Fig. 4. TRPM7 is a key mechanosensor that directs decision-making in channels of lower hydraulic resistance.

(A) Representative images of calcium signal intensity of (i) control, (ii) TRPM7-KO, and (iii) para-nitroblebbistatin–treated cells once they first reached and occupied the entire intersection of 1×-0.6×-2.2× devices. Following image segmentation, oversaturated pixels indicating high calcium intensity within vacuoles/organelles were blackened, and then each pixel’s intensity value was assigned a color according to ImageJ’s fire heat map for visualization purposes. The borders of the microchannels are depicted by thin white lines. (B) Normalized calcium signal intensity within each protrusion of MDA-MB-231 control, TRPM7-KO, and para-nitroblebbistatin-treated cells in branches of different hydraulic resistances (n = 10 cells from >3 independent experiments). Data represent the means ± SD. Kruskal-Wallis with post hoc Tukey was performed. *P < 0.05, ****P < 0.0001 relative to cells in 1× channel. n.s., not statistically significant relative to 1× channel. (C) Distribution pattern of MDA-MB-231 cells in branch channels of different hydraulic resistances in response to different pharmacological (BaptaAM, 2-APB, and FTY720) or molecular interventions (TRPM7 KO) as compared to the pattern predicted by the branch channel cross-sectional area partition (n > 70 cells from three independent experiments for each condition). Data represent the mean with 95% confidence interval. P < 0.05 for all treatments relative to controls as indicated by χ² test. (D) Phase contrast image of a Ψ-like trifurcating microfluidic device with branches of identical cross-sectional areas but distinct hydraulic resistances. The relative hydraulic resistance of each branch channel is indicated. Scale bar, 50 μm. (E) Distribution pattern of scramble control, TRPM7-KO, and MIIA-KD MDA-MB-231 cells in branch channels of the device shown in (D) (n > 70 cells from three independent experiments). Data represent the mean with 95% confidence interval. P < 0.05 for all treatments relative to controls as indicated by χ² test. (F) Phase contrast image of a Ψ-like trifurcating microfluidic device with branches of identical hydraulic resistances but different cross-sectional areas. The cross-sectional area of each branch channel is indicated. Scale bar, 50 μm. (G) Distribution pattern of scramble control, TRPM7-KO, and MIIA-KD MDA-MB-231 cells in branch channels of the device shown in (F) (n > 60 cells from three independent experiments). Data represent the mean with 95% confidence interval. P < 0.05 for all treatments relative to controls as indicated by χ² test.