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. Author manuscript; available in PMC: 2016 May 1.
Published in final edited form as: Nat Immunol. 2015 Sep 28;16(11):1142–1152. doi: 10.1038/ni.3268

Figure 3. Mst1 and Mst2 regulate the recruitment of mitochondria to phagosomes via the small GTPase Rac.

Figure 3

(a) Mst1fl/flMst2fl/fl (WT), Mst1fl/flMst2fl/flLyz2-Cre (cDKO) and cDKO-Rac1G12V BMDMs were incubated with uncoated, LPS-coated or Pam3CSK4-coated latex beads, and mitochondrial networks (Mito) were immunostained with HSP60 antibodies. Confocal images were acquired. Colocalization of beads (green) and mitochondria (red) are indicated by short arrows. Scale bar, 20 μm.

(b) The distribution of mitochondrial networks (HSP60, red) in WT, cDKO, cDKO-Rac1G12V BMDMs and WT BMDMs treated with cytochalasin D or Rac inhibitor NSC23766 after infection with GFP-E. coli (green). Colocalization of E. coli (green) and mitochondria (red) is indicated by arrows. Scale bar, 20 μm.

(c) WT, Rac1 G12V, cDKO and cDKO-Rac1G12V BMDMs were treated with DMSO or cytochalasin D and immunostained with F-actin antibodies (green). Scale bar, 20 μm.

(d) WT BMDMs and neutrophils were pretreated with DMSO or NSC23766 for 30 min followed by LPS stimulation for 3 h. mROS and cellular ROS were measured by MitoSox red dye and CM-H2DCFDA, respectively, using flow cytometry.

(e) Active (GTP-bound) Rac1 levels in WT, cDKO or cDKO-Rac1G12V BMDMs, or in WT and cDKO neutrophils, were detected in GST-PAK70-106 (GST-PAK) with a pull-down assay. GST-PAK protein was stained by Coomassie blue (CoBlue).

(f) mROS and cellular ROS in WT, cDKO and cDKO-Rac1G12V BMDMs stimulated with LPS for 3 h were measured by MitoSox and CellRox, respectively, using flow cytometry.

Images shown are representative of approximately 100 cells (a, b and c). Data are from one experiment representative of three independent experiments with similar results (a-f).