Fig. 3. RNA-seq reveals critical gene expression changes during LSD1 inhibition in MCC.

a. MKL-1 cells were treated with GSK-LSD1 (0.1 μM) for three days and processed for RNA-seq. NS= Not Significant; Log 2 FC= Fold change cutoff 1.5; P=P-value cutoff 10e-6. The Wald test was performed using the DEseq2 R package⁴² with the p-values adjusted by Benjamini-Hochberg. n=3. b. RNA-seq of six virus-positive MCC and virus-negative UISO cell lines treated with LSD1 inhibitors (GSK-LSD1 for three days or CPI-670242 for one day). n=3. c. The PCA analysis displays global gene expression changes caused by LSD1 inhibition. d. MKL-1, WaGa, and UISO cell lines were treated with GSK-LSD1 (0.05 μM) for three days. The signals were normalized to untreated samples and RPLP0 in each sample. Data are shown as mean of n=3 ± SD; two-sided t-test; * P<0.05, **<0.005, ***<0.0005 and ****<0.00005. e. LSD1 inhibition increases global levels of H3K4me1 and LSD1 target genes. MKL-1 and WaGa cells were treated with GSK-LSD1 (0.05 μM) for six days, and the whole-cell lysates and histone extracts were prepared. LT= MCV LT; ST=MCV ST; VINC=Vinculin. The experiment was performed at least three times. See Unprocessed gels Figure 3. f. Cells were treated with LSD inhibitor GSK-LSD1 (0.05 μM) for three or six days. LSD1 inhibition activates the BMP pathway as assessed by increased levels of phosphorylated SMAD1/5/9 (P-SMAD1/5/9). The experiment was performed at least three times. See Unprocessed gels Figure 3. g. Cells were transduced with either control or LSD1 targeting shRNA for six days and harvested for western blotting. The experiment was performed at least three times. See Unprocessed gels Figure 3. h. LSD1 inhibition perturbs gene expression in the virus-positive MCC cell lines but not in the virus-negative MCC cell lines. Cells were treated with GSK-LSD1 (0.05 μM) for three days. The experiment was performed at least three times. See Unprocessed gels Figure 3.