Journal: bioRxiv
Article Title: Combined inhibition of AIF/CHCHD4 interaction and GLS1 to exploit metabolic vulnerabilities in pediatric osteosarcoma
doi: 10.64898/2026.04.03.716303
Figure Lengend Snippet: a , AIF/CHCHD4 co-immunoprecipitation using CHCHD4 antibody to immunoprecipitate whole protein fraction from HOS cells treated with DMSO or with mitoxantrone during 48h. Immunoprecipitation was followed by Western blotting with AIF and CHCHD4 immunostaining. IgG immunoprecipitated sample was used as negative control. b , Representative Western blot analysis of AIF-siRNA (siAIF) treatment over time (left panel), with siGAPDH as positive control. Based on this, AIF was silenced 48h before exposure to increasing concentrations of mitoxantrone. Vinculin was used as loading control. Cell viability is measured using an LDH assay. Dose-response curves show cell viability at increasing mitoxantrone concentrations after 72h of treatment (n = 3). Data were shown as mean ± standard deviation (SD). c , Western blot analysis showing the expression levels of AIF, CHCHD4 and their substrates Cox17 and MICU1 in response to either 0.0005% DMSO as a negative control or IC 50 of mitoxantrone treatment over time (6, 24, and 48h). Vinculin and VDAC were used as loading controls. Data are presented as the mean ± SD from four independent experiments (n = 4). Statistical significance was assessed using ANOVA with Sidak’s correction for multiple comparisons. Significance levels are indicated as follows: not significant (ns), p < 0.0332 (*), p < 0.0021 (**), p < 0.0002 (***), and p < 0.0001 (****). d , Western blot analysis of mitochondrial electron transport chain complexes in osteosarcoma cell lines HOS and U2OS treated with either 0.0005% DMSO (control) or IC 50 mitoxantrone during 48h. Vinculin was used as loading control. e , Representative transmission electron microscopy images of HOS cells treated with either 0.0005% DMSO as negative control or with IC 50 of mitoxantrone for 48h, showing that the compound induced cristolysis and changes in mitochondrial ultrastructure. m: mitochondrion f , ATP levels were semi-quantified over time in HOS cells pre-treated with either 0.0005% DMSO as a negative control or with IC 50 mitoxantrone for 6, 24, and 48h, using the ATPlite assay. Data are presented as the mean ± SD from four independent experiments (n = 4). Statistical significance was assessed using ANOVA with Sidak’s correction for multiple comparisons. Significance levels are indicated as follows: not significant (ns), p < 0.0332 (*), p < 0.0021 (**), p < 0.0002 (***), and p < 0.0001 (****). g , Seahorse XFe96 Mito Stress Test graph displaying the oxygen consumption rate (OCR; pmol/min/1.000 cells) over time in HOS cells pre-treated with either 0.0005% DMSO as a negative control or with IC 50 mitoxantrone for 24, 48, and 72h (left). The test was conducted using the following compounds: oligomycin (2.5 µM), Carbonyl cyanide-p-trifluoromethoxyphenylhydrazone (FCCP) (1 µM), and rotenone/antimycin A (0.5 µM). Basal respiration and proton leak values were extrapolated from the kinetic graph. OCR measurements were normalized to cell counts determined by nuclei DAPI staining. Data represent the mean ± SD of three independent experiments, each with at least six technical replicates. Statistical analysis was performed using ANOVA with Sidak’s correction for multiple comparisons, with significance levels indicated as follows: ns, not significant; p < 0.0332 (*); p < 0.0021 (**); p < 0.0002 (***);p < 0.0001 (****). h , Mitochondrial membrane potential assessed by flow cytometry using 100 nM TMRM fluorescent probe labelling for 20 min. HOS cells were analyzed at different time points following mitoxantrone treatment. Oligomycin and FCCP were used as positive controls for membrane potential modulation, and unstained cells served as negative controls for gating. Four independent experiments were performed (n = 4). Data represent the mean±SD. Statistical significance was assessed using ANOVA with Sidak’s multiple comparison test correction, with significance levels indicated as follows: ns, not significant; p < 0.0332 (*); p < 0.0021 (**); p < 0.0002 (***);p < 0.0001 (****). i , Histamine-induced mitochondrial Ca 2+ uptake in osteosarcoma cells measured using the fluorescent probe Rhod-2 AM. Cells were treated with 0.0005% DMSO (control) or IC 50 mitoxantrone for 24h, loaded with Rhod-2 AM (4 µM, 30 min) and Hoechst 33342 for nuclear counterstaining, and imaged using a Cytation 1 reader (Agilent). Baseline fluorescence was recorded for 5 min (interval = 2 s) before stimulation with histamine (100 µM) in the presence of extracellular CaCl 2 (200 mM), and changes in Rhod-2 fluorescence were monitored for an additional 30 min. Mean fluorescence intensity was normalized to Hoechst-positive nuclei. Data are presented as the mean ± SD from four independent experiments (n = 4). Statistical significance was assessed using an unpaired two-tailed Student’s t-test. Significance levels are indicated as follows: not significant (ns), p < 0.0332 (*), p < 0.0021 (**), p < 0.0002 (***), and p < 0.0001 (****).
Article Snippet: To knock down AIF expression for evaluating the specificity of mitoxantrone, HOS cells were seeded at densities of 12.500, 18.750, 25.000, 31.250 cells/cm and transfected with ON-TARGETplus Human AIFM1 siRNA (Dharmacon, #L-011912-00-0005) or ON-TARGETplus GAPDH Control Pool (Dharmacon, #D-001830-10-05) for 24h, 48h, 72h and 96h, using 0.2μL, 0.16μL, 0.2μL and 0.08μL of DharmaFECT 1 Transfection Reagent (Dharmacon, #T-2001-03), respectively, according to the manufacturer’s instructions.
Techniques: Immunoprecipitation, Western Blot, Immunostaining, Negative Control, Positive Control, Control, Lactate Dehydrogenase Assay, Standard Deviation, Expressing, Transmission Assay, Electron Microscopy, Staining, Membrane, Flow Cytometry, Comparison, Fluorescence, Two Tailed Test
