adherent atcc hek293 cells  (ATCC)

Journal: Journal of Medicinal Chemistry

Article Title: Development of BromoTag: A “Bump-and-Hole”–PROTAC System to Induce Potent, Rapid, and Selective Degradation of Tagged Target Proteins

doi: 10.1021/acs.jmedchem.1c01532

Figure Lengend Snippet: Design and development of a heterozygous knock-in BromoTag-Brd2 HEK293 cell line. (A) Design of the knock-in construct used in the development of the CRISPR construct. (B) FACS single cell sort of HEK293 cells based on GFP expression. Successive single cells were sorted into individual wells of a 96-well plate. (C) Junction PCR using genomic DNA of an expanded GFP-expressing clone paired against parental HEK293. (D) Western blot demonstrating the selectivity of the polyclonal Brd4BD2 L387A. antibody.

Article Snippet: The HEK293 human embryonic kidney adherent cell line (ATCC, Manassas, VA, USA) was cultured in Dulbecco’s modified Eagle’s medium (DMEM) (Invitrogen, Carlsbad, CA, USA) supplemented with 10% (v/v) fetal bovine serum (FBS) (Thermo Fisher, Waltham, MA, USA) and 1% (v/v) penicillin/streptomycin (pen/strep) (#15140122, Thermo Fisher, Waltham, MA, USA) at 37 °C, 5% CO 2 , and 95% humidity.

Techniques: Knock-In, Construct, CRISPR, Expressing, Western Blot

Journal: Journal of Medicinal Chemistry

Article Title: Development of BromoTag: A “Bump-and-Hole”–PROTAC System to Induce Potent, Rapid, and Selective Degradation of Tagged Target Proteins

doi: 10.1021/acs.jmedchem.1c01532

Figure Lengend Snippet: First-generation IBET-762-based B&H–PROTACs are inactive against BromoTag-Brd2 due to the proposed steric clash in the MZ1-like ternary complex. (A) Western blot data for BET protein levels after the treatment of PROTAC over 6 h in heterozygous BromoTag-Brd2 HEK293 cells. (B) Alignment of the ternary complex between Brd4 BD2 (green, surface representation), MZ1 ( 1 , stick, gray carbons), and VHL (cyan, surface representation) (PDB code: 5T35), with ET ( 6 , 8-OMe, stick, pink carbons, 4QEW) and 9-ME-1 ( 7 , 9-OMe, stick, blue carbons, 5O3C) to show the potential clash with VHL by the bulkier 8/9-methoxyphenyl group. His110 is highlighted (stick, cyan carbons).

Article Snippet: The HEK293 human embryonic kidney adherent cell line (ATCC, Manassas, VA, USA) was cultured in Dulbecco’s modified Eagle’s medium (DMEM) (Invitrogen, Carlsbad, CA, USA) supplemented with 10% (v/v) fetal bovine serum (FBS) (Thermo Fisher, Waltham, MA, USA) and 1% (v/v) penicillin/streptomycin (pen/strep) (#15140122, Thermo Fisher, Waltham, MA, USA) at 37 °C, 5% CO 2 , and 95% humidity.

Techniques: Western Blot

Journal: Journal of Medicinal Chemistry

Article Title: Development of BromoTag: A “Bump-and-Hole”–PROTAC System to Induce Potent, Rapid, and Selective Degradation of Tagged Target Proteins

doi: 10.1021/acs.jmedchem.1c01532

Figure Lengend Snippet: Biological evaluation of second-generation B&H–PROTACs in BromoTag-Brd2 HEK293 cells. Western blot data for BET protein levels monitored from 10 μM to 1 nM compound treatment over 6 h in heterozygous BromoTag-Brd2 HEK293 cells. Bands are normalized to tubulin and negative control ( cis -MZ1) to derive DC 50 values that enable the rank order of each PROTAC.

Article Snippet: The HEK293 human embryonic kidney adherent cell line (ATCC, Manassas, VA, USA) was cultured in Dulbecco’s modified Eagle’s medium (DMEM) (Invitrogen, Carlsbad, CA, USA) supplemented with 10% (v/v) fetal bovine serum (FBS) (Thermo Fisher, Waltham, MA, USA) and 1% (v/v) penicillin/streptomycin (pen/strep) (#15140122, Thermo Fisher, Waltham, MA, USA) at 37 °C, 5% CO 2 , and 95% humidity.

Techniques: Western Blot, Negative Control

Journal: Journal of Medicinal Chemistry

Article Title: Development of BromoTag: A “Bump-and-Hole”–PROTAC System to Induce Potent, Rapid, and Selective Degradation of Tagged Target Proteins

doi: 10.1021/acs.jmedchem.1c01532

Figure Lengend Snippet: Biological evaluation of AGB1 ( 46 ), AGB2 ( 47 ), and AGB3 ( 48 ) in BromoTag-Brd2 HEK293 cells. (A) Western blot data for BET protein levels monitored from 10 μM to 1 nM compound treatment over 6 h in heterozygous BromoTag-Brd2 HEK293 cells. (B) Time course western blot data of Brd2 levels in heterozygous BromoTag-Brd2 HEK293 cells upon 500 nM treatment of 46 and 47 and 1 μM treatment of 48 over 36 h. (C,D) Plots to calculate (C) DC 50 and ( D ) t 1/2 values for compounds enabling determination that AGB1 is the best choice for further validation. Western blots from (A,B) were normalized to tubulin and compared to a vehicle control (DMSO) to derive pDC 50 or t 1/2 values that enable rank order of each PROTAC.

Article Snippet: The HEK293 human embryonic kidney adherent cell line (ATCC, Manassas, VA, USA) was cultured in Dulbecco’s modified Eagle’s medium (DMEM) (Invitrogen, Carlsbad, CA, USA) supplemented with 10% (v/v) fetal bovine serum (FBS) (Thermo Fisher, Waltham, MA, USA) and 1% (v/v) penicillin/streptomycin (pen/strep) (#15140122, Thermo Fisher, Waltham, MA, USA) at 37 °C, 5% CO 2 , and 95% humidity.

Techniques: Western Blot, Biomarker Discovery, Control

Journal: Journal of Medicinal Chemistry

Article Title: Development of BromoTag: A “Bump-and-Hole”–PROTAC System to Induce Potent, Rapid, and Selective Degradation of Tagged Target Proteins

doi: 10.1021/acs.jmedchem.1c01532

Figure Lengend Snippet: Cellular mechanistic characterization of AGB1 ( 46 ) degradation activity. (A) Western blot illustrating the on -target degradation activity of 46 is dependent on the activity of CRL2 VHL and proteasome and on BromoTag target engagement. BromoTag-Brd2 HEK293 cells were treated with 200 nM 46 (3 h) following pretreatment (1 h) with the proteasome inhibitor MG132, neddylation inhibitor MLN4924, VHL inhibitor VH298, or BromoTag inhibitor ET-JQ1-OMe or DMSO vehicle. (B) Western blots demonstrating the recovery of BromoTag-Brd2 post-removal of 200 nM 46 after a 3 h treatment in heterozygous BromoTag-Brd2 HEK293 cells. Control experiments for no-wash and vehicle treatments are included. Bands are normalized to tubulin protein levels and compared to a vehicle control (DMSO) to quantify the final protein levels of BromoTag-Brd2. (C) Effect on antiproliferation of 46 compared to MZ1 and non-degrader controls 52 and cis -MZ1. Staurosporine was used as a positive control for cytotoxicity. MV-4-11, 22Rv1, and HEK293 cells were treated with varying concentrations of compound, and after 24, 48, and 48 h, respectively, the cells were subjected to the Promega CellTiter-Glo cell viability assay. The pEC 50 values (±S.E.M) are mean from N = 2 for MV-4-11 and 22Rv1 cells and N = 3 for HEK293 cells from data normalized from vehicle control (DMSO).

Article Snippet: The HEK293 human embryonic kidney adherent cell line (ATCC, Manassas, VA, USA) was cultured in Dulbecco’s modified Eagle’s medium (DMEM) (Invitrogen, Carlsbad, CA, USA) supplemented with 10% (v/v) fetal bovine serum (FBS) (Thermo Fisher, Waltham, MA, USA) and 1% (v/v) penicillin/streptomycin (pen/strep) (#15140122, Thermo Fisher, Waltham, MA, USA) at 37 °C, 5% CO 2 , and 95% humidity.

Techniques: Activity Assay, Western Blot, Drug discovery, Control, Positive Control, Viability Assay

Journal: Journal of Medicinal Chemistry

Article Title: Development of BromoTag: A “Bump-and-Hole”–PROTAC System to Induce Potent, Rapid, and Selective Degradation of Tagged Target Proteins

doi: 10.1021/acs.jmedchem.1c01532

Figure Lengend Snippet: Proteomics of AGB1 ( 46 ) and cis -AGB1 ( 52 ) treated heterozygous BromoTag-Brd2 HEK293 cells. Scatterplot depicting the effect of 46 (blue) and 52 (red) treatment on the proteome of heterozygous BromoTag-Brd2 HEK293 cells treated with 1 μM of compound for 2 h. Brd2 expression is highlighted for both treatment conditions. The data plotted is log 2 of the normalized fold change in abundance against −log 10 of the p value per protein identified from TMT mass spectrometry analysis produced from three independent experiments.

Article Snippet: The HEK293 human embryonic kidney adherent cell line (ATCC, Manassas, VA, USA) was cultured in Dulbecco’s modified Eagle’s medium (DMEM) (Invitrogen, Carlsbad, CA, USA) supplemented with 10% (v/v) fetal bovine serum (FBS) (Thermo Fisher, Waltham, MA, USA) and 1% (v/v) penicillin/streptomycin (pen/strep) (#15140122, Thermo Fisher, Waltham, MA, USA) at 37 °C, 5% CO 2 , and 95% humidity.

Techniques: Expressing, Mass Spectrometry, Produced