Figure 2.

Rab37 mediates IL-6 secretion in macrophages in a GTPase-dependent manner. (A) Heatmap depicting the secretin level of cytokines/proteins in CM derived from untreated or LLC-CM treated BMDMs from Rab37 WT and KO mice. IL-6 is indicated by an arrow. (B, C) ELISA was performed to validate the level of IL-6 in CM from Rab37 WT and KO BMDMs (b) or in CM from EV, Rab37^WT, Rab37^Q89L or Rab37^T43N RAW264.7 cells (C). (D, E) Confocal microscopy images of Rab37 (green), IL-6 (red) and nucleus staining (blue) in Rab37 WT and KO BMDMs (D) or in EV, Rab37^WT, Rab37^Q89L or Rab37^T43N RAW264.7 cells (E). Z-stack images are shown. Scale bars: 10 µm. (F) Vesicles of EV or Rab37^WT RAW264.7 cells expressing V5-tagged Rab37, were collected by centrifugations and immunoprecipitated (IP) with anti-V5 and vesicle lysates were blotted for V5-Rab37 and endogenous IL-6. (G-I) Ultrastructural localization of Rab37 (10 nm of gold, red arrow) and IL-6 (20 nm of gold, yellow triangle) illustrated by immuno-EM images of control THP-1 (G) or A549-CM treated THP-1 macrophages (H). PM: plasma membrane. Scale bars: 500 nm. Enlarged images shown in insets of the representative regions. (I) Rab37/IL-6 colocalized vesicles were significantly increased in the A549-CM treated THP-1 compared to those in the control THP-1 cells. An average of 15-25 vesicles per cell and a total of 10 cells were collected. Percentages are expressed in relation to the total number of Rab37 specific vesicles. (J-M) Selected frames from time-lapse confocal movies of GFP-tagged EV, Rab37^WT, Rab37^Q89L, or Rab37^T43N RAW264.7 cells co-transfected with RFP-tagged IL-6 cells. Enlarged images of the boxed areas with time intervals in seconds are shown (bottom). Arrow indicates trafficking vesicle. Scale bars: 10 µm. Data represent mean ± SD. * p < 0.05; ** p < 0.01; *** p < 0.001, Student's t-test.