Fig 2: BNIP-2 knockdown suppresses RhoA activity and retards cell polarization during early cell spreading.(A) Endogenous RhoA activity of MDA-MB-231 cells was examined in the presence of different amounts of BNIP-2. Lysates of MDA-MB-231 cells transfected with BNIP-2–targeting siRNA (siBNIP-2) or transiently expressing gradually increasing amount of GFP-BNIP-2 (illustrated by blue triangle) were used for pulldown with immobilized glutathione S-transferase (GST)–RBD (GST-RBD) of rhotekin and then Western blotted with RhoA, BNIP-2, and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) antibodies. Blue arrow denotes the GFP-BNIP-2, while purple arrow denotes the endogenous BNIP-2. The ratio of active RhoA to total RhoA is normalized to untransfected in lane 2 and labeled at the bottom. (B) Snapshots of shVector control and BNIP-2 knockdown cells during spreading on collagen I–coated plastic-bottom plates. Representative cells spreading at 0, 20, 40, and 54 min are displayed. (C) Cell aspect ratios are quantified and plotted for control and BNIP-2 knockdown cells during 60-min spreading. Data are means ± SEM of two independent experiments.

Fig 3: BNIP-2 binds GEF-H1 and scaffolds for RhoA/GEF-H1 signaling.(A) MDA-MB-231 cells stained for BNIP-2 and a-tubulin. Boxes A and B highlight the regions where BNIP-2 colocalizes with microtubules. (B) BNIP-2 interacts with GEF-H1 under physiological conditions. HEK293T (top) or MDA-MB-231 (bottom) cell lysates were incubated with BNIP-2 antibody or immunoglobulin G (IgG) control for immunoprecipitation and then Western blotted with GEF-H1 and BNIP-2 antibodies. The blue arrow denotes heavy chains. (C) BNIP-2 uses the C terminus to interact with GEF-H1. Lysates of HEK293T cells transiently expressing HA-GEF-H1 and GFP-vector, BNIP-2-FL, or different mutants were immunoprecipitated with anti-HA beads and then Western blotted with GFP or HA antibodies. The blue arrow denotes heavy chains. (D) BNIP-2 uses the RBD-like region in BCH domain to interact with GEF-H1. Lysates of HEK293T cells transiently expressing HA-tagged BNIP-2 or BNIP-2-?RBD with FLAG-tagged GEF-H1 were immunoprecipitated with anti-HA beads and then Western blotted with FLAG or HA antibodies. (E) Knockdown of BNIP-2 reduces interaction between RhoA and GEF-H1 in HEK293T cells. Lysates of shVector control and BNIP-2 knockdown HEK293T cells transiently expressing HA-GEF-H1 and FLAG-RhoA were immunoprecipitated with anti-FLAG beads and then Western blotted with FLAG, HA, BNIP-2, or a-tubulin antibodies. (F) Knockdown of BNIP-2 reduces interaction between RhoA and GEF-H1 in MDA-MB-231 cells. Lysates of control and knockdown MDA-MB-231 cells were incubated with GEF-H1 antibody or IgG control for immunoprecipitation and then Western blotted with GEF-H1, RhoA, BNIP-2, or GAPDH antibodies. (G) BNIP-2 has a scaffolding effect for RhoA and GEF-H1 interaction. Lysates of knockdown HEK293T cells transiently expressing HA-tagged GEF-H1, FLAG-tagged RhoA, and increasing amount of HA-BNIP-2 (illustrated by blue triangle) were immunoprecipitated with anti-FLAG beads and Western blotted with FLAG, HA, or BNIP-2 antibodies. The blue asterisk denotes the endogenous BNIP-2, and the purple asterisk denotes the HA-tagged BNIP-2.

Fig 4: BNIP-2 association with KLC-1 is required for its GEF-H1 binding and migration control.(A) BNIP-2 associates with KLC-1. Schematic representation of BNIP-2-WT with the kinesin-binding motif and BNIP-2-5A mutant, which is unable to bind KLC-1, where WE-WED motif in the N terminus is mutated to five alanines. Bottom: MDA-MB-231 cells were transfected with mCherry-KLC-1 and GFP-BNIP-2-WT, GFP-BNIP-2-5A, or GFP-vector for live imaging. Maximum Z-projected images are presented. Scale bars, 10 µm. (B) KLC-1 binding motif of BNIP-2 is required for interaction with GEF-H1. Lysates of HEK293T cells transiently expressing FLAG-tagged BNIP-2-WT, BNIP-2-5A, or empty vector with HA-tagged GEF-H1 were immunoprecipitated with anti-FLAG beads and then Western blotted with FLAG or HA antibodies. FLAG-V stands for FLAG-vector. (C) BNIP-2-5A mutant cannot rescue cell motility increased by BNIP-2 knockdown. Representative images of transwell migration assay on MDA-MB-231 shVector control cells, shBNIP-2 cells, and BNIP-2 knockdown cells rescued with BNIP-2-5A mutant. Scale bars, 100 µm. Right: Statistical analysis for shVector control cells, shBNIP-2 cells, and BNIP-2 knockdown cells rescued with BNIP-2-WT or BNIP-2-5A mutant. Cell number per area was counted from randomly choosing sites and averaged. Final results presented here were normalized to the number of control cells (equals 1). Data are means ± SEM of three independent experiments, P < 0.01. n.s., not significant.

Fig 5: BNIP-2 is required for GEF-H1–driven RhoA activation upon microtubule disassembly.(A) Nocodazole treatment increases BNIP-2 and GEF-H1 interaction. MDA-MB-231 cells were treated with dimethyl sulfoxide (DMSO) (D) or 1 µM nocodazole (N) for 30 min and lysed. Lysates were incubated with BNIP-2 antibody or IgG control, incubated with Protein A/G PLUS-Agarose beads for immunoprecipitation, and then Western blotted with GEF-H1, BNIP-2, or GAPDH antibodies. (B) shVector control and BNIP-2 knockdown MDA-MB-231 cells were treated with DMSO or 1 µM nocodazole for 30 min, followed by immunostaining for a-tubulin and GEF-H1. Maximum Z-projected images are presented. (C) BNIP-2 is required for nocodazole-induced RhoA activation. MDA-MB-231 cells transfected with siControl and siBNIP-2 were treated with DMSO or 1 µM nocodazole for 30 min, lysed for RBD assay, and then Western blotted with RhoA, BNIP-2, or GAPDH antibodies. Quantification of Rho activity is displayed at the bottom with nocodazole normalized against DMSO for each cell line. (D) BNIP-2 and ROCK are required for nocodazole-induced cell rounding. shVector control and BNIP-2 knockdown MDA-MB-231 cells with or without 2-hour pretreatment with 20 µM Y-27632 (Y) were treated with DMSO or 1 µM nocodazole for 30 min and fixed for phalloidin stainning. Maximum Z-projected images are presented. Right: Statistical analysis for cell area. Data presented are means ± SEM, P < 0.0001. n.s., not significant.