Fig 2: Rab43 regulates the transport of CD91 to the cell surface. (A) Localization of endogenous CD91 in Rab43-C and Rab43-cKO bone marrow-derived macrophages (BMDMs). The Rab43-C and Rab43-cKO BMDMs were stained with anti-CD91 antibody. Red, CD91; blue, DAPI. Scale bars = 10.0 μm. (B) Co-localization of endogenous CD91 with the ER marker Calnexin. The Rab43-C and Rab43-cKO BMDMs cultured in glass-bottom dishes were stained with anti-CD91 antibody and anti-Calnexin antibodies. Red, CD91; green, Calnexin; blue, DAPI. Scale bars = 10.0 μm. (C) Co-localization of endogenous CD91 with the Golgi body. The Rab43-C and Rab43-cKO BMDMs cultured in glass-bottom dishes were fixed with 4% formaldehyde solution for 10 min. Dual Detection Reagent was used to stain the Golgi body for 1 h. The cultured cells were then incubated with anti-CD91 antibody. Red, CD91; green, Golgi body; blue, DAPI. Scale bars = 10.0 μm.

Fig 3: rHMGB1 suppressed CD91 expression on the cell surface. The bone marrow-derived macrophages (BMDMs) were treated with 2 μg/ml rHMGB1 for 12 h. (A) The CD91 level on the cell surface was detected using flow cytometry. *P < 0.05 versus the phosphate-buffered saline (PBS)-treated group. (B) Rab43-C or Rab43-cKO BMDMs were incubated with CD91-blocking antibody or IgG for 1 h, and then engulfment of apoptotic cells was measured by flow cytometry. *P < 0.05 versus the Rab43-C/IgG group. (C) Confocal laser-scanning microscopy (CLSM) to determine the subcellular localization of CD91. The BMDMs were stained with the anti-CD91 antibody. Red, CD91; blue, DAPI. Scale bars = 10.0 μm. (D) Co-localization of endogenous CD91 with the ER marker Calnexin. The BMDMs cultured in glass-bottom dishes were stained with anti-CD91 antibody and anti-Calnexin antibodies. Red, CD91; green, Calnexin; blue, DAPI. Scale bars = 10.0 μm. (E) Co-localization of endogenous CD91 with the Golgi body. The BMDMs were fixed with 4% formaldehyde solution for 10 min. A sufficient volume of Dual Detection Reagent was added to cover the monolayer cells (1:100 dilution). After blocking for 1 h, the cells were stained with anti-CD91 antibody. Red, CD91; green, Golgi body; blue, DAPI. Scale bars = 10.0 μm.

Fig 4: Effects of Rab43 knockout on the cell surface transport of endogenous CD91. (A) The CD91 proteins on the cell surface were analyzed using flow cytometry. The bone marrow-derived macrophages (BMDMs) were isolated from Rab43-C and Rab43-cKO mice. *P < 0.05 versus the Rab43-C group. (B) The CD91 levels on the cell surface of BALF samples. The Rab43-C and Rab43-cKO mice were treated with LPS for 1 (upper panel) and 3 days (lower panel). The macrophages (F4/80+CD11c+CD11b−) in the BALF samples were also analyzed. *P < 0.05 versus the Rab43-C group. (C) The expression of total, cytoplasmic, and membrane CD91 in Rab43-C and Rab43-cKO BMDMs measured using western blot analyses. *P < 0.05 versus Rab43-C group. ns, not statistically significant.