Additional file 1 of BETi enhance ATGL expression and its lipase activity to exert their antitumoral effects in triple-negative breast cancer (TNBC) cells
Creators: name: Rossi, Teresa
name: Zamponi, Raffaella
name: Chirico, Mattea
name: Pisanu, Maria Elena
name: Iorio, Egidio
name: Torricelli, Federica
name: Gugnoni, Mila
name: Ciarrocchi, Alessia
name: Pistoni, Mariaelena
Publisher: name: figshare
Year: 2023
Subjects: subject: Biophysics
subject: Biochemistry
subject: Cell Biology
subject: Genetics
subject: FOS: Biological sciences
subject: Molecular Biology
subject: Physiology
subject: Cancer
subject: Hematology

description: Additional file 1: Supplementary Fig. 1. A. CFSE analysis of MDA-MB231 and HS578t after 24–48-72 hours of 1uM JQ1 treatment. Fluorescence decay is relative to the fluorescence at 24 hours of treatment B. RT-qPCR analysis of RNA-seq selected genes validation in Hs578t after 6 h treatment with JQ1 1 μM. C. Left, representative flow cytometry curves of BODIPY 500/510 fluorescence signal in MDA-MB 231 and Hs578t after 24 hours of 1 μM BETi treatment Right, flow cytometry analysis of LDs content stained with BODIPY 500/510 in MDA-MB 231 and Hs578t cell lines after 6 and 24 h of 1 μM BETi treatment (MFI = median fluorescence intensity). Supplementary Fig. 2. A. NMR analysis of metabolite content in Hs578t after 24 hours of 1 μM BETi treatment. B. Protein expression analysis of ATGL in all BC cell lines for 24 hours with 1 μM of BETi. C. Cell proliferation assay of MDA-MB231 and Hs578t measured by IncuCyte after adding different concentrations of FAs. OA: Oleic Acid; PA: Propionic Acid. D. CFSE analysis of MDA-MB231 and HS578t after 24–48-72 hours of 100uM and 200uM of oleic acid (OA, left) or 1 mM and 5 mM of propionate (PA, right) treatment. Fluorescence decay is relative to the fluorescence at 24 hours of treatment. E. Percentage of cell death in MDA-MB231 and Hs578t cells treated with 3 days with ATGListatin and stained with Annexin V. Representative images are selected for western blot. bACT served as a loading control. Supplementary Fig. 3. A. Cell proliferation assay of MDA-MB231 and Hs578t measured by IncuCyte after silencing ATGL. B. Flow cytometry evaluation of LDs content stained with BODIPY 500/510 in MDA-MB231 and Hs578t after 48 and 72 hours of siRNA transfection (siCNT vs siATGL). C. Immunoblotting for ATGL and p21 in MDA-MB231 and Hs578t cells after 48 hours of siRNA transfection (siCNT vs siATGL). D. Protein expression analysis of CPT1a after a combination of siRNA approach (siATGL for 48 h) and BETi treatment (1 μM for the last 24 hours) in Hs578t. Representative images are selected for the western blot. bACT served as a loading control. Supplementary Fig. 4. A. Validation of RNA-seq analysis of MDA-MB231 treated for 6 hours with 1 μM of JQ1 (FoxO signaling pathway). B. Time-course (30 minutes to 24 hours) protein expression analysis of ATGL and FoxO1 in MDA-MB231 and Hs578t after 1 μM OTX-015 treatment. C. Protein expression analysis of FoxO1 in BT549 and MCF7 cell lines after 6 hours of 1 μM BETi. D. RT-qPCR analysis of FoxO1 and G0G2 in MDA-MB231 and Hs578t after 24 hours treatment with 1 μM of BETi. Representative images are selected for the western blot. bACT served as a loading control.