Additional file 1 of Functional apoptosis profiling identifies MCL-1 and BCL-xL as prognostic markers and therapeutic targets in advanced thymomas and thymic carcinomas
Creators: name: Müller, Denise
name: Mazzeo, Paolo
name: Koch, Raphael
name: Bösherz, Mark-Sebastian
name: Welter, Stefan
name: von Hammerstein-Equord, Alexander
name: Hinterthaner, Marc
name: Cordes, Lucia
name: Belharazem, Djeda
name: Marx, Alexander
name: Ströbel, Philipp
name: Küffer, Stefan
Publisher: name: figshare
Year: 2021
Subjects: subject: Biochemistry
subject: Cell Biology
subject: Genetics
subject: FOS: Biological sciences
subject: Molecular Biology
subject: Pharmacology
subject: 39999 Chemical Sciences not elsewhere classified
subject: FOS: Chemical sciences
subject: Immunology
subject: FOS: Clinical medicine
subject: Developmental Biology
subject: Cancer
subject: Hematology
subject: 60506 Virology

description: Additional file 1: Figure S1. Survival analysis of IHC staining of BCL-2 family proteins in TH and TC. (A-C) Kaplan-Meier analysis of TP53 (cutoff = 0.25), BCL-2 (cutoff = 0.125), and NOXA (cutoff = 1.25), for all subtypes did not show a significant impact on patient OS, (D) of low vs. high MCL-1 in type B2 (n=12) and B3 (n=9) TH and TC (n=5), (E) BCL-2 in type A (n=5), AB (n=19), and B1 (n=2) TH, (F) BCL-2 in type B2 (n=13) and B3 (n=9) TH and TC (n=7), (G) TP53 in type B2 (n=15) and B3 (n=9) TH and TC (n=8), (H) NOXA in type A (n=5), AB (n=18) and B1 (n=2) TH and (I) NOXA in type B2 (n=10) and B3 (n=9) TH and TC (n=5) did not show any significant impact on patient OS. Figure S2. Analysis of BCL-2 family members and specific inhibitors in 1889c and HCC15. (A) Sanger sequencing revealed a TP53 mutation (c.738G&gt;T, p.M246I) in 1889c. (B) IC50 concentrations of 1889c and HCC15 after a single treatment with Cisplatin and Etoposide (0.1-10μM, 72h) and Sunitinib and Sorafenib (0.1-10μM, 48h). (C, D) Dose-response curve and (E) IC50 concentrations of 1889c and HCC15 after a single treatment with AZD5991, A-1155463, ABT-199, and ABT-263 (0.01-1μM, 24h). Relative cell survival of (F) 1889c and (G) HCC15 after a 24h single treatment with AZD5991, A-1155463, ABT-199, and ABT-263. (H) IC50 concentrations of 1889c after a 24h combined treatment with AZD5991 (0.01-1μM) and A-1155463 (0.01μM, 0.025μM, 0.05μM), ABT-199 (0.01μM, 0.025μM, 0.05μM) and ABT-263 (0.1μM, 0.25μM, 0.5μM). (I) Dose-response matrix of 1889c for the combinational treatment with AZD5991 and A-1155463 and (J) for AZD5991 and ABT-263. (K) IC50 curves of 1889c after a 24h single treatment with AZD5991 and ABT-199 (0.01-1μM) and the combination of AZD5991 with ABT-199 (0.05μM). (L) Cell viability assay and (M) Western Blot analysis of 1889c cells treated for 24h with AZD5991 (0.5μM), A-1155463 (0.05μM), ABT-263 (0.5μM), and staurosporine (0.25μM) alone or after 3h pretreatment with the pan-caspase inhibitor zVAD-fmk (100μM). Figure S3. qPCR analysis and BH3 profiling of patient tissue. (A) Correlation analysis of IHC staining of BCL-xL with the relative copy number of BCL-xL (n=32, 5 A, 13 AB, 1 B1, 5 B2, 4 B3, and 4 TC). (B) Kaplan-Meier analysis showed no significant impact of BCL-xL copy number on patient OS. (C-G) BH3-profiling of primary patient tissue of healthy thymus (NT), two type B3 (TH-1 and 2), one type B2 (TH-3), and one type AB (TH-4) TH. (H-K) BH3-profiling of primary patient tissue of healthy lung (NL) and three SCCL samples. Table S1. Clinicopathological parameters of primary patient tissue samples. Table S2. Antibodies and chemical compounds. Table S3. Primer sequences, plasmids, and siRNAs.