atr kinase inhibitor ve 821  (MedChemExpress)

Journal: bioRxiv

Article Title: ATR facilitates the degradation of Api5 through the ubiquitin-proteasome pathway via FBXW2 to regulate apoptosis upon DNA damage

doi: 10.1101/2021.08.08.455545

Figure Lengend Snippet: ( A) MCF7 cells were treated with 50 J/m 2 UV and ATRi (20μM VE821 for 12 hrs) were lysed 12 hrs post UV and levels of Api5, pRPA and RPA were analysed using western blotting. (B) The fold change of Api5 was quantified after normalisation with GAPDH. (C) Immunofluorescence assay was performed on MCF7 cells treated with 50 J/m 2 UV and ATRi (20μM VE821 for 12 hrs) using Api5 specific antibody and (D) Api5 localisation was manually analysed. mCherry-tagged Api5 over-expressing MCF7 cells treated with 50 J/m 2 UV were lysed 12 hrs post treatment and immunoprecipitation was performed using (E) GFP and (F) ATR-specific antibody. Western blotting analysis was performed to analyse the presence of ATR and GFP respectively.

Article Snippet: Camptothecin (C9911), Etoposide (E1383), Protease inhibitor cocktail (P2714), dimethyl sulfoxide (DMSO), propidium iodide (P4170) Chloroquine (C6628), MG132 (M7449), Cyclohexamide (C1988), polybrene (Hexadimethrine bromide; H9268), thiazolyl blue tetrazolium (MTT; M5655) and dimethyl sulfoxide (DMSO; D8418) were purchased from Sigma-Aldrich; romidepsin (FK228, Depsipeptide) was from Seleck Chemicals, USA and the selective ATR kinase ATR inhibitor (VE 821) was purchased from Axon Medchem, USA.

Techniques: Western Blot, Immunofluorescence, Expressing, Immunoprecipitation

Journal: Frontiers in Cell and Developmental Biology

Article Title: ATR Kinase Is a Crucial Player Mediating the DNA Damage Response in Trypanosoma brucei

doi: 10.3389/fcell.2020.602956

Figure Lengend Snippet: Role of ATR kinase in unperturbed cell progression through the S phase. (A) Curve plots show the viability of T. brucei PCFs before and after RNAi induction for ATR silencing with tetracycline (Tet). The data represent the averages of three independent experiments, and error bars the standard deviations. (B) RT-PCR analysis for the relative quantification of ATR transcripts after gene silencing by RNAi induction with tetracycline. (C) Scheme shows the experimental strategy used for the detection of parasites that progress through the S phase by detecting the thymidine analogs incorporated in non-induced and induced cells for 48 h for ATR silencing. (D,E) Bar plots representing the percentage of cells in each group that progressed through the S phase in the non-induced or induced population for 48 h for ATR silencing. Bar plot graphs show the average of three independent experiments, each consisting of n = 300. The means of the percentage of intra-S cells in the induced and non-induced populations after ATR silencing were compared, and significant differences were determined by t- test. Significance values are shown as **** P ≤ 0.0001, *** P ≤ 0.001, ** P ≤ 0.01.

Article Snippet: Exponentially growing parasites were subjected to different concentrations of ATR kinase inhibitor (VE-821, from Sigma Aldrich) or ATM kinase inhibitor (KU55933, from Sigma Aldrich) to determine the optimal concentration of each kinase inhibitor, as indicated by its failure to impair long-term cell viability, by dose-response curves.

Techniques: Reverse Transcription Polymerase Chain Reaction

Journal: Frontiers in Cell and Developmental Biology

Article Title: ATR Kinase Is a Crucial Player Mediating the DNA Damage Response in Trypanosoma brucei

doi: 10.3389/fcell.2020.602956

Figure Lengend Snippet: Functions of ATR in cell progression through the S phase. (A–C) Experimental strategy used (left) and the quantification (right) of the parasites that progress through the S phase in the WT, ATR-silenced and ATR-inhibited populations after IR irradiation. Bar plots show the percentage of parasites in each group (cells leaving, entering or in the intra-S phase) and kinetics of thymidine analog incorporation over time for each population after IR irradiation. (A) WT population was exposed to IdU for 30 min and then irradiated with 50 Gy of IR. Then, the cells were collected at the indicated times and pulsed with CIdU for 30 min before each measurement time. (B) Cells engineered to silence ATR (ATR RNAi) were induced 48 h before being exposed to IdU for 30 min. Then, the cells were pulsed with CIdU as in (A) . (C) WT population was pulsed with IdU as in (A) . Then, the cells were irradiated and cultured in the presence of the ATR inhibitor VE-821 and collected at predetermined times after CIdU pulse as in (A) . The data represent the average of three independent experiments, each consisting of n = 300, and the error bars represent the standard deviations.

Article Snippet: Exponentially growing parasites were subjected to different concentrations of ATR kinase inhibitor (VE-821, from Sigma Aldrich) or ATM kinase inhibitor (KU55933, from Sigma Aldrich) to determine the optimal concentration of each kinase inhibitor, as indicated by its failure to impair long-term cell viability, by dose-response curves.

Techniques: Irradiation, Cell Culture