Additional file 1 of SF3B1 inhibition disrupts malignancy and prolongs survival in glioblastoma patients through BCL2L1 splicing and mTOR/ß-catenin pathways imbalances
Creators: name: Fuentes-Fayos, Antonio C.
name: Pérez-Gómez, Jesús M.
name: G-García, Miguel E.
name: Jiménez-Vacas, Juan M.
name: Blanco-Acevedo, Cristóbal
name: Sánchez-Sánchez, Rafael
name: Solivera, Juan
name: Breunig, Joshua J.
name: Gahete, Manuel D.
name: Castaño, Justo P.
name: Luque, Raúl M.
Publisher: name: figshare
Year: 2022
Subjects: subject: Biochemistry
subject: Genetics
subject: FOS: Biological sciences
subject: Molecular Biology
subject: Pharmacology
subject: 39999 Chemical Sciences not elsewhere classified
subject: FOS: Chemical sciences
subject: 69999 Biological Sciences not elsewhere classified
subject: Cancer

description: Additional file 1: Figure S1. Single-cell bioinformatic analyses to characterize intra-tumor cell populations (a) Quality control (QC) metrics plots showing the number of features, counts, and percentage of mitochondrial features in the single-cell RNAseq dataset and, (b) their features-counts/mithocondrial percentage relationship. (c) JackStraw Plot comparing the distribution of P-values for each principal component (PC) with a uniform distribution (dashed line). (d) Ranking of PC based on the percentage of variance. (e) Top3 markers were used to characterize each cluster of cells identified in the single-cell RNAseq dataset. (f) Non-hierarchical heatmap generated using the expression levels of SF3B1 across the different TME cells and tumor-like cells subtypes. (g) Validation of Ponceau staining as a suitable internal control in U-87/U-118 MG cell lines in the two experimental conditions (control vs. pladienolide B) used in this study. Left images: Comparison of the signal obtained with Ponceau staining and western blot with anti-Beta-Actin (ACTB) and anti-Beta-tubulin (TUBB) in GBM cell models (U-87/U-118 MG) treated with pladienolide B and control. Right-top panel: Results showing that Ponceau, ACTB, and TUBB are not altered across experimental conditions (control vs. pladienolide B). Right-bottom panel: Results showing that the signal of TUBB or ACTB normalized by Ponceau staining is not altered across experimental conditions (control vs. pladienolide B). ns indicates non-statistically significant differences across different conditions. Figure S2. Somatic mutation rate of SF3B1 as well as of commonly mutated genes in glioma samples (IDH1, TP53, ATRX, PTEN, IDH2) obtained from the TCGA-dataset (n = 476) (a), and MSKCC-dataset (n = 841) (b). Mutation count, glioma subtype, and grade are also shown. Kaplan-Meier survival curves for glioma patients with mutated and wildtype SF3B1 obtained from the CGGA-dataset (c), TCGA-dataset (d), and MSKCC-dataset (e). mRNA levels from SF3B1 in control and GBM samples in our cohort of patients (f) and the external Rembrandt cohort (g). Asterisks (*P + 2 (cell line resistance)] and their association with SF3B1 expression of different GBM cell lines. (b) Heatmap generated by the number of drugs [Hits, Zscore &gt; − 2 (cell line sensitivity)] and their association with SF3B1 expression of different GBM cell lines. (c) Pathway annotation of different drugs hit in (a) and (b). Figure S4. Determination and accuracy of in vitro models and pladienolide B dose. (a) Comparison of the mRNA levels of SF3B1 between non-tumor brain tissues, GBM tissues, and GBM cell lines U-87 MG and U-118 MG. (b) Dose-response of pladienolide B in GBM cell lines (U-87 MG and U-118 MG; n = 4) and (c) in primary patient-derived GBM cells. (d) IC50 of pladienolide B in vitro in the two GBM cell lines and primary patient-derived GBM cells. (e) Stratification of the primary GBM cell cultures used in the present study (n = 6) based on the percentage of reduction in the proliferation rate in response to pladienolide B (from lower to higher reduction; top-heatmap) and on the SF3B1 expression levels in the same primary GBM cell cultures (bottom-heatmap). (f) Correlation between SF3B1 expression levels and the effect of pladienolide B (48 h of incubation) in terms of proliferation rate in the primary GBM cell cultures used in the present study. Asterisks (*P ± 0.800). Enrichment analysis was performed based on KEGG Pathways Analysis. Table S6. Specific primers for human transcripts used in this study were specifically designed and used in qPCR-based microfluidic assays. The official name of the genes, NCBI accession number of the transcripts, primers sequences, and product sizes of the amplification products are included. Table S7. Antisense Oligonucleotide (ASO) sequences for human BCL2L1 were used in this study to specifically promote the splicing of Bcl-xL variant. The 3′-5′ ASO sequence [2′ O-methoxy-ethyl (MOE) residues are shown in bold)], 5′-3′ BCL2L1 Targeted Region, and oligo sizes are included. Table S8. Drug resistance hits together with its corresponding annotated targeted pathway in SF3B1 high-expression cell lines using the Genomics of Drug Sensitivity in Cancer dataset. Table S9. Drug sensitive hits together with its corresponding annotated targeted pathway in SF3B1 low-expression cell lines using the Genomics of Drug Sensitivity in Cancer dataset.