Bossi E, Zanella D, Gornati R, Bernardini G.

	Figure 1: Calcein as a cytoplasmic “metal detector”. (A): two electrode voltage clamp of a representative rDMT1 transfected oocyte; inward currents are induced by 100 μM MnCl2, FeCl2 and CoCl2 (Vh = −40 mV, pH 5.5). (B): means and standard errors of the transport currents obtained from 40 oocytes, five batches. (C–E): Plots of fluorescence decay (Ft/F0) with corresponding images of Calcein-injected oocytes (upper series: non-transfected (NT) and lower series: rDMT1 transfected) exposed to 100 μM MnCl2 (C), FeCl2 (D), and CoCl2 (E) at pH 5.5 from 3 to 10 oocytes, from 2 to 4 oocytes batches.
	Figure 2: Calcein quenching in oocytes exposed to cobalt NPs. (A): Representative image series of non-transfected (NT) Calcein-injected oocytes exposed to CoCl2 or different cobalt NPs for 0, 10, 20 and 30 min. (B): Means of the fluorescence decay of 5 to 25 oocytes (obtained from 2 to 5 different batches). Decay is expressed as the fluorescence intensity at time 30 min over fluorescence intensity at time 0 (F30/F0). Note that quenching is statistically significant in NT oocytes exposed to bare Co3O4 NPs and in rDMT1 expressing oocytes exposed to CoCl2 (positive control); moreover, the endocytosis blocker Dynasore does not change the quenching effect of Co3O4 NPs. Bars are ± SE; stars indicate a statistically significant (One-way ANOVA, P < 0.05) difference with non exposed oocytes.
	Figure 3: Endocytosis is not involved in NP quenching activity. In the inset, few fully grown oocytes are visible; note the presence of a pigmented pole (denominated animal pole) and of an unpigmented one (denominated vegetal pole). After fixation, a spherical cap is hand sliced with a razor blade, placed on a glass slide under a coverslip and observed with a 63X oil immersion objective from above. (A–C): Bright field (left) and the corresponding FITC filter (right) images of oocytes exposed to 1 mg/mL Lucifer Yellow CH. (A) control oocyte; (B) oocyte exposed to 0.1 mg/mL Co3O4 NPs and (C) oocyte incubated 24 h with 40 μM Dynasore and exposed to 0.1 mg/mL Co3O4 NPs. In bright field images, pigment granules, which are present in the cortex of the oocyte, are clearly visible as dark brown dots indicating that we are observing the oocyte animal pole. In the corresponding FITC filter images, no fluorescent vesicles are visible indicating no endocytotic activity.
	Figure 4: Two electrode voltage-clamp of Xenopus oocytes. Inward currents elicited by solutions and NP suspensions in NT (A) and rDMT1 transfected (B–D) representative oocytes. Oocytes were clamped at a holding potential of −40 mV and exposed to MnCl2 (1) and CoCl2 (2) solutions, to Co3O4 (3) and Co (4) NP suspensions and to their surnatants obtained at time 0 h (a) and 24 h (b). (E): Current mean values (±SE) obtained by subtracting from the current in the presence of the substrate, the current in its absence and normalizing to the Mn2+ current, a reference for divalent metal transporters in electrophysiological studies 6 to 12 oocytes from 4 batches.
	Figure 5: Measure of membrane resistence of Xenopus oocytes. Ramp (from −85 to + 55 mV) protocols (A) were applied in non transfected oocytes starting from the holding potential of −25 mV. Representative currents elicited by the protocol in not-exposed oocytes (black line in B), exposed to Co3O4 NP suspensions (red line in C) and perfused with ionophore A23187 (blue line in D). Current mean values at −80, −60, −40, −20, 0 and +20 mV (± SE) obtained from at least 12 oocytes (from 2 different batches) are plotted in (E).
	Figure 1. Calcein as a cytoplasmic “metal detector”.(A): two electrode voltage clamp of a representative rDMT1 transfected oocyte; inward currents are induced by 100 μM MnCl2, FeCl2 and CoCl2 (Vh = −40 mV, pH 5.5). (B): means and standard errors of the transport currents obtained from 40 oocytes, five batches. (C–E): Plots of fluorescence decay (Ft/F0) with corresponding images of Calcein-injected oocytes (upper series: non-transfected (NT) and lower series: rDMT1 transfected) exposed to 100 μM MnCl2 (C), FeCl2 (D), and CoCl2 (E) at pH 5.5 from 3 to 10 oocytes, from 2 to 4 oocytes batches.
	Figure 2. Calcein quenching in oocytes exposed to cobalt NPs.(A): Representative image series of non-transfected (NT) Calcein-injected oocytes exposed to CoCl2 or different cobalt NPs for 0, 10, 20 and 30 min. (B): Means of the fluorescence decay of 5 to 25 oocytes (obtained from 2 to 5 different batches). Decay is expressed as the fluorescence intensity at time 30 min over fluorescence intensity at time 0 (F30/F0). Note that quenching is statistically significant in NT oocytes exposed to bare Co3O4 NPs and in rDMT1 expressing oocytes exposed to CoCl2 (positive control); moreover, the endocytosis blocker Dynasore does not change the quenching effect of Co3O4 NPs. Bars are ± SE; stars indicate a statistically significant (One-way ANOVA, P < 0.05) difference with non exposed oocytes.
	Figure 3. Endocytosis is not involved in NP quenching activity.In the inset, few fully grown oocytes are visible; note the presence of a pigmented pole (denominated animal pole) and of an unpigmented one (denominated vegetal pole). After fixation, a spherical cap is hand sliced with a razor blade, placed on a glass slide under a coverslip and observed with a 63X oil immersion objective from above. (A–C): Bright field (left) and the corresponding FITC filter (right) images of oocytes exposed to 1 mg/mL Lucifer Yellow CH. (A) control oocyte; (B) oocyte exposed to 0.1 mg/mL Co3O4 NPs and (C) oocyte incubated 24 h with 40 μM Dynasore and exposed to 0.1 mg/mL Co3O4 NPs. In bright field images, pigment granules, which are present in the cortex of the oocyte, are clearly visible as dark brown dots indicating that we are observing the oocyte animal pole. In the corresponding FITC filter images, no fluorescent vesicles are visible indicating no endocytotic activity.
	Figure 4. Two electrode voltage-clamp of Xenopus oocytes.Inward currents elicited by solutions and NP suspensions in NT (A) and rDMT1 transfected (B–D) representative oocytes. Oocytes were clamped at a holding potential of −40 mV and exposed to MnCl2 (1) and CoCl2 (2) solutions, to Co3O4 (3) and Co (4) NP suspensions and to their surnatants obtained at time 0 h (a) and 24 h (b). (E): Current mean values (±SE) obtained by subtracting from the current in the presence of the substrate, the current in its absence and normalizing to the Mn2+ current, a reference for divalent metal transporters in electrophysiological studies 6 to 12 oocytes from 4 batches.
	Figure 5. Measure of membrane resistence of Xenopus oocytes.Ramp (from −85 to + 55 mV) protocols (A) were applied in non transfected oocytes starting from the holding potential of −25 mV. Representative currents elicited by the protocol in not-exposed oocytes (black line in B), exposed to Co3O4 NP suspensions (red line in C) and perfused with ionophore A23187 (blue line in D). Current mean values at −80, −60, −40, −20, 0 and +20 mV (± SE) obtained from at least 12 oocytes (from 2 different batches) are plotted in (E).

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