Fig 2: NKCC1 regulates RhoGTPase activity. a) Analysis of RhoA and Rac1 activation levels after NKCC1 knockdown in GBM 965 by immunoprecipitation of their GTP active forms. Densitometry of the RhoA-GTP and Rac1-GTP band is shown below, n = 2. b) Cell speed of GBM 612 cells using bumetanide, ROCK inhibitor Y-27632 and combination. c) Cell speed of GBM 965 control and NKCC1-KD cells using different concentrations of the inhibitor Y-27632, n = 3. d) Western blot of showing the effects of ROCK inhibition on the MLC2 (ROCK substrate) in control and NKCC1-KD cells on MCF10A cells, n = 2. Bars represent mean ± SEM. **P-value < 0.01; *** P-value < 0.001.

Fig 3: NKCC1 genes and proteins are expressed in SH-SY5Y neural cells. Gene and protein expressions were analyzed using RT-PCR and western blotting techniques, respectively. (a) RNA was isolated from SH-SY5Y cells and was reverse transcribed, then the cDNA from RT was used for real time PCR (qPCR) to quantify RNA levels. Single band on the right side is HT-29 control cells in both panels a and b. NKCC1 gene expression was detected between 150–200 bp size, for SH-SY5Y cells (a, Top). The bottom lanes show that total RNA was run and the clear 18S and 28S ribosomal RNA bands show that the RNA was intact with no degradation. Total RNA was also used as the loading control for both SH-SY5Y cells and HT-29 Cells and for the evaluation of NKCC1 gene expression difference between SH-SY5Y and HT-29 cell lines (b) Cell lysate was collected in RIPA buffer and analyzed using western blots for NKCC1 protein expression. Data indicate that both SH-SY5Y and HT-29, control cells show NKCC1 expression. For western blot experiments, both proteins of interest, NKCC1 and the loading control protein, β-Actin, were probed on the same PVDF membrane i.e., same membrane were incubated with NKCC1 and β-Actin antibodies. Similarly, for semi-quantitative RT-PCR, both the gene of interest (NKCC1) and reference gene (β-Actin) were probed together using respective primers and analyzed on the same agarose gel.

Fig 4: NKCC1 protein levels were increased by upregulation of mineralocorticoid receptors. (a) Cells were treated with eplerenone (EPL—20 µM), a specific mineralocorticoid receptor inhibitor. Cell lysates were collected in PBS buffer and analyzed for total lysate and membrane fraction for NKCC1 protein expressions using western blot techniques. Equal amounts of lysate protein were used for the isolation of cell membrane proteins (according to the lysate protein concentration measured by spectrophotometer). β-actin was used to achieve equal loading for total lysate, but β-actin is not found in membrane fractions. The blue bar is the control, untreated cells and remaining are various colors with concentration of ALD from 1 nM to 10 µM along with EPL. a) There was no difference between control and treated samples (ALD + EPL) i.e., ALD did not upregulate NKCC1 protein expression in the presence of inhibitor. Bar graphs show mean ± SD from 3 independent runs. (b) Similar to protein expression, there was also no difference in gene expression for NKCC1 following ALD treatment in the presence of eplerenone from 1 nM to 10 µM. Bar graph data are presented as mean ± SEM for three independent runs.

Fig 5: Inhibiting ion‐channel activity significantly reduces cell migration speed in high viscosity media. A) Representative immunofluorescence images of NKCC1, NHE1, NaK and F‐Actin of cells in DMEM and 1% MC. B) Cell speed upon inhibition of NKCC1 via 30 µm Bumetanide (DMEM+DMSO Ctrl N = 182; 1% MC DMSO Ctrl N = 411; DMEM 30 µm Bum N = 171; 1% MC 30 µm Bum N = 263). C) Cell speed upon inhibition of NHE1 via 20 µm EIPA (DMEM+DMSO Ctrl N = 182; 1% MC DMSO Ctrl N = 411; DMEM 20 µM EIPA N = 293; 1% MC 20 µm EipA N = 346). D) Cell speed upon dual‐ inhibition of NKCC1 and NHE1 via 30 µm Bumetanide and 20 µm EIPA (DMEM+DMSO Ctrl N = 182; 1% MC DMSO Ctrl N = 411; DMEM Dual N = 264; 1% MC Dual N = 461). E) Cell speed upon inhibition of NaK via 25 µm and 50 µm Ouabain (DMEM+DMSO Ctrl N = 182; 1% MC DMSO Ctrl N = 411; DMEM 25 µm Ouabain N = 202; 1% MC 25 µm Ouabain N = 288; DMEM 50 µm Ouabain N = 248; 1% MC 50 µm Ouabain N = 331). F) Cell speed upon NKCC1 knockdown from shRNA (DMEM Scr Ctrl N = 74; 1% MC Scr Ctrl N = 224; DMEM S1 N = 74; 1% MC S1 N = 301; DMEM S2 N = 71; 1% MC S2 N = 217; DMEM S3 N = 77; 1% MC S3 N = 133). G) Cell speed for NKCC1 knockdown cells with 20 µm EIPA (DMEM Scr Ctrl N = 14; 1% MC Scr Ctrl N = 17; DMEM S1 N = 13; 1% MC S1 N = 166; DMEM S2 N = 117; 1% MC S2 N = 188; DMEM S3 N = 84; 1% MC S3 N = 160). H) Cell Speed for NKCC1 and NHE1 double knockdown (dKD) (DMEM Scr Ctrl N = 107; 1% MC Scr Ctrl N = 126; DMEM dKD1 N = 125; 1% MC dKD1 N = 126; DMEM dKD2 N = 116; 1% MC dKD2 N = 123). *p < 0.05, **p < 0.005 and ***p < 0.0005 across all plots. Error bars represent standard deviation of the data.