RhoH inhibits NMHC IIA-mediated granules and mitochondria binding to F-actin.

(A) Enriched granules and mitochondria from mature HoxB8 neutrophils expressing HA-RhoH or the corresponding EV were incubated for 30 min in the presence or absence of F-actin. After centrifugation, the pellet (P) and supernatant (S) were evaluated by immunoblotting (left). The ratios of F-actin in the pellet (P) to the supernatant (S) were quantified (right). (B) Docking analysis for predicting the binding sites between RHOH and NMHC IIA (upper). Sequence alignment of RAC1, CDC42, RHOA, and RHOH (lower). (C) Immunoblot analysis of Co-IP with anti-HA antibody in cell lysates from activated HoxB8 neutrophils expressing WT or mutated HA-RhoH. Quantification of the precipitated NMHC IIA was shown below the representative images. (D) Enriched granules and mitochondria were incubated for 30 min in the presence or absence of F-actin. The pellet (P) and supernatant (S) were evaluated by immunoblotting (left). The ratios of F-actin in the pellet (P) to the supernatant (S) were quantified (right). (E–G) Mature HoxB8 neutrophils expressing WT or mutated HA-RhoH or EV were treated as indicated. (E) Neutrophil degranulation was determined by flow cytometry. (F) Extracellular DNA fibers were analyzed by confocal microscopy. Scale bars, 10 μm. (G) Quantification of dsDNA released into the supernatants. (A, C, D, F) Data are representative of 3 independent experiments. Values are means ± SD. Two-tailed Student t test (A, D); 1-way ANOVA with Dunnett’s multiple comparisons test (C); 2-way ANOVA with Tukey’s multiple comparisons test (E, G). The underlying numerical data for Fig 5A, 5C–E, and 5G can be found in S1 Data. The uncropped immunoblots for Fig 5A, 5C, and 5D can be found in S1 Raw images. Co-IP, co-immunoprecipitation; dsDNA, double-stranded DNA; EV, empty vector; NMHC IIA, non-muscle myosin heavy chain IIA; WT, wild-type.