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. Author manuscript; available in PMC: 2024 Aug 8.
Published in final edited form as: Oral Oncol. 2023 Sep 2;146:106562. doi: 10.1016/j.oraloncology.2023.106562

Figure 5. Analysis of Mediators Downstream of IFNγ and FGF2 that Upregulate PD-L1 in HNSCC.

Figure 5.

UM-SCC-14a cells were pre-treated with Fedratinib (5 μM) (A), BGJ398 (5 μM) (B) or vehicle control (DMSO) for three hours as indicated. Then, IFNγ (10 ng/mL) or FGF2 (30 ng/mL) was added for an additional three-hour treatment. RNA was harvested and expression of genes of interest analyzed by qPCR. C, GSEA of RNAseq data showed significant upregulation of IFNγ response gene set in UM-SCC-14a cells after 24-hour treatment with IFNγ but not FGF2. D, GSEA of RNAseq data showed significant upregulation of MYC family genes in UM-SCC-14a cells after 24-hour treatment with IFNγ or FGF2. E, Differential expression (Log2[DE]) of target genes in IFNγ- or FGF2-treated (24 hours), relative to untreated, UM-SCC-14a cells shows distinct but overlapping transcriptional programs. F, qPCR validation of MYC gene family upregulation in IFNγ- and FGF2-treated (24 hours), relative to untreated, UM-SCC-14a cells. G, UM-SCC-14a cells were pre-treated with tunicamycin (50 ng/mL) or vehicle control (DMSO) for six hours. Then, IFNγ (10 ng/mL) or FGF2 (30 ng/mL) was added for an additional three-hour treatment. Protein was harvested and PD-L1 expression was assessed by western blot. H, UM-SCC-14a cells were treated with IFNγ (10 ng/mL), FGF2 (30 ng/mL), or vehicle control (DMSO) for 72 hours. Protein was harvested and lysate was treated with PNGase or control (RIPA buffer). PD-L1 expression was assessed by western blot.