Figure S1Kinetics of ATP and LDH release from RAW264.7 cells induced by 1.5 µM Mt-II in mKRB, with or without an ecto-ATPase inhibitor, ARL67156 (100 M) or of EGTA (1.5 mM). Values are mean ± SD; n =4.

Expression profiling of the purinergic receptors in RAW264.7 cells. Bars represent expression values of purinergic receptors in the three control replicates reported in GEO DataSet Record GDS3549 (Bok et al. 2009).

Figure S2

Figure S3N13 cells are sensitive to Mt-II intoxication similarly to macrophages. Cells were incubated with Mt-II in mKRB medium (see experimental section) for one hour, and cell vitality was determined. Values are mean ± SD; n =4-5.

Table S1Anion channel inhibitors and purinergic antagonists protect N13 cells from Mt-II intoxication

Inhibitor [M] Target Protection (%) SD Inhibitor [M] Target*Protection

(%)SD

Flufenamic ac 10

Pannexins and connexins

none Suramin 10 P2X, P2Y; Gα protein 99 2***

Niflumic acid 10 none NF-449 10 P2X1,1+5,2+3,3,2,4; Gsα 96 4***

Carbenoxolone 100 none NF-023 20 P2X1,3,2,4; Go/iα 82 3***

Mefloquine 30 none PPADS 10 P2X1,2,3,5; P2Y2,4 98 3***

DIDS 30

Maxi-anion channel and VSOR

45 3** BBG 20 P2X7,2,4,1,3 85 4***

NPPB 100 16 5* RB2 20 P2X2,1,2+3; P2Y12,1, 6 97 4***

SITS 100 30 2** Clopidogrel 20 P2Y12,13,GRP17 52 3**

Gd3+ 50Maxi-an ch

96 3*** MRS2211 30 P2Y13,1,12 98 3***

Phloretin 100 VSOR 28 5** MRS2500 30 P2Y1 none

O-ATP 300 P2X7 91 5*** MRS2395 30 P2Y1>>P2Y13 none

5-BDBD 30 P2X4 none MRS2578 20 P2Y6>>P2Y1,2,4,11 20 9*

Protection of N13 cells from Mt-II induced cytotoxicity by inhibitors of ATP channels, of the purinergic receptors. Inhibitor concentration that was non toxic to cells was used. Data reported are the average values obtained in a minimum of four independent experiments. Statistically significant differences were determined via a one-tailed Student’s t test (*p < 0.05; **p < 0.01;***p>0.001).

Figure S4N13 cells intoxicated with Mt-II show a rapid peak of cytosolic [Ca2+], followed by a more extended increase which precedes cell bursting, occurring randomly on the plate. J744 cells show a similar behaviour (not shown). N13 cells have been pre-loaded with fura-2 and treated with 3.6 M Mt-II (arrow). The effect on [Ca 2+ ] was followed as a change in the fura-2 fluorescence ratio (340/380 nm) in the cells that were identified with different colored ROIs in the inset. The panel is representative of experiments carried out under the same conditions in at least four different cell preparations. Video S4 shows gray scale fluorescence images (ratio 340/380 nm, acquisition 1 frame/10 seconds.

Figure S5ATP (4 mM) induces on N13 cells synchronized spikes of cytosolic [Ca2+], followed by a prolonged but lower rise. J744 show a similar behaviour (not shown). N13 cells were pre-loaded with fura-2 and treated with 4 mM ATP (arrow). The effect on [Ca2+] was followed as a change in the fura-2 fluorescence ratio (340/380 nm) in the cells that were identified with different colored ROIs in the inset. The panel is representative of experiments carried out under the same conditions in at least four different cell preparations. Video S5 shows gray scale fluorescence images (ratio 340/380 nm, 1 frame/10 seconds).

Figure S6 ARAW cells intoxicated with Mt-II (1.5 M in mKRB) in presence of annexin-V-fluorescein. Membrane blebs induced by intoxication with Mt-II (at least 10 minutes after intoxication) show exposition of phosphatidylserine, as evidenced by the green fluorescence (central panels). Phosphatidylserine exposition induced by the toxin comes before cell membrane rupture, as tested by the lack of staining with propidium iodide (right panel), except for one cell. All experiments were repeated at least three times.

Figure S6 BControl of the experiment reported in Figure S6 A, the same volume of mKRB, but without the toxin, was added to RAW cells treated with annexin-V-fluorescein/propidium iodide.