Figure 3. Transferrin is a transcriptional target of SREBF2.

(A) Knockdown of SREBF1 (SREBF1-KD), SREBF2 (SREBF2-KD) or both (SREBF1&2-KD) in Mel-167 cultured CTCs, using antisense oligonucleotides (ASO), demonstrating both specificity for each individual gene and effective dual targeting. Y-axis, relative mRNA fold change normalized to Actin. Statistical significance was assessed by two-sided Welch’s t-tests. ** P < 0.01; *** P < 0.001; *** P < 0.0001.

(B) Quantification of soft agar colony numbers formed by Mel-167 CTCs transfected with control, SREBF1-KD, SREBF2-KD or SREBF1&2-KD ASO sequences. Y-axis, relative colony number normalized to control. Statistical significance was assessed by two-sided Welch’s t-test. * P < 0.05; n.s, not significant.

(C) Heatmap representation of SREBF2 ChIP-seq in Mel-167 CTCs, showing enrichment of SREBF2 binding sites within the transcriptional start sites (TSS) of genes comprising three pathways: SREBP_TARGET, FERROPTOSIS and IRON_ION_HOMEOSTASIS. For each pathway, the first two columns represent replicate experiments, and the third shows the input reads. The GSEA pathway enrichment plots and assessment of statistical significance of SREBF2 ChIP-seq are shown in Table S4. The heatmap color scale (Y-axis) represents the read intensities in bins per million mapped reads (BPM: set the maximum value at 3).

(D) Venn diagram showing the top 10 genes at the intersection of SREBF2 bound promoters in CTCs and genes with increased expression in CTCs, compared with primary and metastatic melanoma (TCGA) and standard tumor-derived melanoma lines (CCLE). The mean fold change in individual gene expression is listed below the Venn diagram, with transferrin (TF) as the top hit.

(E) Integrative Genomic Viewer (IGV) plot showing SREBF2 ChIP-seq peaks in the TF gene promoter region (framed in red). Two experimental repeats (rep 1, 2) are shown. Input genomic DNA serves as control. The scales in bins per million mapped reads (BPM) of peak window for each sample are shown in brackets, and the genomic structure of the TF gene is shown below the IGV plot.

(F) In vivo binding of SREBF2 to the TF gene promoter, as shown by ChIP-qPCR analysis in Mel-167 melanoma CTCs. Top: Schematic representation of expected qPCR products (#1, 2, 3, 4) spanning regions of the TF gene promoter including those containing the two predicted SREBP binding sites (#2, 3), which are shown in blue and red (35). Bottom: ChIP-qPCR performed using anti-FLAG antibody to precipitate FLAG-SREBF2-DNA complexes. Y axis shows relative fold enrichment of TF gene promoter fragments (normalized to control IgG antibody), with strong in vivo binding of SREBF2 to fragments #2 and #3 that contain the SREBP consensus sequences, but not to neighboring fragments (#1 and #4) or to unrelated sequences (a, b). Data are normalized to 2% of total genomic DNA input.

(G) Suppression of TF mRNA expression in Mel-167 CTCs, following treatment with ASOs targeting SREBF1 and SREBF2 alone or together, compared with control (see Figure 3A for knockdown efficiency and specificity), demonstrating that SREBF2 is the primary regulator of TF expression, with modest enhancement by combined SREBF1&2 KD.

(H) Induction of TF mRNA expression by SREBF2 in Mel-167 CTCs, demonstrated by real-time q-PCR analysis, 48 hours following doxycycline-mediated inducible expression of SREBF2. SREBF2 also mediates a modest increase in SREBF1 mRNA. Data are normalized to actin. Y-axis shows relative fold change in SREBF2-expressing cells compared with uninduced controls. Statistical significance was assessed by two-sided Welch’s t-test. P = 0.003 for TF; P = 0.0002 for SREBF2; P = 0.0009 for SREBF1. **P < 0.01; ***P < 0.001.

(I) Induction of TF protein by doxycycline-inducible FLAG-tagged SREBF2, quantified by Western blot analysis in Mel-167 CTCs. Actin is shown as loading control.