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. 2016 Sep 8;5:e16432. doi: 10.7554/eLife.16432

Figure 1. IFI6 is transcriptionally upregulated by NRASQ61K via MAPK pathway.

(A) Relative IFI6 mRNA expression in MEL-ST/NRASQ61K cells compared to empty vector-expressing MEL-ST cells. (B) Box plots for IFI6 mRNA expression in indicated melanoma gene expression datasets show significantly higher IFI6 mRNA expression in patient-derived cutaneous melanoma samples (2) compared to normal skin controls (1). (C) Box plot for IFI6 mRNA expression in Barretina cell line dataset. Lane 13 shows average of IFI6 mRNA expression in melanoma cell lines. (D) MEL-ST cells expressing empty vector or indicated HRAS mutants were analyzed by RT-qPCR. The relative expression of IFI6 mRNA in HRAS mutant-expressing MEL-ST cells compared to empty vector-expressing MEL-ST cells. (E) Relative expression of indicated proteins was evaluated by immunoblotting in MEL-ST cells expressing an empty vector or indicated HRAS mutants. (F) MEL-ST cells expressing empty vector or MEK-DD were analyzed for IFI6 mRNA expression by RT-qPCR. IFI6 mRNA expression in MEK-DD expressing MEL-ST cells relative to empty vector is shown. (G) MEL-ST cells expressing empty vector or MEK-DD were analyzed for indicated proteins by immunoblotting. (H) Analysis of patient-derived melanoma samples (n = 20) reveals co-expression of IFI6 with MAPK target genes. (I) Box plot for indicated melanoma gene expression dataset shows significantly higher IF6 mRNA expression in patient-derived NRAS-mutant melanoma samples (2) compared to NRAS wild-type melanoma samples (1). (J) Schematic presentation of NF-κB DNA binding site on the IFI6 promoter. (K) MEL-ST/NRASQ61K cells were analyzed for NF-κB enrichment using the ChIP assay.% NF-κB enrichment in comparison to IgG on the ACTIN or IFI6 gene promoter is shown. (L) MEL-ST cells expressing empty vector or NRASQ61K with NS or NF-κB shRNAs were analyzed for IFI6 mRNA expression by RT-qPCR. Relative IFI6 mRNA in comparison to empty vector expressing MEL-ST cells is shown. (M) MEL-ST cells expressing empty vector or NRASQ61K with NS or NF-κB shRNAs were analyzed for IFI6 protein levels by immunoblotting. ACTINB served as the loading control. In all panels, data are presented as mean ± SEM, and *p<0.05 and ***p<0.0005.

DOI: http://dx.doi.org/10.7554/eLife.16432.002

Figure 1.

Figure 1—figure supplement 1. NRASQ61K transcriptionally upregulates interferon-stimulated genes.

Figure 1—figure supplement 1.

Heatmap shows altered gene expression in MEL-ST cells expressing either an empty vector or NRASQ61K. Upregulated genes are shown in red, and downregulated genes are shown in green. The top five upregulated genes shown on the right include IFI6, IFI27, and MX1.
DOI: http://dx.doi.org/10.7554/eLife.16432.003
Figure 1—figure supplement 2. Regulation of MX1 and IFI6 by NRASQ61K.

Figure 1—figure supplement 2.

Relative mRNA expression of MX1 and IFI27 in MEL-ST cells expressing NRASQ61K or empty vector was analyzed by RT-qPCR. Data are presented as mean ± SEM; ***p<0.0005.
DOI: http://dx.doi.org/10.7554/eLife.16432.004
Figure 1—figure supplement 3. Monitoring regulation of IFI6 by BRAFV600E and NF1 loss.

Figure 1—figure supplement 3.

(Top left) Relative expression of IFI6 mRNA in MEL-ST cells expressing BRAFV600E or empty vector was analyzed by RT-qPCR. (Top right) Relative expression of the indicated proteins in BRAFV600E- and empty vector-expressing MEL-ST cells was analyzed by immunoblotting. (Bottom left) Relative expression of NF1 mRNA in MEL-ST cells expressing NF1 or non-specific (NS) shRNA was analyzed by RT-qPCR. (Bottom middle) Relative expression of IFI6 mRNA in MEL-ST cells expressing NF1 or NS shRNA was analyzed by RT-qPCR. (Bottom right) Expression of the indicated proteins in MEL-ST cells expressing NF1 or NS shRNA was analyzed by immunoblotting. Data are presented as mean ± SEM; *p<0.05.
DOI: http://dx.doi.org/10.7554/eLife.16432.005
Figure 1—figure supplement 4. STAT1 is not necessary for IFI6 expression in MEL-ST/NRASQ61K cells.

Figure 1—figure supplement 4.

(Left) IFI6 promoter analysis using rVista 2.0 reveals DNA binding sites for STAT1. (Right) MEL-ST/NRASQ61K cells were analyzed for STAT1 enrichment using the ChIP assay.% STAT1 enrichment (%) relative to IgG on the ACTIN or IFI6 gene promoter is shown.
DOI: http://dx.doi.org/10.7554/eLife.16432.006
Figure 1—figure supplement 5. Oncogenic NRASQ61K transcriptionally upregulates IFI6 via transcription factor NF-κB.

Figure 1—figure supplement 5.

(Top Left) mRNA expression of NF-κB was analyzed in NRASQ61K-transformed MEL-ST cells expressing either NF-κB or non-specific (NS) shRNA. Relative expression of NF-κB mRNA in cells expressing NF-κB shRNA in comparison to NS shRNA expressing cells is shown. (Top Right) Immunoblot analysis to determine NF-κB protein levels in MEL-ST/NRASQ61K cells expressing either NF-κB or NS shRNA. (Bottom Left) mRNA expression of STAT1 was analyzed in NRASQ61K-transformed MEL-ST cells expressing either STAT1 or non-specific (NS) shRNA. Relative expression is shown. (Bottom Middle) Immunoblot analysis to determine STAT1 protein levels in NRASQ61K-transformed MEL-ST cells expressing indicated shRNA. (Bottom Right) RT-qPCR analysis to determine IFI6 mRNA levels in MEL-ST cells expressing either empty vector or NRASQ61K along with STAT1 or NS shRNA. In all panels, data are presented as mean ± SEM; *p<0.05.
DOI: http://dx.doi.org/10.7554/eLife.16432.007
Figure 1—figure supplement 6. IKKβ is necessary for NF-κB activation and NRASQ61K-induced IFI6 upregulation.

Figure 1—figure supplement 6.

(Top left) IKKβ mRNA levels in MEL-ST/NRASQ61K cells expressing IKKβ shRNA or non-specific (NS) shRNA was analyzed by RT-qPCR. Relative mRNA expression of IKKβ in IKKβ shRNA expressing cells in comparison to NS shRNA expressing cells is plotted. (Top right) Relative expression of the indicated proteins in MEL-ST cells expressing IKKβ shRNA or NS shRNA was analyzed by immunoblotting. (Bottom left) MEL-ST cells expressing IKKβ shRNA or NS shRNA were transfected with an NF-κB–responsive firefly luciferase reporter along with control TK-renilla luciferase reporter construct. Relative firefly luciferase activity under the indicated conditions is shown. (Bottom Right) % enrichment of NF-κB measured via ChIP on ACTINB or IFI6 promoters in MEL-ST cells expressing an empty vector or NRASQ61K along with NS shRNA or IKKβ shRNAs. Data are presented as mean ± SEM; *p<0.05, and **p<0.005.
DOI: http://dx.doi.org/10.7554/eLife.16432.008