Base calling was done by Illumina NextSeq Control Software (NCS) v2.0 and demultiplexed to FastQ format with bcl2fastq v1.9.0 (Illumina Inc.). Reads were aligned to hg38 using STAR (v2.5.2b) with options –twopassMode Basic –quantMode GeneCounts. The data were filtered for a minimum of ten counts per million in at least one sample. Differential expression analysis was carried out using Deseq2 (v1.24.0) with apeglm (v1.6.0) applied to account for absolute magnitude of gene expression. Significant genes were determined using a cutoff of q value < 0.0511. Heatmaps were generated using pheatmap package (v1.0.12) in R (v3.6.1). Analysis was performed on three biological replicates of each sample. All samples were collected at the same time, and RNA was isolated on the QiaCube (Qiagen) in batches after sample randomization to avoid batch effect. The defined EWS-WT1 gene signature consists of genes with a log-fold change of 2 or greater in both cell lines following silencing. c Western blot of EWS-WT1 silencing confirms repression (ERG) and induction (CEBPD) of targets. Lysates collected at 16, 24, 30, and 48 h of exposure. Western blot probed with EWSR1 (11910, Cell Signaling) and H3 (2650, Cell Signaling), WT1 (sc-7385 Santa Cruz Biotechnology), ERG (ab92513, Abcam), and CEBPD (ab65081, Abcam) antibodies.
Fig 2: ABCA1 expression is correlated with CEBPD expression and promotes against TMZ-induced cell death in GSCs.A The relative ABCA1 expressions are higher in various grades of human glioma tissues. Expression of ABCA1 was analyzed in tumor tissues of glioma patients compared with normal tissues from the GEO database. B The mRNA expression of ABCA1 positively correlates to CEBPD in glioma samples. C The CEBPD-ABCA1 axis mediates TMZ resistance in GSCs. The knockdown control (siNegative) or CEBPD-knockdown (siCEBPD) U87MG cells were cultured in stem cell medium for 2 days to form spheroids, pretreated with ABCA1 antagonists (400 μM DIDS and 10 μM Probucol) for 24 h, and then treated with 50 μM TMZ for 72 h. The caspase 3/7 activity was detected by the Caspase-Glo® 3/7 Reagent. The summary data are presented as the mean ± SEM; the numbers in the bars represent the sample sizes; one-way ANOVA; *p < 0.05, **p < 0.01, ***p < 0.001, ns: no significant.
Fig 3: CEBPD promotes spheroid formation by regulating stemness-related factors and ABCA1 expression.A U87MG cells transiently expressing HA or HA-CEBPD were cultured in stem cell medium to form spheroids for 3 days and 6 days. B U87MG cells transiently expressing GFP or GFP-CEBPD were cultured in stem cell medium to form spheroids for 6 days. Total RNA were harvested and examined by RT-qPCR to detect the expression of SOX2, OCT4, NANOG, KLF4, and ABCA1. C Total protein lysates were harvested from (A) and examined by western blot to detect the expression of SOX2, OCT4, NANOG, KLF4, and ABCA1. D LacZ-knockdown or CEBPD-knockdown U87MG cells were cultured in stem cell medium to form spheroids for 3 days and 6 days. E Total RNA and F total protein lysates were harvested from shLacZ-U87MG or shCD-U87MG spheroid cells and examined by RT-qPCR and western blot to detect the transcription and expression of SOX2, OCT4, NANOG, KLF4, and ABCA1. The summary data are presented as the mean ± SEM; the numbers in the bars represent the sample sizes; one-way ANOVA and Student’s t-test; *p < 0.05, **p < 0.01, ***p < 0.001. HA: hemagglutinin, HA/CD: HA-tagged CEBPD, GFP: green fluorescent protein, GFP/CD: GFP-tagged CEBPD.
Fig 4: CEBPD can directly bind to the SOX2, OCT4, NANOG, and ABCA1 promoter regions to regulate gene expression.A Schematic representation of reporter constructs with the SOX2, OCT4, NANOG, and ABCA1 promoters. The approximate locations of the putative CEBPD-binding motifs are indicated by ovals. B U87MG cells were co-transfected with pcDNA3-HA or pcDNA3-HA/CEBPD and SOX2, OCT4, NANOG, and ABCA1 reporter vectors and then examined by reporter assay. C CEBPD binds to the SOX2, OCT4, NANOG, and ABCA1 promoters in vivo. Chromatins from U87MG monolayer or spheroid cells were isolated, and ChIP assays were performed with the indicated antibodies. The precipitated DNA was amplified by PCR using specific primers. The summary data are presented as the mean ± SEM; the numbers in the bars represent the sample sizes; Student’s t-test; **p < 0.01, ***p < 0.001.
Fig 5: Relative CEBPD expression is higher in GBM and is associated with a poor survival rate.A GEO database analyses showing higher CEBPD expression in GBM tissues than in normal brain tissues. B CEBPD expression is higher in various grades of human glioma tissues. C Analysis of Kaplan–Meier survival curves of high-risk GBM patients and low-risk GBM patients from TCGA. D Representative immunohistochemistry staining of CEBPD in GBM and normal brain tissues. The CEBPD H-score value is higher in GBM samples than in normal samples. Scale bar, 100 μm. The summary data are presented as the mean ± SEM; the numbers in the bars represent the sample sizes; one-way ANOVA and Student’s t-test; *p < 0.05, ***p < 0.001.
Supplier Page from Abcam for Anti-CEBP Delta/CEBPD antibody