Additional file 1 of Targeting of mitochondrial fission through natural flavanones elicits anti-myeloma activity
Creators: name: Torcasio, Roberta
name: Gallo Cantafio, Maria Eugenia
name: Veneziano, Claudia
name: De Marco, Carmela
name: Ganino, Ludovica
name: Valentino, Ilenia
name: Occhiuzzi, Maria Antonietta
name: Perrotta, Ida Daniela
name: Mancuso, Teresa
name: Conforti, Filomena
name: Rizzuti, Bruno
name: Martino, Enrica Antonia
name: Gentile, Massimo
name: Neri, Antonino
name: Viglietto, Giuseppe
name: Grande, Fedora
name: Amodio, Nicola
Publisher: name: figshare
Year: 2024
Subjects: subject: Biochemistry
subject: Cell Biology
subject: Molecular Biology
subject: Physiology
subject: FOS: Biological sciences
subject: Pharmacology
subject: Chemical Sciences not elsewhere classified
subject: Developmental Biology
subject: Cancer
subject: Hematology

description: Additional file 1: Figure S1. Hes and Nar content in Tacle® extract after 7-week storage under controlled ambient conditions (1 ℃ and 90–95% RH). Values are expressed as means ± SD on three different measurements. Figure S2. Interaction of Hes and Nar with key amino acid residues of the Drp1 active site. Ligand, Structure, Pose, Binding Energy and Interactions (Hydrogen and hydrophobic bonds) of both Hes and Nar compounds are reported; docking score values are expressed in kcal/mol. Figure S3. WB analysis of MFN2 protein levels in AMO cells, 48h after treatment with increasing doses of Hes or Nar. Normalization was performed using GAPDH as loading control. Figure S4. Representative traces of respiration and O2 concentration in H929 cells, 48h after treatment with A) Hes (250µM) or (B) Nar (250µM). O2 flux per volume in the two chambers was measured simultaneously by Oroboros O2k Instrument, performing consecutive injections of Oligomycin (2µM), FCCP (0.5µM) and Antimycin A (2µM), according to SUIT 003 D009 protocol. Traces were analyzed by DatLab 7 software. Figure S5. Half-maximal inhibitory concentration (IC50) of Hes (left) and Nar (right) determined for AMO, AMO-BZB, H929, H929-BZB, and H929-CFZ cell lines. The IC50 were calculated using GraphPad Prism software from three independent experiments. Figure S6. FACS analysis of Annexin V/7-AAD stained AMO and AMO-BZB cells exposed for 48h to different concentrations of (A) Hes or (B) Nar. Representative dot plots of the data from an independent biological replicate (n=3) are shown. Figure S7. Fluorescence microscopy analysis of lipid droplets in AMO cells, 24hafter Hes (250µM) or Nar (250µM) treatment, labeled with BODIPY 493/503 probe. Representative images are reported (40x magnification). Histogram bars reported the number of lipid droplets ± SD in at least 100 cells from three different fields. *p&lt;0.05. Figure S8. Representative traces of respiration and O2 concentration in H929 cells, 48h after treatment with Tacle (2mg/ml). O2 flux per volume in the two chambers was measured simultaneously by Oroboros O2k Instrument, performing consecutive injections of Oligomycin (2µM), FCCP (0.5µM) and Antimycin A (2µM), according to SUIT 003 D009 protocol. Traces were analyzed by DatLab 7 software. Figure S9. WB analysis of S616 p-Drp1 and Drp1 (A), ATF4, IRE1α (B), Caspase 3 (C) and DGAT2 (D) in lysates from AMO cells exposed for 48h to Tacle extract. GAPDH was used as protein loading control. E) Fluorescence microscopy analysis of lipid droplets in AMO cells, 24h after Tacle extract (2 mg/ml) exposure, labeled with BODIPY 493/503 probe. Representative images (40x magnification) are reported. Histogram bars represent the number of lipid droplets ± SD in at least 100 cells from three different fields. *p&lt;0.05. Figure S10. A) Fluorescence microscopy analysis of lipid droplets in AMO cells stably expressing DNM1L, or the empty vector, labeled with LipidTOX Red probe, 24h after Hes (250µM), Nar (250µM) or Tacle extract (2 mg/ml) exposure. Representative images (100x magnification) are reported. Histogram bars reported the number of lipid droplets ± SD in at least 100 cells from three different fields. *p&lt;0.05. B) Triglycerides measurement using Triglycerides Glo assay in DNM1L overexpressing AMO cells, 24h after Hes (250µM), Nar (250µM) or Tacle extract (2 mg/ml) exposure. Luminescence was evaluated at 48h. *p&lt;0.05. Figure S11. H&amp;E staining (20x magnification) of vital kidney, spleen, liver and heart tissue retrieved from mice, two weeks after treatments. Representative images are reported.