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Supplementary figures and tablesSupplementary Figure S1. Construction of TMAs from DMPM tumor specimens.(A) Representative picture of a TMA slide containing 4 cylindrical core tissue biopsies (? = 0.6 mm) collected from at least 2 paraffin blocks derived from the same patient (different color-coding represents each patient).(B) Distribution of patient samples according to SF3B1 scoring. The pathological examination was performed by two experienced pathologists, who were blinded to the clinical patient outcome. Immunostaining intensity was classified into six grades considering both the staining intensity and percentage of positive cells, as following: 0 (absent), 1 (weak), 2 (moderate), 3 (strong). We attributed one, two, or three additional points if the percentage of positive cells was less than 25%, 25% to 50%, or greater than 50%, respectively. According to the observed distribution, the median (= 3.5) was chosen as cut-off for discriminating between low and high SF3B1 expression..Supplementary Figure S2. PB induced early DNAJB1 intron retention and MCL-1 splicing after 4 hours treatment at 30 nM. Schematic splice variants and primer annealing locations (black triangles) are shown on the left.Supplementary Figure S3. Number of significant splicing events of each type detected by rMATS. Exon skipping events (ES - considered as “exclusion of exon”) were found mainly in treated samples (?Ψ>0, when Ψ values are higher in control than treated samples).Similarly, retained intron events (note: RI - considered as “inclusion of intron” and therefore is characterized by ?Ψ<0, because Ψ values are higher in treated than control samples) were also detected mainly in treated samples, as expected. Alternative 3` and 5` splice site selection events (A3SS and A5SS) were detected at lower rates compared to ES and RI. ? Ψ = Inclusion Level Difference between average Ψ of control and average Ψ of PB-treated samples.Supplementary Figure S4. BCL-X splicing assessed by RT-PCR after 24 hours PB exposure at increasing drug concentrations. Schematic splice variants and primer annealing locations (black triangles) are shown on the left.Supplementary Figure S5. PB inhibited cell proliferation of DMPM cells in a dose-dependent fashion. Proliferation of DMPM cells treated with 1, 10 and 30 nM PB at 24 and 48 hours. Absolute cell count was measured by flow cytometry and normalized by using fluorospheres. Bars represent percentage of 7-AAD negative cells relative to untreated controls ± SEM of at least 2 independent experiments. Asterisks indicate statistical significance compared to untreated cells (***P<0.001, **P<0.01, *P<0.05, Student`s t-test).Supplementary Figure S6. Aberrant splicing of RON (MST1R) induced by PB. Sashimi plot (IGV) on the upper panel shows exon 11 skipping in untreated and treated samples. The plot on the bottom panel shows the full-length gene structure. Transcript lacking exon 11 (ΔRON) is reported in the reference track at the bottom of both plots. With differential splicing analysis ΔRON transcripts were found in PB-exposed samples, but junction counts were too low to reach statistical significance probably due to abundance of GC-rich regions throughout the transcript (ID= 12657, FDR = 0.0485, sum counts = 143; Supplementary Table 5, “ES” Tab).Supplementary Figure S7. PB exposure induced aberrant splicing of focal adhesion genes PIP5K1A and TES (GO:0005925).Supplementary Figure S8. Monitoring of body weight of E7107-treated mice compared to vehicle-treated controls. Data points represent body weight (grams) ± SD.Supplementary Figure S9. Detection of Gaussia Luciferase (expressed as relative light units, RLU) in blood (every 2 weeks) during the in vivo experiments in mice treated with E7107 compared to vehicle-treated mice. Each box extends from the 25th to 75th percentiles of RLU values. Whiskers extend from smallest to the largest value.Supplementary Figure S10. Splicing patterns of selected genes assessed by RT-PCR in orthotopic tumor samples after administration of E7107. Two animals bearing MesoII-derived tumors received one dose of E7107 (5 mg/kg) and were sacrificed 4 and 24 hours after treatment together with one animal from the vehicle group used as negative control. Tissue isolation, RNA extraction and RT-PCR are described in methods section.Supplementary Figure S11. Splicing patterns of selected genes assessed by RT-PCR after in vitro administration of E7107 for 24 hours. ................
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