Fig. 2: NSC-260594/XMH95 induces apoptosis in highly aggressive LMS cells.

A Venn diagrams showing the number of transcripts upregulated in SK-UT-1 cells after 6 and 18 hours of treatment with 10 µM of XMH95. B Bar plots of functional enrichments using the GSEA and the Molecular Signatures Database (MSigDB) tools. The analysis was performed for genes upregulated after XMH95 treatment at 6 and 18 hours. Only the top terms are indicated. The detailed data are shown in Tables S3 and S4. C Bar plots of functional enrichments using the GSEA and the Molecular Signatures Database (MSigDB) tools. The analysis was performed for genes downregulated after XMH95 treatment at 6 and 18 hours. Only the top terms are indicated. The detailed data are shown in Tables S5 and S6. Gene Ontology (GO), Chemical and Genetic Perturbations (CGP), Canonical Pathways (CP). D Regulation of the cell cycle machinery in response to XMH95. Heatmaps reporting the expression levels (log2 fold change relative to untreated cells) of genes encoding for the cell cycle machinery (left) and BCL2 family members (right) in response to XMH95 at 6 and 18 hours of treatment. E Expression levels of the indicated BH3-only genes in HUtSMC and SK-UT-1 cells treated with 5 µM of XMH95. RNAs were extracted at the indicated times and processed for qRT-PCR. Data are relative to DMSO-treated cells. Mean ± SD; n = 3 or 4. *p < 0.05, **p < 0.01, ***p < 0.001 relative to DMSO-treated cells. F BIK, PMAIP1/NOXA and BBC3/PUMA expression in HUtSMC and SK-UT-1 cells. Expression values are shown as normalized counts obtained after DESeq2’s median of ratios normalization. G SK-UT-1 cells were treated for the indicated times with XMH95 [10 µM]. Cellular lysates were generated and immunoblot performed using the indicated antibodies. Actin was used as loading control. H Immunoblot analysis of SK-UT-1 cells engineered to express BCL-XL or Hygro as control by retroviral infection. Immunoblot was performed using the indicated antibodies. Actin was used as loading control. I Cell death in the SK-UT-1 cells expressing BCL-XL or Hygro genes. Cells were treated with the indicated concentrations of XMH95. Cell death was calculated 48 hours later as the percentage of cells positive to Trypan blue staining. Data are presented as mean ± SD (n = 3). Etoposide [5 µM] was used as control. *p < 0.05, **p < 0.01, ***p < 0.001, relative to the corresponding SKT-UT-1-Hygro cells (0). J HUtSMC and SK-UT-1/BCL-XL cells were treated for the indicated times with XMH95 [10 µM]. Cellular lysates were generated and immunoblot performed using the indicated antibodies. Actin was used as loading control. Lysates from SK-UT-1 cells treated for 36 with10µM of XMH95 were used as reference for γH2AX signal.