Fig 2: knockdown of CEBPD inhibited invasion capacity of GBM cells.A WB assays showing proteins levels of CEBPD after shCEBPD lentiviruses infection in U87 (up panel) and U251 (bottom panel) cells, in normoxia (20% O2) or hypoxia (1% O2) conditions. B In vitro CCK8 assays showing the effect of CEBPD knockdown on growth curves for U87 (up panel) and U251 cells (bottom panel). C In vitro colony formation assays of U87 (up panel) and U251 (bottom panel) cells with shCtrl or shCEBPD infection. D, E Transwell invasion assays of U87 (D) and U251 (E) cells infected with shCtrl or shCEBPD lentiviruses in normoxia (20% O2) or hypoxia (1% O2) conditions. **p < 0.001, *p < 0.05, compared with corresponding normoxia (20% O2) group; #p < 0.05 compared with shCtrl group in normoxia (20% O2); !p < 0.05 compared with shCtrl group in hypoxia (1% O2). Right images in (D, E): Fold increase of number of hypoxia induced invasion cells after shCtrl or shCEBPD treatment in U87 (D, right image) and U251 (E, right imag) cells. Repeated data for each group were obtained by calculating the ratio of each hypoxia data to the averaged value of normoxia data. *p < 0.05, compared with shCtrl group. Experiments were repeated in triplicate.

Fig 3: 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 4: CEBPD-regulated proteins in both normoxia and hypoxia conditions.A TMT quantitative proteomic data, showing heatmap of protein levels in shCEBPD KD and shCtrl U87 cells, in both normoxia (20% O2) and hypoxia (1% O2) conditions. Proteins that are significantly down-regulated at least 1.2-folds after shCEBPD KD were chosen as the CEBPD-regulated proteins. The proteins were sorted in descending order from top to bottom according to their folds changes. B KEGG pathway analysis and Gene Ontology (GO) analysis of CEBPD positively regulated proteins in both normoxia and hypoxia conditions 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 for CEBPD-regulated protein in normoxia. The black bars, which are covered by the colored bars, represent the corresponding values for CEBPD-regulated proteins in hypoxia. The black parts that are not covered by the colored ones indicated that, for the specific item, the value of the item has a greater value in hypoxia condition than in normoxia condition. The black and red integers in the right panel indicate counts of proteins regulated by CEBPD in normoxia and hypoxia conditions, respectively. C Detailed proteins that are belong to the key KEGG pathways affected by CEBPD knockdown in both normoxia and hypoxia conditions. Proteins labeled with red are those which are also belong to the PI3K/Akt signal pathway. D Quantitative analysis of hypoxia impact on the protein levels that are belong to the key KEGG pathways, in shCtrl (up panel) or shCEBPD KD (bottom panel) conditions. *p < 0.05, compared with normoxia (20% O2) group. E Fold changes of hypoxia induced protein levels of the CEBPD-regulated genes in shCtrl and shCEBPD KD groups. *p < 0.05 compared with the shCtrl group. Repeated data for each group were obtained by calculating the ratio of each hypoxia data to the averaged value of normoxia data.

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.