u2os cell lines  (ATCC)

Journal: The Journal of Biological Chemistry

Article Title: Biosensor-guided discovery of peptide inhibitors targeting the ribosomal protein uS5-PDCD2 chaperone interaction

doi: 10.1016/j.jbc.2026.111415

Figure Lengend Snippet: Inhibition of the uS5-PDCD2 interaction using residues 21 to 50 of uS5. A , Western blot analysis of total extracts (Input, Top ) and anti-Flag purifications (IP:Flag, Bottom ) prepared from HeLa cells that stably expressed Flag-PDCD2 and that were previously transfected with constructs encoding either GFP alone (lane 1), wild-type (WT, lane 2) or F29Y (MT, lane 3) versions of uS5 21-50 -GFP. The blots were analyzed for GFP, Actin, uS5, and Flag-PDCD2. B , quantification of relative uS5 levels copurified from anti-Flag precipitates normalized to Flag-PDCD2 levels. Values were expressed relative to the GFP control vector, which was set to 1.0. The data and error bars represent the average and SD from at least three independent experiments. p -value is indicated, as determined by a one-way ANOVA with Dunnett’s multiple comparisons test. ( C ) U2OS cells that conditionally express Flag-PDCD2 were transfected with either GFP control vector (panels a-d), wild-type (panels e-h) or F29Y (panels i-l) versions of uS5 21-50 -GFP. At the time of transfection, doxycycline was added to the media to induce the expression of Flag-PDCD2. 48 h post-transfection, cells were fixed and simultaneously analysed by direct fluorescence (b, f and j) and immunostaining for Flag-PDCD2 (c, g and k). DNA staining with DAPI shows the nucleus of each cell (a, e and i). Scale bars correspond to 20 μm. D , quantification of nucleus-to-cytoplasmic ratios of Flag-PDCD2 shows a significant decrease in cells that expressed the wild-type version of uS5 21-50 -GFP. More than 60 cells were analyzed for each condition from three independent immunofluorescence experiments. Statistical differences were calculated using a one-way ANOVA with Dunnett’s multiple comparisons test. p -value is indicated. E , Peptide sequences corresponding to residues 21 to 50 of uS5, showing conserved phenylalanines in blue (WT) and substitutions to alanine residues in the mutant in red (MT). F , schematic of the peptide competition experiments using purified uS5-GFP/PDCD2 complex. See text for details. G , Western blot analysis of an uS5-GFP immunoprecipitate that were washed, divided, and treated with increasing concentrations of either wild-type ( Top , WT) or mutant ( Bottom , MT) uS5 21-50 peptides (lanes 2–5) or with no peptide (lane 1). Blots were analyzed for uS5-GFP and endogenous PDCD2. ( H ) Quantification of PDCD2 levels copurified from anti-GFP precipitates normalized to uS5-GFP levels. The values were then set to 1.0 for the control purification in the absence of peptide. Solid lines mark the average binding from three independent replicates, with error bars corresponding to standard deviations. Statistical differences were calculated using a one-way ANOVA with Dunnett’s multiple comparisons test. p -value is indicated.

Article Snippet: Human MOLT-4, HEK293T, HeLa, and U2OS cell lines were acquired from ATCC.

Techniques: Inhibition, Western Blot, Stable Transfection, Transfection, Construct, Control, Plasmid Preparation, Expressing, Fluorescence, Immunostaining, Staining, Immunofluorescence, Mutagenesis, Purification, Binding Assay

Journal: Clinical Cancer Research

Article Title: A Novel Potent and Selective GCN2 Inhibitor, APL-4098, Has Antileukemic Activity through Dysregulation of Mitochondrial Function

doi: 10.1158/1078-0432.CCR-25-1444

Figure Lengend Snippet: APL-4098 is a potent inhibitor of GCN2. A, APL-4098 molecular structure. B, Crystal structure of APL-4098 bound to human GCN2. Backbone of the GCN2 catalytic site (blue ribbon) and binding of APL-4098, with the extensive interaction network in the ATP site shown with dashed red lines (hydrogen bonds) and yellow lines (hydrophobic contacts). C, Biochemical, ADME, and PK profile of APL-4098. *All data generated from a rat intravenous/oral study: 1 mg/kg i.v. and 3 mg/kg orally. 1 Data generated from the i.v. study and 2 data generated from the oral study. D, APL-4098 inhibition of GCN2 kinase activity, measured as the level of eIF2α phosphorylation, using LanthaScreen FRET-based assay. Plot shows mean ± SEM from three representative experiments conducted in duplicate. E, U2OS cells were treated with APL-4098 in the presence or absence of borrelidin as indicated for 4 hours. Cell lysates were analyzed for phosphorylation of eIF2α using HTRF assay. Plot shows mean ± SEM from four individual experiments conducted in duplicate. F, U2OS cells were grown in glutamine-depleted medium and treated with APL-4098 as indicated for 4 hours. Cell lysates were analyzed by Western blotting.

Article Snippet: The osteosarcoma cell line U2OS (HTB-96, RRID: CVCL_0042) was obtained from ATCC and cultured in DMEM high glucose (Gibco, #41965-039) with 10% FBS (Gibco, #10270-106), 1 mmol/L sodium pyruvate (Gibco, #11360-039), 1% nonessential amino acids (Gibco, #11140-035), and antibiotics (penicillin/streptomycin 100 U/mL and 100 μg/mL; Gibco, #15140-122).

Techniques: Binding Assay, Generated, Inhibition, Activity Assay, Phospho-proteomics, HTRF Assay, Western Blot

Journal: Clinical Cancer Research

Article Title: A Novel Potent and Selective GCN2 Inhibitor, APL-4098, Has Antileukemic Activity through Dysregulation of Mitochondrial Function

doi: 10.1158/1078-0432.CCR-25-1444

Figure Lengend Snippet: APL-4098 is a selective inhibitor of GCN2. A, Eurofins KINOMEScan selectivity panel assay Treespot results for APL-4098 tested at 1 μmol/L. B, Eurofins biochemical K d concentration–response assay. C, U2OS cells were treated with APL-4098 or positive control dabrafenib (bioRxiv 2024.08.14.607626) in the presence of BtdCPU for 4 hours. Cell lysates were analyzed for phosphorylation of eIF2α using HTRF assay. Plot shows mean ± SEM from three individual experiments conducted in duplicate. D, U2OS cells were treated with APL-4098 or positive control GSK2606414 in the presence of thapsigargin for 4 hours. Cell lysates were analyzed for phosphorylation of eIF2α using HTRF assay. Plot shows mean ± SEM from three individual experiments.

Article Snippet: The osteosarcoma cell line U2OS (HTB-96, RRID: CVCL_0042) was obtained from ATCC and cultured in DMEM high glucose (Gibco, #41965-039) with 10% FBS (Gibco, #10270-106), 1 mmol/L sodium pyruvate (Gibco, #11360-039), 1% nonessential amino acids (Gibco, #11140-035), and antibiotics (penicillin/streptomycin 100 U/mL and 100 μg/mL; Gibco, #15140-122).

Techniques: Concentration Assay, Positive Control, Phospho-proteomics, HTRF Assay