Fig. 6: MLKL re-expression partially restores necroptotic sensitivity and adipocyte differentiation in 3T3-L1 cells.

Experimental setup: Functional analyses were performed on 3T3-L1 preadipocytes genetically ablated for Mlkl (Mlkl-KO). Transient MLKL re-expression was achieved using nucleofection method. a Western blot analysis of MLKL protein level following transient re-expression in Mlkl-KO cells via nucleofection. b Necroptotic sensitivity restored in transiently re-expressing Mlkl-KO cells upon stimulation with TNF, BV6 (IAP inhibitor), and ZVAD (pan-caspase inhibitor). c Phase contrast images of 3T3-L1 control cells from induction (day 0) to ten days post-induction. Day 0 corresponds to five days after transfection, when cells had reached two-day post-confluence period and differentiation cocktail was added. Throughout differentiation, control cells undergo morphological changes and accumulate lipid droplets. In contrast, only a few cells appear differentiated in the 3T3-L1 Mlkl-KO following MLKL re-expression (Scale bar, 50 µm). d Western blot analysis of MLKL expression in Mlkl-KO cells re-expressing MLKL at days 1, 7 and 15 days post-nucleofection. e Western blot analysis of MLKL protein level during differentiation (day 0, day 5, and day 10) in control 3T3-L1, Mlkl-KO cells re-expressing MLKL and Mlkl-KO cells. f Immunofluorescence staining of MLKL and Lipid staining (LipidTox Neutral red) in control, Mlkl-KO and Mlkl-KO re-expressing MLKL cells after induction of differentiation. Images were acquired using a ×40 objective. (Scale bar, 20 µm). g Relative mRNA expression of Fabp4 in WT cells, Mlkl-KO cells, and Mlkl-KO cells re-expressing MLKL, as determined by RT-qPCR. Results are expressed as mean ± SD. Statistical analysis: statistical differences were analyzed using multiple t tests. Normality was verified with the Shapiro–Wilk test and homoscedasticity with the F-test. *p < 0.05; **p < 0.01; ***p < 0.001.