Suppl Figure 1-6, Suppl Table S0 and Suppl Methods from A Unique Morphological Phenotype in Chemoresistant Triple-Negative Breast Cancer Reveals Metabolic Reprogramming and PLIN4 Expression as a Molecular Vulnerability
Authors: Sirois Isabelle , Aguilar-Mahecha Adriana , Lafleur Josiane , Fowler Emma , Vu Viet , Scriver Michelle , Buchanan Marguerite , Chabot Catherine , Ramanathan Aparna , Balachandran Banujan , Légaré Stéphanie , Przybytkowski Ewa , Lan Cathy , Krzemien Urszula , Cavallone Luca , Aleynikova Olga , Ferrario Cristiano , Guilbert Marie-Christine , Benlimame Naciba , Saad Amine , Alaoui-Jamali Moulay , Saragovi Horace Uri , Josephy Sylvia , O'Flanagan Ciara , Hursting Stephen D. , Richard Vincent R. , Zahedi René P. , Borchers Christoph H. , Bareke Eric , Nabavi Sheida , Tonellato Peter , Roy Josée-Anne , Robidoux André , Marcus Elizabeth A. , Mihalcioiu Catalin , Majewski Jacek , Basik Mark 
Published: April 2023

<p>Supplementary Figure 1 a. Doxorubicin resistance of DOXO-R C8 cells was further validated using the cell viability assay (SRB) following 48 hrs treatment with DOXO, n=4 independent experiment, 3 replicates/experiment. **p<0.01 vs parental. b DOXO-R cells are not cross resistant to cisplatin. Cell viability measured by alamar blue following treatment with cisplatin for 48hrs, n=4 independent experiment, 3 replicates/experiment. c ABCB1 mRNA levels are not increased in DOXO-R cells compared to parental cells. Gene expression data showing LOG2FC values of C1, C8, CD and CF colonies compared to parental cells for ABCB1 gene. LOG2FC do not reach significance, i.e. LOG2FC >0.6 and pval<0.05. d DOXO-R cells do not express more ALDH+ve cells than parental cells. % of ALDH+ve cells quantified by flow cytometry, n=3 independent experiments, *p<0.05 vs parental. e Doubling time (hrs) of parental (P) and DOXO-R C1 and C8 cells measured by alamar blue, n=4 independent experiment, 3 replicates/experiment. *p<0.05 vs parental, n.s. = not significant. f Light microscopy of parental PTX selected colonies (PTX-CD and PTX-CF) showing that PTX colonies are different from parental cells but similar to DOXO-R C1 and C8 with neuron-like projections described in Fig. 2a. g Electron micrographs of PTX-CD and CF cells showing increased LDs (left panel, arrows), neuron-like projections (middle panel, arrow heads) and cytoplasmic regions filled with numerous small mitochondria (right panel, white circles), similar to DOXO-R C1 and C8 cells described in Fig. 2a. h Phalloidin staining showing PTX-CF cells but not parental cells exhibit PGCCs with neuron-like projections as shown in DOXO-R C1 and C8 cells in Fig. 2b. Scale bars 10, 5 and 2 µm. For a-d, data represent means +/- SEM. Student t-test was done for c-e. For a and b, two-way anova analysis with multiple comparison test (Dunnet) was performed. Supplementary Figure 2. Example of DOXO-R PGCC with neuron-like projection at high cell confluence visible by light microscopy at lower magnification (4x). Supplementary Figure 3. Omics analysis of chemoresistant TNBC cells and tumors. Supplementary Figure 4. a,b PPARG and PLIN4 mRNA quantification by RT-PCR following transient silencing of PPARG using 2 siRNAs (2 and 5) and siRNA ctrl in DOXO-R cells (n=3 independent experiment, *p<0.05, **p<0.01). Supplementary Figure 5. KM plots. Supplementary Figure 6. a,b Cell viability of MDA-MB-436 and CRC T786 (5000 cells/well) measured by alamar blue following treatment with DOXO in F-medium for 72hrs (n{greater than or equal to}3). Supplementary Table S0. Summary of the molecular characteristics identifying the DOXO-R phenotype in chemoresistant TNBC cells and tumors.</p>