br Methods br Results We have

Methods
Results We have previously used ß-hex assays in HeLa tankyrase inhibitor to study the role of lysosomal exocytosis on transition metal clearance and the effects of transition metals on lysosomal exocytosis [26], [29], [40]. ß-hex is a lysosomal enzyme, the delivery of which to the extracellular medium is commonly used as a measure of lysosomal exocytosis. In the previous studies we performed a detailed analysis and verification of this process using LAMP1 exposure and siRNA for the components of the protein complex responsible for the lysosomal fusion with the plasma membrane [26]. Tert-Butyl hydroperoxide (tBHP), a stable form of hydrogen peroxide is commonly used to induce oxidative stress [48], [49], [50]. In our hands, tBHP changed the delivery of ß-hex activity to the extracellular medium bathing HeLa cells in a concentration-dependent manner. Fig. 1A shows that cells treated with 10–100μM tBHP for 1h increased the rate of the lysosomal exocytosis. In this set of experiments, untreated cells released 2.81±0.32% of their total ß-hex context (measured by lysing cells with CHAPS) into the medium during the 1h exposure. Cells treated with 50μM tBHP released 3.87±0.13% of total enzyme, while cells treated with 100μM tBHP released 4.20±0.09% of the total ß-hex (3 individual samples per condition per experiment, 6 experiments, p<0.05 for 10, 50 and 100μM tBHP). An independent series of experiments Fig. 1B shows that the tBHP-induced increase was eliminated by siRNA-mediated knockdown of VAMP7, which was implicated in the lysosomal exocytosis [51], [52] (3 measurements per condition per experiment, 6 experiments, p<0.05 for 10, 50 and 100μM tBHP, p<0.05 for all VAMP7-siRNA points). TRPML1 has been implicated in lysosomal exocytosis [32], [38]. Accordingly, TRPML1 activator ML-SA1 [32] stimulated lysosomal exocytosis (Fig. 1C, 3 measurements per condition per experiment, 3 experiments, p<0.05 for 10, 50 and 100μM ML-SA1). siRNA-mediated knockdown of TRPML1 eliminated the tBHP-dependent increase in the lysosomal exocytosis rate as well (Fig. 1D, 3 measurements per condition per experiment, 6 experiments, p<0.05 for 10, 50 and 100μM tBHP). TRPML1 mRNA levels following the knockdown are shown in Supplementary Fig. S1. These data suggest that (a) TRPML1 activation by oxidative stress contributes to the increase in ß-hex exocytosis in response to oxidative stress, and (b) such an increase occurs according to the common paradigm of lysosomal exocytosis: Ca2+ release through TRPML1 actuating or facilitating VAMP7-dependent fusion events. Interestingly, the trend of lysosomal exocytosis activation by oxidative stress reversed when tBHP concentration was raised over 200μM. Fig. 2A shows that while in cells treated with 100μM tBHP, ß-hex exocytosis averaged 180% of its levels in untreated cells (n=9, p<0.05), increasing tBHP concentration to 300μM dropped the exocytosis to about 100% of control cells. At 400μM tBHP ß-hex exocytosis averaged 60% of its levels in control cells (Fig. 2A, n=9, p<0.05). The total ß-hex cellular activity measured after the lysing of the cells using Triton X-100 did not show the same degree of suppression (Supplementary Fig. S2). The lysosomal exocytosis decline in response to oxidative stress was detected in the mouse primary cortical neuron cultures as well. In this set of experiments, ß-hex exocytosis rate in cells treated with 400μM tBHP averaged 67% of its levels in untreated cells (n=6, p<0.05) (Fig. 2B). We conclude that at high levels oxidative stress suppresses lysosomal exocytosis in both transformed and primary cells. As an additional treatment we used the mitochondrial uncoupler rotenone [53], [54], known to increase the mitochondrial electron-leak causing oxidative stress. Fig. 2B shows that rotenone suppressed levels of lysosomal exocytosis as well (72% of control levels, n=9, p<0.05). These tBHP and rotenone concentrations are routinely used to study the effects of oxidative stress in neuronal cultures in vivo and in vitro[19], [49], [50], [55].