Fig. 4: SMART responds to necroptosis, but not apoptosis, in MEFs.

a, b primary MEFs (pMEFs) (a) and immortalized MEFs (iMEFs) (b) were untreated or stimulated with TBZ (10 ng/mL TNF, 0.5 μM BV6, 20 μM zVAD) for 12 h. MEFs were generated from SMART Tg mice, and MEFs with less than ten passages were used as pMEFs. Cell death was determined with the LDH release assay. Results are mean ± SD of triplicate samples. c Western blots of lysates from untreated iMEFs from Mlkl^−/− or wild-type mice, and untreated or IFNβ (2000 IU/mL)-treated pMEFs from SMART Tg mice. Blots were probed with anti-MLKL and anti-actin antibodies. d pMEFs were pretreated with IFNβ, and then stimulated with TBZ for 4 h. Cell viability was determined with the LDH release assay. Results are mean ± SD of triplicate samples. e–h pMEFs were untreated or stimulated with the indicated combination of agents (10 ng/mL TNF, 0.5 μM BV6, 20 μM zVAD, 5 μM GSK’872), and the FRET/CFP ratio was calculated. Pseudocolored images show cellular changes in the FRET/CFP ratio values in response to the indicated stimulations (e, g). FRET/CFP responses are color-coded according to the color scales (right). White arrowheads and white arrows indicate cells undergoing necroptosis (e) and apoptosis (g), respectively. Scale bars, 20 μm. Maximum changes of the FRET/CFP ratio are shown (f, h). Results mean ± SE (n = 10 cells per condition). Each dot indicates an individual cell. Statistical analyses were performed by the unpaired two-tailed Student’s t-test (a, b, d, h) or one-way ANOVA with Tukey’s multiple comparison test (f). All results are representative of at least two independent experiments.