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Advanced Neurology TRPM7 signaling in glioblastoma

A B C

D E F

Figure 5. CsA and naltriben co-treatment affects AKT and ERK phosphorylation levels similarly to CsA-only treatment. Cells were treated with vehicle,
CsA, and/or naltriben (10 or 25 µM, respectively) 24 h before protein collection for Western blot. Optic density was analyzed with ImageJ. (A) Representative
images and (B and C) statistical analysis of p-AKT and t-AKT levels. One-way ANOVA and Tukey post hoc test were used to assess significant differences
(**p < 0.01 compared to control; #### p < 0.0001 compared to 25 µM naltriben group; n = 7/group). (D) Representative images and (E and F) statistical
analysis of p-ERK and t-ERK levels. One-way ANOVA and Tukey post hoc test were used to assess statistical significance (no significance between any
groups; n = 4/group). Note that although there were trends toward reduction in the 10 µM CsA and co-treatment groups when compared to the control,
we did not observe significance (p > 0.05).
Abbreviations: CsA: Cyclosporine A; ANOVA: Analysis of variance.

human TRPM7 protein and reverse IP experiments to
precipitate endogenous or overexpressed can. Importantly,
reciprocal co-immunoprecipitation assay should be
employed to validate the interaction between Flag-TRPM7
and the calcineurin A-subunit. Furthermore, investigators
can employ approaches such as PLA assay or confocal
microscopy to examine the subcellular dispositions of
TRPM7 and calcineurin. Finally, potential mediators
of this interaction can be investigated using in silico
protein docking simulations or affinity purification-mass
spectrometry approaches, potentially testing different
Figure 6. Graphical summary created with Biorender.
concentrations of Ca to investigate the Ca dependency
2+
2+
may mediate this binding since CaM has been shown to of this interaction.
interact with both TRPM7 and calcineurin [31,33] . Consistent The inhibition of either TRPM7 or calcineurin has
with a previous finding , we showed that calcineurin A previously been shown to decrease the rate of migration
[25]
was precipitated alongside His-CaM. However, TRPM7 and invasion of GBM cells [17,20,23] . The AKT and ERK
was not found in the pull-down protein mixture in the pathways, which play important roles in GBM function,
2+
absence of Ca (Figure 2). This suggested that either have been reported to act downstream of both TRPM7
Ca is crucial for this interaction, or calmodulin may and calcineurin [26,34] . Thus, in U251 cells, we applied the
2+
not be required for the TRPM7/calcineurin interaction. TRPM7 agonist naltriben together with the calcineurin
Nonetheless, there are limitations to our current inhibitor CsA to determine whether TRPM7 activation can
experimental approach. Future co-IP studies may consider reverse the CsA-induced reduction in GBM cell function
using anti-TRPM7 antibodies to precipitate endogenous and signaling. Consistent with previous findings, results

Volume 2 Issue 3 (2023) 7 https://doi.org/10.36922/an.334