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Fig. S1. The HlgC or HlgB subunits separately fail to induce glutamate release or free intracellular [Ca2+] changes in granular neurones.

a. Dose–response curves of glutamate released by granular neurones challenged for 10 min with the indicated concentrations of leukotoxin HlgC/HlgB (image), HlgC subunit alone (image) or HlgB subunit alone (image). The green line shows the glutamate measured after a 10 s pulse of 60 mM KCl depolarization.

b. averaged traces of records obtained in the presence of 2 nM HlgC/HlgB leukotoxin (51 cells, blue line), 2 nM HlgC subunit alone (60 cells, green line) and 2 nM HlgB subunit alone (31 cells, dark red line). The boxes show the distribution values of [Ca2+] peak amplitude (vertical box) and peak latency (horizontal box) from the control records.

Fig. S2. Confocal microscopy records of living granular neurones incubated in 12 μM Ethidium bromide and Leukotoxin HlgC/HlgB. Optical sections of 1.68 μm of cells bathing in HBSS containing Ethidium bromide (12 μM) were observed on a Leica SP5-II confocal microscope (fields of magnification × 63, initial pixel size 79.4 nm). Images of 1024 × 1024 pixels were acquired using 500–545 nm (green channel), 589–750 nm (red channel) and Nomarsky-DIC. Line 1: sample images after 3 min incubation in 12 μM Ethidium Bromide; 1A shows the living cells autofluorescent signal in the green channel, 1B is the Ethidium Bromide red fluorescence signal and 1C shows the recorded cells. Line 2 shows the same cells 15 min after the addition in the bath of HlgC/HlgB-Alexa-488 tagged leukotoxin (4 nM final); 2A green channel, 2B red channel and 2C Nomarsky-DIC image. Line 3 shows the same two cells after 20 more min incubation in 8 nM final concentration of HlgC/HlgB-Alexa-488 tagged; 3A green fluorescence, 2B Ethidium Bromide red fluorescence and 2C Nomarsky-DIC image.

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