Figure 1. MCU Has a Crucial Function in Phagosomal Killing of Fungal Pathogens.

(A) Schematic depicting the selection of siRNA targets for Ca²⁺ channel screen in myeloid cells.

(B) Schematic depicting the C. albicans killing experiment in RAW264.7 cells.

(C) Scatterplot showing Z scores of C. albicans killing in relationship to relative gene expression. Ion channel genes that were screened are shown as individual points. Z score calculations are described in STAR Methods. Normalized expression value is the average expression level across four macrophage populations (ImmGen database). Knockdown of Mcu (marked) resulted in a significant deficit in C. albicans killing relative control.

(D) Killing of C. albicans by macrophages transfected with control siRNA and Mcu-targeting siRNA. Individual replicates from the siRNA screen are shown (n = 4). Error bars represent SEM; p < 0.0001 according to Welch’s t test, two tailed.

(E) Schematic showing the breeding strategy used to generate Mcu(M)^−/− mice.

(F) Gene expression analysis (qPCR) of Mcu mRNA in WT (n = 6, mice) and Mcu^−/− (n = 6, mice) bone-marrow-derived macrophages (BMDMs). Error bars represent SEM; p < 0.0001 according to Welch’s t test, two tailed.

(G) Protein quantification from immunoblots of whole-cell lysates from WT (n = 7, mice) and Mcu^−/− (n = 7, mice) BMDMs. Error bars represent SEM; p = 0.0014 according to Welch’s t test, two tailed. See Figure S1I for immunoblots.

(H) Mitochondrial Ca²⁺ (mCa²⁺) uptake assay using mitochondria isolated from WT and Mcu^−/− BMDMs as reported by the reduction in extramitochondrial Ca²⁺ signal, after addition of 45 μM Ca²⁺ to the isolated mitochondria.

(I) Quantification of Ca²⁺ uptake in mitochondria isolated from WT (n = 4) and Mcu^−/− (n = 4) BMDMs. mCa²⁺ uptake is calculated as percentage of maximum. Error bars represent SEM; p < 0.0001 according to Welch’s t test, two tailed.

(J) mCa²⁺ uptake assay using digitonin-permeabilized BMDMs. 15 μM Ca²⁺ was added to 500,000 digitonin-permeabilized BMDMs in a 96-well plate, and Ca²⁺ Green-5N fluorescence was monitored (Excitation/Emission 505/535 every 2 s for 6 min). 5 μM FCCP was added as positive control to uncouple mitochondria and release Ca²⁺ at the end of each run.

(K) C. albicans killing by WT (n = 10) and Mcu^−/− (n = 10) BMDMs. Error bars represent SEM; p = 0.0015 according to Welch’s t test, two tailed.

(L) Killing of S. cerevisiae by WT (n = 10) and Mcu^−/− (n = 10) BMDMs. Error bars represent SEM; p < 0.0001 according to Welch’s t test, two tailed.

(M) LDH release by WT and Mcu^−/− BMDMs in response to C. albicans MOI1. LDH release is reported as a percentage of maximum release (black). Error bars represent SEM; no significant difference was detected between WT and Mcu^−/− at baseline (n = 4, each group) or 6 h (n = 6, each group) according to ordinary one-way ANOVA with multiple comparisons.

(N) LDH release by WT and Mcu^−/− BMDMs in response to zymosan. LDH release is reported as a percent of maximum release (black). Error bars represent SEM; no significant difference was detected between WT and Mcu^−/− at baseline (n = 4, each group) or 6 h (n = 6, each group) according to ordinary one-way ANOVA with multiple comparisons.