cell lines u2os cells atcc htb  (ATCC)

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

Journal: Nature Communications

Article Title: Versatile and sensitive detection of mono- and poly(ADP-ribosyl)ation reveals XRCC1-dependent remodelling of PARP1 signalling

doi: 10.1038/s41467-026-71311-4

Figure Lengend Snippet: A Immunoblot analysis of in vitro ADP-ribosylated PARP1 with (mono-ADPr) or without (poly-ADPr) PARG using Poly-/ Pan-/ Mono-ADPr antibodies. Detection was done in parallel resulting in comparable signal intensities. Red colour represents saturated signal. N = 3. B Dot-blot analysis of in vitro auto-poly-ADPr of PARP1 in 1:2 dilution series using Poly-/ Pan-ADPr antibodies. Detection was done in parallel resulting in comparable signal intensities. Red colour represents saturated signal. N = 3. C Immunoblot analysis of in vitro ADP-ribosylated PARP1 with (Ser-ADPr) or without (Asp/Glut-ADPr) HPF1 using Poly-ADPr antibodies. Detection was done in parallel resulting in comparable signal intensities. N = 3. D Immunoblot analysis of SDS cell extracts from 2 mM H2O2-treated wild-type (WT) U2OS cells with and without 1 μM Olaparib treatment with the indicated antibodies. Detection was done in parallel resulting in comparable signal intensities. Red colour represents saturated signal. N = 3. E Immunoblot analysis of SDS cell extracts from untreated wild-type (WT) hTERT RPE1 cells with and without 1 μM Olaparib treatment for 1 h with the indicated antibodies. Detection was done in parallel, resulting in comparable signal intensities. N = 3. F Immunofluorescent staining of mono-/ poly-/ pan-ADPr in 2 mM H2O2-treated WT U2OS cells with and without 1 μM Olaparib treatment using the indicated antibodies. Detection was done on individual basis, signal intensities between different antibodies are not comparable. Signals are normalised to WT untreated conditions. Error bars represent SEM. Combined analysis of 3 Biological Replicates. Representative Images from 1 Biological Replicate. Scale bar, 10 μM. Source data are provided as a Source Data file.

Article Snippet: U2OS cell lines were obtained, authenticated by STR profiling and confirmed mycoplasma-free by ATCC cell line authentication service.

Techniques: Western Blot, In Vitro, Dot Blot, Staining

Journal: Nature Communications

Article Title: Versatile and sensitive detection of mono- and poly(ADP-ribosyl)ation reveals XRCC1-dependent remodelling of PARP1 signalling

doi: 10.1038/s41467-026-71311-4

Figure Lengend Snippet: A Schematic illustration of the affinity maturation process. Illustrations generated in Adobe Illustrator. Schematics adapted from Dauben et al. . B ELISA analysis of antibody specificities using the indicated antibodies (2 μg/mL) and biotinylated peptides (61 nM). Bars represent the arithmetic mean of 3 independent experiments. Error bars represent SD. C Left: Dot Blot analysis of isolated RNA from U2OS cells before and after glucose starvation. Right: Enzymatic digestion control of isolated RNA. Detection was done in parallel, resulting in comparable signal intensities. N = 3. D Immunoblot analysis of SDS cell extracts from 2 mM H2O2-treated wild-type (WT) U2OS cells with and without 1 μM Olaparib treatment with the indicated antibodies. Detection was done in parallel, resulting in comparable signal intensities. Red colour represents a saturated signal. N = 3. E Immunofluorescent staining of mono-ADPr in 2 mM H2O2-treated WT U2OS cells with and without 1 uM Olaparib treatment using the indicated antibodies. Detection was done in parallel, resulting in comparable signal intensities. Signals are normalised to WT untreated conditions. Error bars represent SEM. Combined Analysis of 3 Biological Replicates. Representative Images from 1 Biological Replicate. Scale bar, 10 μM. F Immunoblot analysis of H2SO4 cell extracts from 2 mM H2O2-treated wild-type (WT) U2OS cells with and without 1 μM Olaparib treatment with H3S10/S28-ADPr site-specific antibodies. Detection was done in parallel, resulting in comparable signal intensities. N = 3. G Immunoblot analysis of SDS cell extracts from 2 mM H2O2-treated wild-type (WT) U2OS cells with and without 1 μM PARG inhibition treatment with the indicated antibodies. Detection was done in parallel, resulting in comparable signal intensities. Red colour represents a saturated signal. N = 3. Source data are provided as a Source Data file.

Article Snippet: U2OS cell lines were obtained, authenticated by STR profiling and confirmed mycoplasma-free by ATCC cell line authentication service.

Techniques: Generated, Enzyme-linked Immunosorbent Assay, Dot Blot, Isolation, Control, RNA Detection, Western Blot, Staining, Inhibition