LRRK2 and Rab10 coordinate macropinocytosis to mediate immunological responses in phagocytes (2020)
Figure 1

Figure 1. Rab10 expression regulates macropinocytosis.

A. Raw 264.7 macrophage cells were treated with DMSO (0.05%), 1 μM control antisense oligonucleotide (ASO), or Rab10-directed ASO (1 μM), for four days. Immunoblots are representative of three independent experiments, with similar results obtained.

B. Calculated reduction of Rab10 (n=3 biologically independent experiments). Each dot in the plot presents one independent experiment. Group are shown. Error bars represent ± SEM. Significance was assessed by one-way ANOVA with Tukey's post hoc test, with **** representing P <0.0001.

C. Raw 264.7 cells were treated with the indicated ASO for four days, or 50 μM EIPA (5-[N-ethyl-N-isopropyl]amiloride) for 20 min, prior to incubation for 60 min with fluorescent (tetramethylrhodamine, TRITC)-labeled dextran (70 kDa, shown as red color), transferrin, or IgG-conjugated agarose beads. Representative photomicrographs (from 30 images analyzed for each condition, from three biologically independent experiments) are shown. Cells were washed, fixed, and stained with DAPI (shown as blue color). Scale bars show 200 μm.

D-F. Relative fluorescent signals were calculated as a percent of control-ASO treated cells to indicate (D) dextran or (E) transferrin uptake level. (F) For quantification of phagocytosis, the average number of IgG-agarose beads internalized were calculated as a percent of control-ASO treated cells. 30 images across three independent experiments were quantified for each condition, with each dot in the plot representing results from one image. The group means were calculated and shown. Error bars represent ± SEM. Significance was assessed by one-way ANOVA with Tukey's post hoc test, with *** representing P<0.0001, N.S P>0.05.

G. Primary mouse bone marrow-derived macrophage cells (BMDM) from adult male C57BL/6J mice were treated with murine cytokines to polarize cells towards a monocytic lineage (mBMDM), dendritic lineage (mBMDC), or microglia-like lineage (mMicroglia, see Methods section). Cells were incubated with TRITC-dextran for 30 min prior to washing, fixing, and staining with DAPI. Representative photomicrographs from 30 images analyzed for each condition from n=3 biologically independent experiments are shown. Scale bars show 200 μm.

H-J. Relative fluorescent signals were calculated as a percent of control-ASO treated cells. 30 images across 3 independent experiments were quantified for each condition, with each dot in the dotted plot representing 1 image quantified. The group means were calculated and shown. Error bars represent ± SEM. Significance was assessed by one-way ANOVA with Tukey's post hoc test, with ****representing P <0.0001, N.S P>0.05.

K. Human monocytes purified from venous blood draws from healthy adult male volunteers were treated with human cytokines towards a monocytic lineage (hMDM), dendritic lineage (hMDC), or microglia-like lineage (hMicroglia). Cells were incubated with TRITC-dextran for 30 min prior to washing, fixing, and staining with DAPI. Representative photomicrographs from 30 images analyzed for each condition from n=3 biologically independent experiments are shown. Scale bars show 200 μm.

L-N. Relative fluorescent signals were calculated as a percent of control-ASO treated cells. 30 images across 3 independent experiments were quantified for each condition, with each dot in the plot representing one image quantified. The group means were calculated and shown. Error bars represent ± SEM. Significance was assessed by one-way ANOVA with Tukey's post hoc test, with **** representing P <0.0001.