Fig. 2. TRPM7 is the key fluid shear sensor, which mediates the reversal of fibroblast migration direction.

(A and B) Fluo-4 Direct fluorescence in vehicle control, 2-aminoethoxydiphenyl borate (2-APB)–, or fingolimod hydrochloride (FTY720)–treated dermal fibroblasts in a 2D-like channel imaged every 6 s for 1 min under static conditions followed by exposure to shear flow. Scale bars, 50 μm. (B) Quantification of Fluo-4 Direct fluorescence (ΔF/F₀) from (A), where ΔF = F − F₀, F = fluorescence at a given time point, and F₀ = average fluorescence intensity under static conditions. Data represent means ± SEM for n > 25 cells pooled from three independent experiments. *P < 0.05, two-way ANOVA followed by Tukey’s post hoc test. Percentage of cells that reverse migration direction following treatment with (C) 2-APB (100 μM), (D) FTY720 (2 μM) or vehicle control. (E) Time course of whole-cell TRPM7-like cationic currents recorded at +100 and −100 mV in wild-type primary fibroblasts exposed to flow condition (10 μl/s) followed by exposure to 10 μM FTY720. (F) Current-voltage relationships of whole-cell cationic currents recorded under static, flow, and flow + FTY conditions. (G) Mean current densities measured under static and flow conditions in wild-type primary fibroblasts (n = 6). P = 0.02 paired t test between static versus flow. (H) Percentage of scramble control (SC) or TRPM7-knockdown cells that reverse migration direction under static or flow conditions. (I) Schematic of microfluidic device after replacement of the medium in the channel opposite to the seeding channel with medium containing naltriben or vehicle control. (J) Percentage of fibroblasts that reverse migration direction under static conditions at the end of the microchannels in response to naltriben or vehicle control as shown in (I). Data represent means ± SD from n ≥ three independent experiments. P values calculated one-way ANOVA followed by Tukey’s post hoc test unless otherwise stated in the legend. See also figs. S2 and S3.