Fig. 1: Mg²⁺ deficient diet decreases M1 macrophage cells and promote tumor growth.

Schematic overview of mice diet study, on day 0, mice were put on an Mg²⁺ deficient diet and an Mg²⁺ normal diet. On day 3, 3×10⁶ Cal27 cells were injected into the mice, and tumor growth was measured every week. On day 28, mice were sacrificed for tumor collection (A). Tumor volumes were measured using a digital caliper every 7 days using a Xenograft tumor model protocol. The data shown are representative of three independent experiments with similar results. Tumor volumes are depicted in the picture. Bar graphs depict average ± SD for relative values, NS = non-significant (Student’s t-test) (B). The pictures shown below are isolated solid tumors from mice, and (C) shows microscopy analysis showing H&E staining of tumors from the Mg²⁺ deficient diet and Mg²⁺ normal diet. D Flow cytometer analysis on nude mice tumors from Mg²⁺ deficient diet, and Mg²⁺ normal diet was performed to measure the levels of surface markers to demonstrate the efficacy of cell infiltration. Data show low arginase and CD206⁺ M2 markers and high CD80⁺ and iNOS M1 markers to illustrate successful M1 infiltration on Mg²⁺ normal diet and low levels of both markers when mice were given Mg²⁺ deficient diet. Bar graphs show the percentage of M1 and M2 markers (average ± SD for relative values, ***p ≤ 0.001 (Student’s t-test)) on different diets, from three independent experiments (E). Expression analysis showing the level of expression of arginase 1 and iNOS of mice placed on Mg²⁺ sufficient diet (normal) or Mg²⁺ deficient diet. F Representative whole-cell recording showing outward currents at +100 mV from macrophages isolated from tumor cells (n = 6) in mice placed on Mg²⁺ sufficient diet or Mg²⁺ deficient diet. IV curves (acquired when currents reach peak) under various conditions are also shown. A bar graph (average ± SE) showing the quantification of the current is also shown. G VGEF in tumors from the Mg²⁺ deficient diet and Mg²⁺ normal diet. The data shown are representative of three independent experiments. ***p ≤ 0.001. Bar graphs represent quantification ( ± SD) of tumor cytokines present from the Mg²⁺ deficient diet and the Mg²⁺ normal diet. The data shown are representative of three independent experiments. ***p ≤ 0.001, NS = non-significant (Student’s t-test). H Quantification (bar graphs ± SE) of serum Mg²⁺ levels in Mg²⁺ deficient diet and Mg²⁺ supplemented diet. ****p ≤ 0.005 (Student’s t-test).