Fig. 6: Metabolic rescue of increased RIPK1 activation in NEK1 deficient cells.

a Nek1^+/+ and Nek1^Kat2J/Kat2J MEFs were pre-treated with 20 mM acetyl-l-carnitine for 18 h and then treated with1 ng/mL TNF + 500 nM 5z7 to induce RDA for 3, 4, and 6 h. Cell death was measured by ToxiLight. Mean ± SD, n=3 biological independent samples. One-way ANOVA with Dunnett’s test. b Nek1^+/+ and Nek1^Kat2J/Kat2J MEFs were pre-treated with indicated concentrations of sodium octonate for 18 h and then treated with 1 ng/mL TNF + 500 nM 5z7 to induce RDA for 6 h. The % of cell death was measured by ToxiLight. Mean ± SD. n = 3 biological independent repeats. One-way ANOVA with Dunnett’s test. c–e The sizes of lysosomes in Nek1^+/+ and Nek1^Kat2J/Kat2J MEFs were determined with a biomarker LAMP1 by immunostaining (c) and quantified with ImageJ for sizes (d) and density (e). n = 6 cells per group. Bar = 10 µm.Data are presented as mean ± SEM. Unpaired two-tailed Student’s t-test. f, Whole-cell lysates of Nek1^+/+ and Nek1^Kat2J/Kat2J MEFs were analyzed by western blotting with indicated antibodies. Uncropped blots in the Source Data file. g Nek1^+/+ and Nek1^Kat2J/Kat2J MEFs were stained with 50 nM MitoTracker-Green for 30 min and imaged by fluorescent microscopy. The length of mitochondria was quantified with ImageJ. Mean ± SD, n = 12 cells for each group. Bar = 10 µm. Unpaired two-tailed Student’s t-test. h, Nek1^+/+ and Nek1^Kat2J/Kat2J MEFs were treated with indicated concentration of acetyl-l-carnitine for 24 h, and then treated with 50 nM MitoTracker-Green for 30 min. The images were acquired by confocal microscopy. The average branch lengthes of mitochondria were quantified with ImageJ (n = 5 cells per group). The data are presented as mean ± SD. One-way ANOVA with Dunnett’s test. Bar = 10 µm. i WT and Nek1^Kat2J/Kat2J MEFs were treated with 0, 1 mM, 5 mM, 10 mM, and 20 mM acetyl-l-carnitine for 20 hours, then lysed with SDS buffer. Uncropped blots in the Source Data file. j Nek1^+/+ and Nek1^Kat2J/Kat2J MEFs were pre-treated with 20 mM acetyl-l-carnitine for 18 h and then treated with 10 ng/mL TNF + 50 nM SM164 + 20 µM zVAD.fmk for indicated periods of time. The cell lysates lysed with NP40 buffer and with 6 M urea were analyzed by western blotting using indicated abs. Uncropped blots in the Source Data file.