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. Author manuscript; available in PMC: 2020 Jun 2.
Published in final edited form as: Nat Cell Biol. 2019 Dec 2;21(12):1532–1543. doi: 10.1038/s41556-019-0431-1

Extended Data Fig. 1. SLC12A2 acts as a ‘brake’ on apoptotic cell clearance.

Extended Data Fig. 1

(a) qPCR of Slc12a2 and Slc12a4 during efferocytosis. Data shown as mean ± SEM ***p < .001, n=4 independent experiments.

(b) Slc12a2 or control LR73s were fed CypHer5E-labeled apoptotic Jurkat cells for 2h. Data are from n=6 independent experiments. Data shown as mean ± SEM. ***p < .001. Validation of Slc12a2 deletion by qPCR for two independent small guides.

(c) (Left panel) Slc12a2 siRNA-treated LR73 phagocytes were incubated with CypHer5E-labeled apoptotic Jurkat cells for 2h. Data represent n=3 independent experiments. Data shown as mean ± SEM. *p < .05, ns = not significant. (Right panel) siRNA targeting efficiency of Slc12a2 in LR73 phagocytes via qPCR. ***p < .001.

(d, e) (d) LR73 phagocytes or (e) peritoneal macrophages were pre-treated for 1h with bumetanide then incubated with CypHer5E-labeled apoptotic Jurkat cells for 2h (e), or 1h (f). Data represent n=3 (d, ***p < .001, mean ± SEM) or n=2 independent experiments (e).

(f) Control or Slc12a2-deficient LR73 phagocytes were pre-treated for 1h with bumetanide then incubated with CypHer5E-labeled apoptotic Jurkat cells for 2h. Data shown as mean ± SD, n=3 independent experiments. ns = not significant.

(g) Control or Slc12a2-deficient LR73 phagocytes were cultured with apoptotic cells, with or without Annexin V for 2h. Data are n=3 independent experiments. Data shown as mean ± SEM. ***p < .001.

(h) qPCR determination of the knockdown efficiency of Slc12a2 in ER-Hoxb8 immortalized bone marrow-derived macrophages used in Fig. 2b. n=2 independent experiments.

(i) Mice were injected i.p. with the SLC12A2 inhibitor bumetanide. After 1h, CypHer5E-labeled apoptotic Jurkat cells were injected. Peritoneal cells were collected and engulfment by CD11b+ F4/80+ macrophages assessed by flow cytometry. Data represent n=2 independent experiments.

(j) SLC12A2-deficient macrophages were cultured for 3h with apoptotic cells prior to imaging. Efferocytosis was assessed by fluorescent microscopy. Images represent four independent experiments. scale bar= 20 microns. Statistics source data are provided in Source Data Extended Data Figure 1.