RhoH interacts with NMHC IIA.

(A) Coomassie blue staining of HA pulldown proteins in HoxB8 neutrophils after stimulation with the indicated triggers. The proteins interacting with RhoH were identified by mass spectrometry analysis. (B, C) Immunoblot analysis after Co-IP with anti-HA antibody in Hoxb8 neutrophils (B) and anti-NMHC IIA antibody in human neutrophils (C). Quantification of the precipitated NMHC IIA (B) or RHOH (C) was shown below the representative images. (D–F) Pretreated human neutrophils with vehicle control or myosin IIA inhibitors for 30 min were subsequently activated with GM-CSF in combination with C5a. (D) Neutrophil degranulation was determined by flow cytometry. (E) Quantification of dsDNA released into the supernatants. (F) NET formation indicated by the colocalization of NE (green) with released DNA (PI, red) was analyzed by confocal microscopy. Scale bars, 10 μm. (G) Protein expression of NMHC IIA in mature HoxB8 neutrophils treated with control (Ctrl) or Myh9 shRNA was analyzed by immunoblot. (H, I) Mature HoxB8 neutrophils were treated as indicated. (H) Neutrophil degranulation was determined by flow cytometry. (I) Quantification of released dsDNA in culture supernatants. (J) Extracellular DNA fibers were analyzed by confocal microscopy. Scale bars, 10 μm. (A–C, F, G, J) Three independent experiments were performed. (B–E, H, I) Values represent means ± SD. (B–E) One-way ANOVA with Dunnett’s multiple comparisons test. (H, I) Two-way ANOVA with Šídák’s multiple comparisons test. The underlying numerical data for Fig 3B–E, 3H, and 3I can be found in S1 Data. The uncropped immunoblots for Fig 3A–C and 3G can be found in S1 Raw images. Co-IP, co-immunoprecipitation; dsDNA, double-stranded DNA; GM-CSF, granulocyte/macrophage colony-stimulating factor; NE, neutrophil elastase; NET, neutrophil extracellular trap; NMHC IIA, non-muscle myosin heavy chain IIA; PI, propidium iodide.