Fig. 4

Fig. 4

Figure 4. Nudt3 is a Zn²⁺-dependent polyPase in vivo

(A) Zn²⁺ decreases nuclear polyP content. HEK293T cells were grown with 100 μM Zn²⁺ in the medium and with the chelators EDTA or NaHS (200 μM) for 48 h. Polyphosphate was detected as in Figure 3A. Representative pictures are in the left panel. Quantification of at least 10 pictures from three independent experiments is in the right panel. A minimum of 500 cells per condition were analyzed. Mean ± SEM is provided.

(B) Mg²⁺ does not affect nuclear polyP content. HEK293T cells were grown in the presence of Zn²⁺ or Mg²⁺ (100 μM) for 24 and 48 h. Polyphosphate was detected, and representative pictures are in the left panel. Quantification of five pictures containing more than 200 cells per condition from one experiment is presented.

(C) Zn²⁺ controls polyP content in the brain cell line SH-SY5Y. Cells were grown in the presence of 100 μM Zn²⁺ for 24 and 48 h. Polyphosphate was detected, and representative pictures are in the left panel. Quantification of three pictures containing a minimum of 100 cells from one experiment.

(D) Zn²⁺ effect on polyP is dependent on Nudt3. Nudt3 was silenced in HEK293T cells and after 24 h, 100 μM Zn²⁺ was added. Polyphosphate was detected after 24 h. Representative pictures are in the left panel. Quantification of 15 pictures from three independent experiments is in the right panel. A minimum of 300 cells per condition were analyzed. Mean ± SEM is provided.

(E) Inositol pyrophosphate (InsP₇) is not affected by Zn²⁺. HEK293T cells were grown in the presence of 100 μM Zn²⁺ or Mg²⁺ for 24 and 48 h. Cells were treated with 10 mM NaF for 1 h to raise the InsP₇ intracellular levels and ensure its visualization in a 35% PAGE gel. InsP₆ was included as a size marker at the left line.

The scale bars represent 10 μm. NT, not treated. See also Figure S2. ∗∗∗p < 0.001; ∗∗p < 0.01; ∗p < 0.05.