Extended Data Fig. 4: MCU deficiency does not affect canonical autophagy.
a-e, Confocal imaging (a and d) and quantification (b and e) of the colocalization of Zymosan (red) with either LC3B puncta (green) (a and b) or LysoTracker (green) (d-e), immunoblotting of LAP-associated molecules in isolated phagosomes or total cell lysates from Mcu fl/fl and Mcu ∆mye BMMs in the presence of LLO (5 nM) (c). Scale bar, 2 µm. f-i, Immunofluorescence staining of LC3B in Mcu fl/fl and Mcu ∆mye BMMs (f and g), or THP-1 MCU-WT and MCU-KO cells (h and i) left untreated or challenged with EBSS for 2 h, or rapamycin (100 nM) for 16 h. LC3B puncta per cell (g and i) were shown. Scale bar, 2 μm. j-l, Immunoblotting of LC3B and p62 in Mcu fl/fl and Mcu ∆mye BMMs (j), or THP-1 MCU-WT and MCU-KO cells (k) left untreated or pretreated with bafilomycin (50 nM) for 1 h, followed by EBSS or rapamycin (100 nM) incubation for another 2 or 16 h, respectively. Immunoblotting of AMPK and mTOR signaling molecules AKT, S6K and S6 in Mcu fl/fl and Mcu ∆mye BMMs challenged with L. monocytogenes (MOI, 10) for indicated periods (l). The averages of n = 3 biologically independent samples are shown. The error bars represent the SEM. Statistical significance was determined using t test (and nonparametric tests).
