Fig 2: ZBTB18 and CTBP regulate the expression of sterol regulatory element–binding protein (SREBP) genes involved in fatty acid synthesis.(A) WB analysis of FLAG-ZBTB18 and CTBP2 expression in SNB19 cells transduced with either FLAG-ZBTB18 or shCTBP2. (B) Venn analysis showing the overlap between regulated genes in ZBTB18 versus empty vector (EV) and genes regulated in shCTBP2 versus shCtr (upper part) or in ZBTB18 shCTBP2 versus EV (lower part). Genes were selected based on adjusted P < 0.05 and absolute fold change > 0.5. (C) Top 10 down-regulated consensus pathways in the overlap between shCTBP2- versus shCtr- and between ZBTB18- versus EV-regulated genes (ZBTB18 versus shCTBP2) from Fisher’s exact test comparing the DEGs (adj. P < 0.05, FC < -0.5) with the whole set of quantified genes. Processes related to SREBP signaling are highlighted. (D) Row-wise z-score heatmap showing the expression of SREBP signaling genes in each triplicate across the four conditions. Both row and column hierarchical clustering are based on the Euclidean distance, and the complete clustering method was used. (E) qRT-PCR validation of selected SREBP gene expression upon ZBTB18 expression and/or CTBP2 silencing in SNB19 cells. n = 3 biological replicates; error bars ± s.d. *P < 0.05, **P < 0.01, and ***P < 0.001 by a t test. Gene expression was normalized to 18S RNA. (F) qRT–PCR analysis of selected SREBP genes in SNB19 cells treated with the CTBP2 inhibitor MTOB. n = 3 biological replicates; error bars ± s.d. *P < 0.05, **P < 0.01, and ***P < 0.001 by a t test. Gene expression was normalized to 18S RNA. (G) WB analysis of ZBTB18 expression in BTSC233 cells transduced with empty vector (EV), ZBTB18, or ZBTB18-mut, using a ZBTB18 antibody. (H) qRT–PCR analysis of selected SREBP genes upon ZBTB18 overexpression and MTOB treatment. Results are presented as the mean of n = 3 biological replicates; error bars ± s.d. *P < 0.05, **P < 0.01, and ***P < 0.001 by a t test. Gene expression was normalized to 18S RNA. (I) Western blot analysis of CTBP1 (top panel) and CTBP2 (bottom panel) expression upon silencing in BTSC233 cells. (J) qRT–PCR analysis of SREBP targets in BTSC233 transduced with shCTBP1- or shCTBP2-expressing lentivirus. Two independent shRNAs to knock down CTBP1 or CTBP2 were used. Results are presented as the mean of n = 3 biological replicates; error bars ± s.d. *P < 0.05, **P < 0.01, and ***P < 0.001 by a t test. Gene expression was normalized to 18S RNA.

Fig 3: ZBTB18 and ZBTB18-mut expression differentially alters the phenotype and the properties of the tumor cells.(A) WB analysis of FLAG-ZBTB18 expression in SNB19 cells transduced with EV, FLAG-ZBTB18, or FLAG-ZBTB18-mut. (B) Micrographs showing the morphology of SNB19 EV controls compared with SNB19-overexpressing ZBTB18 or ZBTB18-mut. Top row is transmitted light, and bottom row is GFP. Scale bar: 100 µm. (C) Quantification of the EdU labeling for proliferating cells of SNB19 cells transduced with EV, FLAG-ZBTB18, or FLAG-ZBTB18-mut. n = 10; error bars ± s.d. *P < 0.05, **P < 0.01, and ***P < 0.001. (D) Quantification of the apoptosis by assessing the activity of caspases 3 and 7 in SNB19 cells transduced with EV, FLAG-ZBTB18, or FLAG-ZBTB18-mut. n = 4; error bars ± s.d. *P < 0.05, **P < 0.01, and ***P < 0.001. (E) Micrographs showing the wound closure process in a migration assay with SNB19 cells transduced with EV, FLAG-ZBTB18, or FLAG-ZBTB18-mut. Scale bar: 400 µm. (E, F) Quantification of the migration assay described in (E). n = 4; error bars ± s.d. *P < 0.05, **P < 0.01, and ***P < 0.001.

Fig 4: ZBTB18 interacts with CTBP1 and CTBP2 in GBM cells.(A) Outline and Coomassie staining analyses of FLAG-ZBTB18 co-IP in SNB19 GBM cells transduced with EV or FLAG-ZBTB18. (B) List of ZBTB18 co-precipitated proteins analyzed by mass spectrometry showing detection of CTBP1 and CTBP2 in the co-precipitated fraction. (C) Western blot (WB) analysis of FLAG co-IP in LN229 cells transduced with empty vector (EV) or FLAG-ZBTB18. (D) Western blot (WB) analysis of CTBP1 and CTBP2 upon co-precipitation with antibodies directed to CTBP1 or CTBP2 in LN229 cells. An anti-CTBP antibody that recognizes both CTBP1 and CTBP2 was used. The asterisk indicates an unspecific band. (E) Mass spectrometry results of endogenous ZBTB18 co-IP (using an anti-ZBTB18 antibody) showing detection of CTBP1 and CTBP2 in the co-precipitated fraction of BTS268. (F) WB analysis of endogenous ZBTB18 co-IP in BTSC475 cells. (G) Mass spectrometry results of endogenous ZBTB18 co-IP (using an anti-ZBTB18 antibody) showing detection of CTBP1 in the co-precipitated fraction of U3082 cells.

Fig 5: CTBP2 and ZBTB18 map to the promoter of sterol regulatory element–binding protein (SREBP) genes.(A) Experimental flowchart of the ChIP-seq analysis. (B) Venn diagram showing the overlap between the three consensus peak sets from EV_CTBP2, ZBTB18_CTBP2, and ZBTB18_FLAG. (C) Enrichment of peaks on promoter regions depicted on a forest plot from Fisher’s test analysis. Dots indicate the odds ratio, and lines indicate the 95% confidence interval. (D) Meta-gene peak density profile showing an overrepresentation of peaks around SREBP gene TSS regions. Y-axis indicates the percentage of peaks in EV_CTBP2, ZBTB18_CTBP2, and ZBTB18_FLAG that overlap the genomic region depicted on the x-axis. (E) Homer analysis showing enrichment of the ZNF238/ZBTB18 DNA binding motif in the Venn diagram groups. (F) UpSet plot showing the overlap of SREBP genes having at least one peak in one of the three conditions: EV_CTBP2, ZBTB18_CTBP2, and ZBTB18_FLAG. (G) Read coverage around biologically relevant selected genes, that is, FASN, GSK3A, INSIG1, SCAP, and SCD. Identified peaks are highlighted under the grey area, and gene TSS regions are drawn on the top of the plot. (H, I) FLAG-ZBTB18 (H) and CTBP2 (I) enrichment at the promoter of the indicated SREBP genes in SNB19 cells transduced with empty vector (EV) of FLAG-ZBTB18. ChIP was performed using control beads alone (IgG), anti-FLAG antibodies, or anti-CTBP2 antibodies. Graphs show representative qRT–PCR results (n = 3 technical replicates) of at least three (FLAG) or two (CTBP2) biological replicates, which are expressed in % of the input as indicated.