Journal: Nature Communications
Article Title: Rapid and direct control of target protein levels with VHL-recruiting dTAG molecules
Figure Lengend Snippet: dTAG V -1 is an in vivo-compatible degrader of FKBP12 F36V -tagged proteins. a Immunoblot analysis of PATU-8902 FKBP12 F36V -KRAS G12V ; KRAS −/− clone treated with DMSO, dTAG V -1, or dTAG V -1-NEG for the indicated time-course. b Immunoblot analysis of 293T WT FKBP12 F36V -KRAS G12V or 293T VHL-/- FKBP12 F36V -KRAS G12V cells treated with DMSO or the indicated dTAG molecules for 24 h. Data in a , b are representative of n = 3 independent experiments. c DMSO-normalized antiproliferation of PATU-8902 LACZ-FKBP12 F36V or FKBP12 F36V -KRAS G12V ; KRAS -/- clones treated with dTAG V -1 or dTAG V -1-NEG for 120 h. Cells were cultured as ultra-low adherent 3D-spheroid suspensions. Data are presented as mean ± s.d. of n = 4 biologically independent samples and are representative of n = 3 independent experiments. d Bioluminescent imaging to evaluate degradation of luciferase-FKBP12 F36V in mice was performed daily as follows: day 0 to establish baseline signal, day 1–3 to monitor luciferase-FKBP12 F36V signal 4 h after vehicle or dTAG molecule treatment (T), day 4 to monitor duration of luciferase-FKBP12 F36V signal 28 h after third and final vehicle or dTAG molecule treatment. Total flux for each mouse is depicted. Data are presented from vehicle ( n = 5 biologically independent mice at day 0–4), dTAG-13 ( n = 5 biologically independent mice at day 0–3; n = 4 biologically independent mice at day 4) or dTAG V -1 ( n = 5 biologically independent mice at day 0–4) treated mice. P values are derived from a two-tailed Welch’s t -test (* P
Article Snippet: Cell lines The following cell lines were employed in this study: 293T (source: ATCC #CRL-3216, media: DMEM with 10% FBS and 1% Penicillin–Streptomycin), 293FT (source: Thermo Fisher Scientific #R70007, media: DMEM with 10% FBS and 1% Penicillin–Streptomycin), PATU-8902 (source: DSMZ #ACC-179, media: DMEM with 10% FBS and 1% Penicillin–Streptomycin), MV4;11 (source: ATCC #CRL-9591, media: RPMI with 10% FBS and 1% Penicillin–Streptomycin) and EWS502 (source: kindly provided by Dr. Stephen L. Lessnick of Nationwide Children’s Hospital and established by Dr. Jonathan A. Fletcher of Harvard Medical School, media: RPMI with 15% FBS and 1% Penicillin–Streptomycin-l -Glutamine).
Techniques: In Vivo, Clone Assay, Cell Culture, Imaging, Luciferase, Mouse Assay, Derivative Assay, Two Tailed Test