hos cells  (Revvity)

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, b , Kaplan-Meier overall survival analysis for patients with osteosarcoma, Ewing sarcoma and in the GSE42352, GSE17679 dataset, respectively. Rhabdomyosarcoma data set was collected from Missiaglia et al., 2012 . Kaplan-Meier curves for a, AIFM1 gene and b, CHCHD4 gene of low-expression versus high-expression groups based on median AIFM1 and CHCHD4 expression, respectively. Kaplan-Meier analysis and automatically computed expression cut-off were generated by R2: Genomics Analysis and Visualization Platform. c , Cell viability of 8 pediatric osteosarcoma cell lines treated with mitoxantrone. Dose-response curves represent cell viability measured using a LDH assay at increasing concentrations of mitoxantrone after 72h of treatment. Individual experiments are shown (n > 3). Data were shown as mean ± SD. IC 50 inhibitory concentration yielding 50% cell viability. 0.0005% DMSO was used as the solvent of mitoxantrone and as control for calculation of percentage of viability. d , Representative image of a 6-well plate showing fixed colonies subjected to crystal violet staining of HOS, HOS-R/DOXO, and HOS-R/MTX cells after treatment with either 0.0005 % DMSO as negative control or mitoxantrone at 5nM (IC 50 of HOS-R/MTX) during indicated time points. e , Cell viability of 11 secondary cultured in vitro PDX samples derived from refractory or relapsed tumors treated with different concentrations of mitoxantrone, was measured using an LDH assay. Dose-response curves represent cell viability at increasing concentrations of mitoxantrone after 72h of treatment and the IC 50 values were determined using GraphPad Prism software. Individual replicates are shown (n > 3). Data were shown as mean ± SD.

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: Expressing, Generated, Lactate Dehydrogenase Assay, Concentration Assay, Solvent, Control, Staining, Negative Control, Cell Culture, In Vitro, Derivative Assay, Software

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

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 , Volcano plot of metabolomic profiling data generated with MetaboAnalyst 6.0. Data were processed using the Statistical Analysis and Enrichment Analysis modules, with log 10 transformation applied for normalization. The thresholds were set at –log 10 (p-value) = 1 for statistical significance and log 2 (fold change) = 1 for differential abundance. b , Seahorse XF Long Chain Fatty Acid Oxidation Stress Test graph displaying the oxygen consumption rate (OCR; pmol/min/1.000 cells) over time in HOS cells. The test was conducted using the following compounds: etomoxir (16µM), oligomycin (2.5 µM), FCCP (1 µM), and rotenone/antimycin A (0.5 µM). The acute response to etomoxir is shown as the difference in OCR measured immediately before and after etomoxir injection, reflecting the contribution of long-chain fatty acid oxidation to mitochondrial respiration. Data represent the mean ± SD from four independent experiments. 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 (****). c , Enrichment analysis of metabolites that differ significantly between 0.0005% DMSO as negative control and IC 50 mitoxantrone-treated HOS cells following 72h of treatment. The enrichment ratio represents the observed number of metabolites in a specific metabolic pathway divided by the expected number. Metabolic pathways are ranked by p-value, with the most significant pathways at the top. d Relative abundance of amino acids showing significant alterations upon IC 50 mitoxantrone treatment compared to 0.0005% DMSO control. Data represent relative metabolite abundance expressed as normalized log 2 -transformed values (areacorrLog2Cen). Data represent the mean ± SD from four independent experiments. 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 (****). e-h , Analysis of different metabolites involved in TCA cycle (pyruvate/oxaloacetate, citrate/isocitrate, fumarate, and α-ketoglutarate) (e); glutathione metabolism (GSH, GSSG and GSH/GSSG ratio) (f); pyrimidine metabolism (uracil, thymidine, and 2-deoxycytidine) (g) purine metabolism (adenosine/guanosine and inosine) (h). Data represent relative metabolite abundance expressed as normalized log 2 -transformed values (areacorrLog2Cen). Data represent the mean ± SD from four independent experiments. 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-j , Mitoxantrone-induced increase in glutamate/glutamine ratio. Glutamate, total glutamate + glutamine, and glutamate/glutamine ratio of controls or mitoxantrone-treated HOS and U2OS cells. Cells were treated with IC 25 of mitoxantrone or DMSO as a negative control for 72h, and levels of glutamate and total glutamate + glutamine were semi-quantified using the Glutamine/Glutamate-Glo™ Assay (Promega, #J8021). Glutamate/glutamine ratio was calculated accordingly. 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 (****). k , Upregulation of glutaminase 1 (GLS1) after mitoxantrone treatment. Western blot analysis showing the expression levels of GLS1 in response to either 0.0005% DMSO as a negative control or IC 50 of mitoxantrone treatment over time (24, 48 and 72h). Vinculin was used as loading control. Data are presented as the mean ± SD from five independent experiments. 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 (****).

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: Metabolomic, Generated, Transformation Assay, Injection, Comparison, Negative Control, Control, Glo Assay, Two Tailed Test, Western Blot, Expressing

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 , Cell viability of 4 pediatric osteosarcoma cell lines HOS, U2OS, SAOS2, 143B treated with different concentrations of telaglenastat, measured using an LDH assay. Dose-response curves represent cell viability at increasing concentrations of telaglenastat after 72h of treatment. 0.0005% DMSO was used as the solvent of mitoxantrone and as control for calculation of percentage of viability. Data were shown as mean ± SD from > 2 individual replicates. b , Illustrative 2D and 3D maps with overall HSA synergy score for mitoxantrone/telaglenastat combination in the osteosarcoma cell lines U2OS, HOS, SAOS2, 143B, obtained using SynergyFinder tool ( https://synergyfinder.fimm.fi/ ). Cells were treated with varying concentrations of both drugs in a matrix form. Cell viability was measured using LDH assay and input onto the SynergyFinder platform. Red, white, and green areas correspond to synergism (score > 10), additivity (-10 < score < 10) and antagonism (score <-10), respectively. c , Effects of mitoxantrone/telaglenastat combination on nucleotide biosynthesis. Combination-treated (IC 25 mitoxantrone, IC 25 telaglenastat) cells were supplemented with nucleoside at 1x (30 μM each of cytidine, guanosine, uridine, adenosine and 10 μM thymidine) or 2x. Cell viability was measured using LDH assay. Data represent the mean±SD from four independent experiments. 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 (****). d , Involvement of AIF in mitoxantrone/telaglenastat synergism through selective AIF silencing and targeting nucleotide biosynthesis. HOS and U2OS cells were treated with siAIF for 48h prior to treatment with IC 25 of telaglenastat and addition of nucleoside at 1x (30 μM each of cytidine, guanosine, uridine, adenosine and 10μM thymidine) or 2x. Data represent the mean±SD from at least four independent experiments. 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 (****). e , Tumor volume curves of U2OS-engrafted mice treated with PBS + HPBCD (Vehicle; n = 10) or mitoxantrone + HPBCD (mono-mitoxantrone; n = 8) or PBS + telaglenastat (mono-telaglenastat; n = 8) or mitoxantrone + telaglenastat (combi-mitoxantrone/telaglenastat; n = 10). Data were shown as mean ± standard error of mean (SEM) and statistical significance was determined by Two-way ANOVA (mixed model). Significance levels are indicated as follows: ns, not significant; p < 0.0332 (*); p < 0.0021 (**); p < 0.0002 (***); p < 0.0001 (****). f , Representative in-vivo bioluminescence images of U2OS-Luc/mkate2-mice treated with PBS + HPBCD (Vehicle), mitoxantrone + HPBCD (mono-mitoxantrone), PBS + Telaglenastat (mono-telaglenastat), or mitoxantrone + telaglenastat (combi-mitoxantrone/telaglenastat).

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: Lactate Dehydrogenase Assay, Solvent, Control, Comparison, In Vivo