Combination Epigenetic Treatment Induces Significant Differential Gene Expression, Related to Figure 2 (A) Quantitation of the differentially expressed genes for each treatment condition (differential gene expression cutoff Log2 fold change over mock > 0
Combination Epigenetic Treatment Induces Significant Differential Gene Expression, Related to Figure 2 (A) Quantitation of the differentially expressed genes for each treatment condition (differential gene expression cutoff Log2 fold change over mock > 0.5, day 8, microarray, 500nM Aza, 100nM MS275, 100nM ITF-2357). (B) Relative RNA expression correlation matrix for inhibitor conditions and cell lines indicated in panel A (microarray, day 8, 500nM Aza, 100nM MS275, 100nM ITF-2357). (C) GSEA enrichment plots for top upregulated pathway (Interferon ) following combination epigenetic treatment with the indicated HDACi. (D) GSEA enrichment plots for top downregulated pathway (DNA replication) following combination epigenetic treatment with the indicated HDACi. Figure S3. = 6 (ITF-2357)). Data are presented as mean SEM. *p value < 0.05 Pamabrom relative to mock, # p value < 0.05 relative to Aza Pamabrom + MS-275. (G) Evaluation of pharmacologic targets of HDAC1/2: histone acetylation and for HDAC6: tubulin acetylation by immunoblotting in A549 cell line. Histone 3 and -Actin used as loading controls. Drug and drug concentrations are indicated in the figure. (24-hour treatment, ITF = ITF-2357, MGCD = MGCD0103, TubA = Tubastatin A, n = 2). (H) Normalized BrdU percent positivity for mock or Aza treated cells in combination with the indicated HDACis (day time 9, 200nM MGCD0103, 2000nM RGFP996, 1000nM Tubastatin A; data are mean SEM, n = 6). (I) Immunoblotting for DNMT1 in A549 and H460 cells infected with vacant vector or shDNMT1 vector. -Actin used as loading control (day time 5, n=2). (J) Log dose response for A549 and H460 cells infected with vacant vector or shDNMT1 vector and treated with HDACi as indicated in number (5-day time HDACi exposure, n=2, data are mean SEM). (K) Average quantities of tumor xenografts from NOD-SCID mice subcutaneously injected with H460 cells (Day time 0 equals onset of treatment, data are mean SEM, n = 4 mock, n = 5 Aza + MGCD0103). (L) Tumor weights for patient derived xenografts from NSG mice and treated with the providers as layed out in the number, mock data will also be presented in Number 1H (28 days of treatment period, n = 6 mock n = 7 Aza + MGCD0103, data are mean SEM) *p value < 0.05 relative to mock acquired by two tail t test. Number S2. Combination Epigenetic Treatment Induces Significant Differential Gene Manifestation, Related to Number 2 (A) Quantitation of the differentially Pamabrom indicated genes for each treatment condition (differential gene manifestation cutoff Log2 collapse switch over mock > 0.5, day DCHS1 time 8, microarray, 500nM Aza, 100nM MS275, 100nM ITF-2357). (B) Relative RNA expression correlation matrix for inhibitor conditions and cell lines indicated in panel A (microarray, day time 8, 500nM Aza, 100nM MS275, 100nM ITF-2357). (C) GSEA enrichment plots for top upregulated pathway (Interferon ) following combination epigenetic treatment with the indicated HDACi. (D) GSEA enrichment plots for top downregulated pathway (DNA replication) following combination epigenetic treatment with the indicated Pamabrom HDACi. Number S3. HDACi Isoform-Specific Induction of Interferon Signaling, Related to Number 3 (A) Manifestation of Interferon stimulated genes induced by Aza and/or the indicated HDACi in various NSCLC cell lines (qRT-PCR, day time 8, 500nM Aza, 100nM ITF-2357, and 200nM MGCD0103). (B and C) Manifestation of Interferon stimulated genes induced by Aza and/or the indicated HDACi in A549 and H23 cells (PCR genecard, day time 8, 500nM Aza, 1000nM Tubastatin A, 2000nM RGFP996). (D and E) Manifestation of Interferon stimulated genes induced by Aza and/or the indicated HDACi in A549 and H23 cells (PCR genecard, day time Pamabrom 8, 500nM Aza, 100nM ITF-2357, 200nM MGCD0103, 300nM SAHA). Number S4. Sequential Aza + HDACi Imparts Significant Amplification of Aza Response, Related to Number 3 (A) Heatmap of relative RNA manifestation for malignancy/testis antigen genes across NSCLC cell lines (microarray, day time 8, 500nM Aza, 100nM MS275, 100nM ITF-2357). (B) Quantification of malignancy/testis antigen transcriptional induction by combination Aza +.