Extended Data Fig. 4. WNK1, OSR1, & SPAK deficiency phenocopies SLC12A2 during apoptotic cell clearance.

(a) Phosphorylation of SLC12A2 and SLC12A4 during efferocytosis. LR73 phagocytes were mixed with apoptotic Jurkat cells for the indicated times, and apoptotic cells thoroughly washed. Total lysates of LR73 cells were analyzed using phosphate affinity electrophoresis (see diagram). 12A2-P and 12A4-P denote the phospho-mobility shift, while 12A2 and 12A4 denote the non-phosphorylated forms. Quantification was based on the intensity and size of the bands followed by subtracting background value. Fold change relative to time point 0 is shown after normalization to ERK2 loading control. Molecular size marker does not show their aciculate sizes due to the modified gels.

(b, c) Validation of Oxsr1 or Stk39 knockdown in LR73 phagocytes. Knockdown efficiency of siRNAs targeting Oxsr1 (OSR1) or (C) Stk39 (SPAK) were determined by qPCR. ***p < .001, n=3 (Stk39) or 4 (Osr1) independent experiments, mean ± SEM.

(d) siRNA targeting of Wnk1 results in increased efferocytosis. (Left) LR73 phagocytes were transfected with siRNAs targeting Wnk1 and incubated with CypHer5E-labeled apoptotic Jurkat cells for 2 h. Data are presented as phagocytosis index and represent n=4 independent experiments. Data shown as mean ± SEM. (Right) Wnk1 knockdown efficiency of two independent siRNAs, determined by qPCR. ***p < .001.

(e) Wnk1 deficiency phenocopies Slc12a2 deficiency in phagocytes during efferocytosis. GFP+ Wnk1-deficient LR73 cells were co-cultured with CypHer5E-labeled apoptotic Jurkat cells for 2 h. ***p < .001. Data are from n=3 independent experiments. Data shown as mean ± SEM.

(f) qPCR validation of CRISPR/Cas9-mediated targeting of Wnk1 in LR73 phagocytes. *p < .001. The drop in mRNA expression due to individual small guide used to target Wnk1 are shown. Statistics source data are provided in Source Data Extended Data Figure 4. Unprocessed blots are provided in Unprocessed Blots Extended Data Figure 3.