Fig 2: MDMX overexpression partially reverses the inhibitory effect of TPEN but not TRPM7 inhibition on MCF-7 cell growth.A, TPEN treatment inhibits MCF-7 cell growth in a zinc-dependent manner. 5 × 104 MCF-7 cells were plated on 35 mm dishes. Twenty-four hours later, the cells were treated with DMSO, TPEN (5 μM), or TPEN plus ZnSO4 (5 μM each) for 24 h. Fresh DMEM medium was then added into each dish and replaced every other day. The cells were fixed and stained 5 days after treatment. B, NSC207895 further inhibits TPEN-repressed MCF-7 cell proliferation. Top panel: 1 × 104 MCF-7 cells were plated on 96-well plates and treated in triplicates with DMSO, TPEN (5 μM), NSC207895 (2.5 μM), or TPEN (5 μM) plus NSC207895 (2.5 μM) for 8 h. Cell proliferation was measured as described in Experimental procedures. The results were expressed as means ± sd of three independent experiments. The asterisk indicates statistical significance (p value <0.05). Bottom panel: MCF7 cells were treated with DMSO, TPEN (5 μM), NSC207895 (NSC; 5 μM), or TPEN (5 μM) plus increasing dosages of NSC207895 (NSC; 1, 2.5, or 5 μM) for 8 h. The cells were harvested and total cell lysates were prepared and subjected to immunoblotting with anti-MDMX and anti-actin antibodies. C, depletion of MDMX by siRNA enhances the inhibitory effect of TPEN on MCF-7 cell proliferation. MCF-7 cells were transfected with control siRNA or siRNA targeting MDMX (siMDMX). Eighteen hours later, the cells were trypsinized, counted, and plated (6 × 103 cells for each well) on 96-well plates in triplicates. After 24 h, cells were treated with DMSO or TPEN (5 μM) for 8 h. Cell proliferation was measured as described in Experimental procedures. The results were expressed as means ± sd of three independent experiments. The asterisk indicates statistical significance (p value <0.05). Knockdown of MDMX by siRNA in MCF-7 cells was confirmed by western blot analysis. D, MDMX overexpression partially rescues the inhibitory effect of TPEN on MCF-7 cells. Top and middle panel: 5 × 104 MCF-7 cells with tetracycline-inducible MDMX overexpression were plated on 6-well plate with or without 2.25 μg/ml of tetracycline and treated with DMSO or 5 μM TPEN for 24 h. Fresh medium was then added and replaced every other day. Five days later, the cells were fixed and stained. Relative colony formation capacity was quantified as described in Experimental procedures based on three independent experiments. The asterisk indicates statistical significance (p value <0.05). Bottom panel: MCF7 cells with tetracycline inducible MDMX overexpression were plated on 6-well plate with or without 2.25 μg/ml of tetracycline. Twenty-four hours later, the cells were treated with DMSO or 5 μM TPEN. 5 μM ZnSO4 was added in one of the TPEN-treated cells 5 h later. After another 5 h incubation, the cells were harvested and total cell lysates were analyzed by immunoblotting with indicated antibodies. E, knockdown of TRPM7 inhibits MCF-7 cell growth. MCF-7 cells were treated with control siRNA or siRNAs targeting TRPM7 (siTRPM7_1 and siTRPM7_2). Twenty-four hour after transfection, 2 × 104 cells were plated in 6-well plate and then fixed and stained 1 week later. F, MDMX overexpression does not rescue TRPM7 ablation-mediated cell growth inhibition in a tetracycline-inducible MDMX overexpression MCF-7 system. 5 × 104 MCF7 cells with tetracycline-inducible MDMX overexpression were plated on 6-well plate in triplicates with or without 2.25 μg/ml of tetracycline. Twenty-four hours later, the cells were transfected with control siRNA or siRNA targeting TRPM7 (siTRPM7_2). Fresh medium was added 6 h after treatment and replaced every other day. Five days later, the cells were fixed and stained with crystal violet (0.05% in 20% ethanol). Relative colony formation capacity was quantified as described in Experimental procedures based on three independent experiments. No statistical significance was observed. G, TRPM7 inhibitor NS8593 does not affect cell growth for tetracycline-inducible MDMX overexpression MCF7 cell system. 5 × 104 MCF7 cells with tetracycline-inducible MDMX overexpression were plated on 6-well plate per well in triplicates with or without 2.25 μg/ml of tetracycline in the absence or presence of NS8593 (30 μM). Medium was replaced every other day. Five days later, the cells were fixed and stained with crystal violet (0.05% in 20% ethanol). Relative colony formation capacity was quantified as described in Experimental procedures based on three independent experiments. No statistical significance was observed. H, TRPM7 inhibitor NS8593 does not affect cell growth for MDMX overexpression stable MCF7 cell system. 5 × 104 MCF7-pcDNA3 and MCF7-Myc-MDMX cells were plated on 6-well plate in triplicates in the absence or presence of NS8593 (30 μM). Medium was replaced every other day. Five days later, the cells were fixed and stained with crystal violet (0.05% in 20% ethanol). Relative colony formation capacity was quantified as described in Experimental procedures based on three independent experiments. No statistical significance was observed. MCF7-Myc-MDMX cells expressing higher levels of MDMX in comparison to MCF-pcDNA3 cells were confirmed by western blot analysis. All the immunoblotting experiments were repeated at least twice to ensure the reproducibility of the results.

Fig 3: The ion channel protein TRPM7 stabilizes ectopically expressed MDMX, induces the appearance of faster moving species of MDMX on SDS-PAGE, and regulates TPEN-induced MDMX degradation through its channel function.A, TRPM7 stabilizes ectopically expressed MDMX and induces the appearance of faster-moving species of MDMX on SDS-PAGE. HEK293T cells were transfected with combination of Myc-MDMX (0.15 μg), Flag-MDM2 (1 μg), and Flag-TRPM7 (1 μg) as indicated. Total cell lysates were analyzed 24 h after transfection. B, channel but not kinase function of TRPM7 is needed for TRPM7-induced appearance of faster-moving species of MDMX. HEK293T cells were transfected with Myc-MDMX (0.2 μg) and different Flag-TRPM7 variants (1 μg; wild-type, E1027Q, or K1646R) as indicated for 24 h (C) TRPM7 channel inhibitor NS8593 counteracts the effect of wild-type TRPM7 on MDMX stability and appearance of faster-moving species. HEK293T cells were transfected with Myc-MDMX (0.2 μg) and different Flag-TRPM7 variants (1 μg; wild-type, E1027Q, or K1646R) as indicated. Twenty-four hours after transfection, DMSO or NS8593 (2 or 10 μM) was added, and cells were incubated for another 12 h before harvesting. D, knockdown TRPM7 by siRNAs attenuates Zn2+-mediated recovery of MDMX in the presence of TPEN. MCF-7 cells were treated with control siRNA or two different siRNAs targeting TRPM7 (siTRPM7-1 and siTRPM7-2). Forty-eight hours later, the cells were treated with 5 μM TPEN for 5 h and then incubated with or without 5 μM ZnSO4 for another 5 h (E) TRPM7 channel inhibitor NS8593 attenuates Zn2+-mediated recovery of MDMX in TPEN-treated MCF-7 cells. MCF-7 cells were treated with 5 μM TPEN for 5 h and then 5 μM ZnSO4 was added in the absence of presence of 50 μM NS8593. The cells were harvested at 1, 3, or 5 h after ZnSO4 and NS8593 treatment. F, overexpression of TRPM7 facilitates Zn2+-mediated recovery of MDMX in TPEN-treated HEK293T cells. HEK293T cells were plated in 60 mm dishes and transfected with Flag-TRPM7 variants (2 μg). Twenty-four hours after transfection, cells were trypsinized and replated into 35 mm dishes. Twenty-four hours after replating, the cells were treated with DMSO, 4 μM, or 5 μM TPEN for 5 h. Then 5 μM ZnSO4 was added into one of TPEN-treated dish. One hour later, the cells were harvested and total cell lysates were prepared and subjected to immunoblotting with anti-Flag, anti-MDMX, and anti-GFP antibodies. G, TRPM7 modulates intracellular levels of Zn2+. HEK293T cells transfected with control siRNA or siRNA targeting TRPM7 (siTRPM7_2) (left panel; 50 nM each; 48 h after transfection) or HEK293T cells transfected with pcDNA3, Flag-TRPM7 (right panel; 5 μg each; 24 h after transfection), or MCF-7 cells (middle panel) were loaded with Zinpyr-1 (5 μM) for 30 min at 37 °C. Then the cells (for HEK293T, 4 × 104 cells; for MCF-7, 3 × 104 cells) were plated, treated as indicated, and the intracellular zinc levels were measured and calculated as described in Experimental procedures. The relative zinc levels were calculated by normalizing to the measurements of DMSO-treated samples. The results were expressed as means ± sd of three independent experiments. The asterisk indicates statistical significance (p value <0.05). All the immunoblotting experiments had been repeated at least twice to ensure the reproducibility of the results.