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. Author manuscript; available in PMC: 2022 Jul 15.
Published in final edited form as: Cancer Res. 2021 Nov 22;82(2):248–263. doi: 10.1158/0008-5472.CAN-21-1991

Figure 5. Notch2-Mutant Adherent Subpopulations Have Enriched Interferon and STING Signaling Compared to Notch-WT Adherent Subpopulations.

Figure 5.

(A) Principal component analysis (PCA) of gene expression from RNA-seq data of 3 Notch-WT (K47, K93, 703 from sgControl RPP) established SCLC adherent cell lines and 3 Notch-Mutant [1(188–2) from sgNotch1 RPP and 2(K60, K62) sgNotch2 RPP] established SCLC adherent cell lines. n=2 biological replicates of each cell line. (B) Unsupervised hierarchical clustering heat map of top 500 upregulated and top 500 downregulated genes in Notch-WT vs. Notch-Mutant adherent cells from the RNA-seq experiment in A. (C) Heat map showing expression of canonical Notch target genes in Notch-WT Adherent vs Notch-Mutant Adherent cells. For B&C, the red to blue color scale indicates FPKM values from large to small. (D-G) GSEA comparing Notch-Mutant adherent cells vs. Notch-WT adherent cells using the cell type signature gene sets (D, E), top 100 neuroendocrine marker gene set (F), and interferon gamma response gene set (G). For D-G, FDR q-values are indicated. (H) Immunoblot analysis of established Notch-Mutant adherent cells and Notch-WT adherent cells with the antibodies indicated. The established suspension counterpart for each cell line is included on the right for comparison. (I) STING IHC of human NOTCH-WT or NOTCH2-Mutant SCLC tumors. Scale Bar=50 μM. (J) Quantification of CXCL10 secretion in the media by ELISA of established adherent SCLC cell lines treated with the STING agonist ADU-S100 or ddH20 for 24 hours. IFN-β was used as a positive control. n=3 biological replicates. (K) Cell counts of established adherent cells treated with ADU-S100 for 3 days normalized to the ddH20 control. n=4 biological replicates. (L) Quantification of CXCL10 secretion in the media by ELISA of K47 Notch-WT adherent SCLC cell line pre-treated with Compound E (1 μM) or DMSO for 24 hours and then ADU-S100 or ddH20 for an additional 24 hours. n=3 biological replicates. For all panels, *=p < 0.05, **=p<0.01, ****=p<0.0001 where indicated.