Fig 1: Procedures of identifying key transcription factors (TFs) of the hypoxia-regulated genes in GBM.A identification of key TFs from hypoxia-regulated proteins (HRPs). Quantitative TMT proteomic analysis identified significantly up-regulated proteins (HRPs) for at least 1.2-fold change under hypoxia. Then systemic TF-targets analysis identified top TFs which regulated the most number of targets belong to HRPs for all human TFs. The targets of each TF were identified with the mutual information-based ARACNe algorithms, as described previously [2]. B Similar procedure was performed in hypoxia-regulated genes (HRGs) in mRNA level, by using the statistically-reliable HRGs (SR-HRGs), which are genes overlapped in at least 3 sets from 5 hypoxia-induced gene sets. Then top TFs regulating the most number of targets belong to SR-HRGs were identified. C Similar procedure was further performed in pseudopalisading cells around necrosis (PAN) specific genes (PANSGs) in GBM tissues from glioblastoma atlas data [26]. PANSGs were those that are specifically significantly highly expressed in PAN area (p < 0.05), and with an averaged fold change greater than 2, compared to the other areas, including leading edge (LE), infiltrating tumor (IT), cellular tumor (CT), and microvascular proliferation (MVP). Then top TFs regulating the most number of targets that are specifically highly expressed in PAN area were identified. D Top TF analysis for Random target genes. Certain number (the same number of genes of the HRPs, HRGs, and PANSGs) of random genes, which are randomly selected from the corresponding background genome (all genes of the high-throughput platform), were produced to analyze the top TFs that regulate the random genes. Totally 1000 random gene sets were analyzed to produce a rank position distribution for each TF. E Rank position distributions of CEBPD and CEBPB in random target gene set analysis. Random targets with 36, 244, or 797 genes, were analyzed, corresponding to the gene numbers of HRPs, HRGs, and PANSGs, respectively. Data were obtained by repeating 1000 times. Rank distributions were analyzed for ARACNe-based TF targets. F Top TF analysis and rank distribution analysis of CEBPD and CEBPB for HRPs, HRGs, and PANSGs, where the targets of each TF were defined by the combined targets of ARACNe and hTFtarget-based targets [35] (ARACNe_hTFtarget_combined). G, H Correlations between top TFs (CEBPD and CEBPB) and HIFs (HIF1a and HIF2a/EPAS1) in TCGA (G) and Rembrandt (H) GBM databases.
Fig 2: CEBPD knockdown inhibited xenograft tumor growth and invasion capacity of GBM cells in vivo.A, B Flank xenograft experiments showing the in vivo growth curves of U87 cells in shCtrl (n = 5) and shCEBPD groups (n = 5). C Intracranial xenografts tumor showing the tumor volumes in shCtrl (n = 5) and shCEBPD KD (n = 5) groups. The bottom binary images showed the tumor areas. D The invasion capacity of U87 cells in vivo in shCtrl and shCEBPD KD groups was evaluated by examining the number of tumor fingers in per field. The bottom binary images showed the tumor areas, where islands and protruded areas were indicated by arrows. *p < 0.05, compared with shCtrl group.
Fig 3: Corroboration of the effect of CEBPD in GBM samples and enrichment analysis of hypoxia-regulated proteins.A Correlations between expression levels of CEBPD and genes in EGFR/PI3K pathways (left image) in different databases (TCGA, Rembrandt, and CGGA). *P < 0.05, **(vertical) P < 10-5, ***(vertical) P < 10-10. In addition, correlations of CEBPD and these genes in each GBM subtype (Proneural, Neural, Classical, and Mesenchymal) were further performed in the TCGA database to test the impact of subtype on the correlations. B Correlations between CEBPD expression level and pathway activities of EGFR/PI3K and HIF1A. C Correlations between pathway activities of CEBPD and EGFR/PI3K related pathways, HIF1A pathways. D Expression levels of HIFs (HIF1A and EPAS1), CEBPD and genes in EGFR/PI3K pathways in high hypoxic samples (HHSs) and low hypoxic samples (LHSs) in the TCGA database. E Coefficients between CEBPD level and EGFR, HIF1A pathway activities in HHSs and LHSs. Digital labels for each bar indicate the coefficients (R, left label) and p values (right label). F KEGG pathway analysis and Gene Ontology (GO) analysis of HRPs in U87 cells. Analyzed GO Categories include Biological process (BP), Cellular component (CM), and Molecular function (MF). The colored bars represent the -log10(p Value) (left panel) and count of protein (right panel) for each enriched items. G Top TF analysis of ECM proteins belong to the HRPs (HRP-ECM), as described in Fig. 1. H Chip-seq analysis of CEBPD binding sites in HRP-ECM proteins. Bars represented number of CEBPD binding sites identified by Chip-seq in the promoters of the indicated proteins.
Fig 4: CEBPD positively regulated ECM-mediated EGFR/PI3K pathway activity, and important genes involved in the pathway and invasion capacity, by directly binding to the promotors of key genes in ECM.A WB results showing the protein levels of CEBPD and key transducers of the EGFR/PI3K pathway, especially phosphorylated EGFR, ERK1/2, AKT, mTOR, STAT3, and essential genes for the invasion capacity, after CEBPD knockdown in normoxia and hypoxia conditions. B, C Correlation coefficients and corresponding p values (labels above each bar) between HIF1a (B) or EPAS1 (C) and genes in EGFR/PI3K/Akt signal pathways, which are regulated by CEBPD and contribute to tumor invasion capacity. Pearson correlation was performed between HIF1a or EPAS1and indicated genes labeled in the X axis, in three GBM databases (TCGA, Rembrandt, and CGGA databases). D Chip-seq analysis of CEBPD binding sites in CEBPD-regulated proteins belong to the EGFR/PI3K pathway. Bars represented number of CEBPD binding sites identified by Chip-seq in the promoters of the indicated proteins. E The DNA fragment of CEBPD binding peaks in the FN1 promoter identified by Chip-seq, which also contained highly reliable CEBPD binding sites as predicted by the JASPAR database [43]. There are 4 highly reliable CEBPD binding consensus sequences (red letters, site 1–4) in the fragment, and the site (1) and (2) had the greatest scores. For each binding site, the sequence, location, and the binding scores (expressed as score/relative score) were labeled; red letters represent positive strand, and yellow shaded letters represent negative strand. Greed shaded letters represent sequences for primers. F mRNA levels of FN1 after shCEBPD treatment in U87 cells in normoxia condition. G Chip-qPCR showing that CEBPD bind to the FN1 promoter region shown in E in normoxic condition. H Luciferase reporter assay showing bind and activation of CEBPD on the promotor of FN1 in normoxia condition. The promoter region of FN1 is shown in (E). For mutation analysis, the putative binding sites (site 1 and 2) with the greatest scores were mutated, where the sequence of “TGTTGCTAAATGA” (as shown in E) were replaced by “CTGCCGCGGCGGG”. (pGL3: pGL3-Basic vector containing blank control; FN1 prom: pGL3-Basic vector containing FN1 promoter sequence; FN1 promMut: pGL3-Basic vector containing FN1 promoter with mutation; pRL: pRL-TK vector containing blank control; CEBPD: pRL-TK vector containing CEBPD sequence; CEBPD_KD: pRL-TK vector containing CEBPD shRNA sequence.) *p < 0.05, ***p < 0.0001 compared to the FN1 promoter + pRL group; ###p < 0.0001 compared to the FN1 promoter + CEBPD group. All of the last 6 groups had p < 0.0001 compared to the first 2 groups. I WB assays of rescue experiments after CEBPD KD in U87 cells. Protein levels of key transducers of the EGFR/PI3K pathway, including p-EGFR, were examined in U87 cells, U87 cells treated with shCtrl (U87 shCtrl) and shCEBPD lentivirus (U87 shKD1, U87 shKD2), or U87-shCEBPD KD cells which are recused with the a5ß1 integrin agonist Pyrintegrin (U87 shKD1+Pyr, U87 shKD2+Pyr) or a recombinated FN1 protein (U87 shKD1 + rFN1, U87 shKD2 + rFN1). The a5ß1 integrin is the receptor for FN1. J Transwell invasion assays of rescue experiments after CEBPD KD for U87 cells. The cell groups were the same as described in (I), and experiments were repeated in triplicate.
Fig 5: CEBPD is highly expressed in GBM and is up-regulated under hypoxia.A The mRNA levels of CEBPD, as revealed by qPCR, in a cohort of glioma samples with different grades (**p < 0.001 compared to Normal brain samples; ##p < 0.001 compared to grade 2 astrocytoma (A); !p < 0.05 compared to grade 3 anaplastic astrocytoma (AA). B Expression of CEBPD mRNA levels in different grades of gliomas in TCGA, Rembrandt, and CGGA datasets. (**p < 0.001 compared to Grade 4 GBM; ##p < 0.001 compared to Grade 3 AA; !p < 0.05, !!p < 0.001, compared to Normal brain samples.) C Representative WB results showing protein levels of CEBPD and HIF1a in different grade gliomas. D, E Representative IHC staining and quantitative staining scores of CEBPD in glioma samples (*p < 0.05, **p < 0.001, compared to grade 2 astrocytoma). F Survival analysis and progression-free analysis of GBM patients in different databases (TCGA, CGGA, and Rembrandt data). G WB results showing CEBPD protein levels in core hypoxic and peripheral less hypoxic areas in GBM samples (n = 3 independent experiments). H IHC staining showing CEBPD stain in hypoxic pseudo-palisading areas in GBM tissues; *, T, indicated pseudo-palisading areas and tumor area, respectively. I CEBPD mRNA levels in different areas in GBM tissues, including leading edge (LE), infiltrating tumor (IT), cellular tumor (CT), pseudopalisading cells around necrosis (PAN), and microvascular proliferation (MVP) areas, in the glioblastoma atlas data [26]; **p < 0.001, compared to PAN. J WB results showing CEBPD protein levels in GBM U87 and U251 cells under hypoxia condition. K Luciferase reporter assay showing bind and activation of HIF1a and HIF2a to the CEBPD promotor region (***p < 0.0001 compared to CEBPD-promotor-HIF-NC group). (pGL3: pGL3-Basic vector containing blank control; CEBPD promotor: pGL3-Basic vector containing CEBPD promoter sequence; pRL: pRL-TK vector containing blank control; HIF1A or EPAS1: pRL-TK vector containing HIF1A or EPAS1 TF sequence). L The DNA fragment with the highly reliable HIF1A or EPAS1 binding sites (Hypoxia-responsive Element, HRE) in the CEBPD promoter, as predicted by the JASPAR database [43]. There are 4 highly reliable HREs (bold letters) in the fragment. For each HRE, the sequence, location, and the binding scores (expressed as score/relative score) were labeled; red letters represent positive strand, while yellow shaded letters represent negative strand. Greed shaded letters represent sequences for primers. M Chip-qPCR showing that HIF1A bind to the CEBPD promoter region shown in (L). A grade 2 astrocytoma; O grade 2 oligodendroglioma; AA grade 3 astrocytoma; GBM grade 4 glioblastoma; peri peripheral.
Supplier Page from Abcam for Anti-CEBP Delta/CEBPD antibody [EPR23518-259]