Figure 4
From: A cellular screen identifies ponatinib and pazopanib as inhibitors of necroptosis
RIPK1 and RIPK3 are targets of ponatinib. (a) Cell viability was determined in HT-29 cells with doxycycline-inducible MLKL S358D expression treated overnight with 2 μg/ml doxycycline and 10 μM NSA, 10 μM Nec-1 or 0.5 μM ponatinib. Cell viability was assessed using a luminescence-based readout for ATP (CellTiter Glo). Data represent mean value±S.D. of three independent experiments performed in triplicates and normalized to untreated control. (b) Microscopy (brightfield, × 10) of HT-29 MLKL S358D cells induced with 2 μg/ml doxycycline overnight and treated with the compounds as indicated. Scale bar, 100 μm. (c) HT-29 cells were treated with 500 nM Smac mimetic, 20 μM z-VAD, 0.5 μM ponatinib (P) or DMSO (D) for a total of 4.5 h in the presence of TNF-α (10 ng/ml) for the time indicated. Cells were lysed and immunoblotted with the indicated antibodies. Data shown are representative of two independent experiments. (d) Direct binding assay for ponatinib and RIPK1. Data represent mean value±S.D. of two independent experiments. (e) In vitro kinase assay using recombinant RIPK3. Phosphorylation of MBP was monitored in presence of 10 μM of the kinase inhibitors stated or (f) ponatinib as indicated. Data represent mean value±S.D. of two independent experiments normalized to DMSO control. (g) CETSA performed in FADD-deficient Jurkat cells treated with 500 nM ponatinib (P) or DMSO (D) control. Cells were lysed by three freeze–thaw cycles and immunoblotted with the antibodies indicated. Data shown are representative of two independent experiments. (h) Quantification of band intensity (ImageJ) of RIPK3 and Tubulin immunoblots shown in g. (i) HT-29 cells with doxycycline-inducible expression of HA-tagged RIPK3 were treated with 0.5 μM ponatinib, 5 μM pazopanib, 10 μM Nec-1 or 10 μm NSA and stimulated overnight with 1 μg/ml doxycycline or for 2 h with 10 ng/ml TNF-α, 500 nM Smac mimetic and 20 μM z-VAD. Cells were lysed and immunoblotted with the indicated antibodies. Data shown are representative of two independent experiments. (j) HT-29 cells with doxycycline-inducible expression of HA-tagged MLKL were treated overnight with 1 μg/ml doxycycline in presence of 0.5 μM ponatinib, 5 μM pazopanib, 10 μM Nec-1 or 10 μM NSA. Cell lysates were subjected to immunoprecipitation and immunoprecipitates (IP) and whole cell extracts (WCE) were analyzed by immunoblotting with the indicated antibodies. Data shown are representative of two independent experiments
