Fig. 3: M-CSF stimulates macropinocytosis in myeloid progenitors in a WNK kinase activity-dependent manner.

a Myeloid progenitors perform macropinocytosis in response to M-CSF. (Left) Representative images of myeloid progenitors incubated with M-CSF and 70kDa-florescent Dextran (magenta). Nuclei were stained with Hoechst (yellow) and the plasma membrane was labeled using CellMask (cyan), then myeloid progenitors were imaged at 100X magnification for 30 min. Scale bar, 2 μm. (Right) Quantitation of 70 kDa Dextran uptake per cell via geometric mean fluorescence intensity (MFI) at t = 0 and 30 min. Data is representative of four independent experiments, n = 8 per time point. Statistical significance was determined via two-tailed paired samples t-test. **p < .01. b Inhibition of WNK kinase activity or macropinocytosis blocks M-CSF-stimulated internalization of 70 kDa Dextran by myeloid progenitors. Experiments were performed as in (a) using myeloid progenitors. (Left) Representative images of myeloid progenitors treated with M-CSF and either WNK463 or EIPA. Scale bar, 2 μm. (Right) Quantitation of 70 kDa Dextran uptake per cell via MFI after 30 min. Data are from four independent experiments. Each dot represents the average MFI per field of view (n = 10 per group). Data are shown as mean ± SEM. Statistical significance was determined via two-tailed independent samples t-test. **p < .01, ****p < .0001. c M-CSF-stimulated internalization of 70 kDa Dextran is lost in WNK1-deficient myeloid progenitors. Experiments were performed as in (a) using bone marrow myeloid progenitors from Csf1r^Cre+; Wnk1^fl/fl (Cre+) or Csf1^Cre–; Wnk1^fl/fl (Cre−) mice. Shown is the quantitation of 70 kDa Dextran uptake via MFI per cell. Data is from four independent experiments with two-three mice (biological replicates) per experiment (n = 5 per condition). Statistical significance was determined via two-tailed independent samples t-test. ****p < .0001. d, e WNK1 boosts M-CSF-stimulated macropinocytosis in myeloid progenitors. Experiments were performed as in (a) using bone marrow myeloid progenitors overexpressing WNK1-mRuby. Shown are confocal microscopy (d) and flow cytometry (e) analysis of 70 kDa Dextran uptake. Data is from four independent experiments. Data are shown as mean ± SEM. Statistical significance was determined via two-tailed independent samples t-test. ***p < .001. f, g Confocal microscopy analysis of WNK1 protein expression and macropinocytosis in M-CSF-treated granulocyte and monocyte progenitors. f (Left) Representative fixed images of granulocyte progenitors (GPs, orange) sorted from WT animals based on Lin− cKit+ Sca-1− CD34+ CD64^High Ly6C+ CSF1R^Low or macrophage/monocyte progenitors (MPs, purple) sorted based on Lin− cKit+ Sca-1− CD34+ CD64^High Ly6C+ CSF1R^High. See Supplementary Fig. 2a for flow cytometry gating strategy. Cells were stained with WNK1 antibody (magenta), plasma membrane dye (PM, blue), DAPI (green), and treated with 1 mg/mL 70 kDa Dextran (yellow), and 100 ng/mL M-CSF for 1 h. Scale bar, 5 μm (Right) Quantitation of WNK1 protein expression and 70 kDa Dextran uptake (g) via MFI per field view area (FOV = μm²). Data are from three independent experiments. n = 12 FOVs per group. Data are shown as mean ± SEM. Statistical significance was determined via two-tailed independent samples t-test. **p < .01, ***p < .001. Source data are provided in a Source Data file included with this manuscript.