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. 2020 Dec 8;16(12):e9819. doi: 10.15252/msb.20209819

Figure 6. Siglec‐F expression drives an endocytic response in BV‐2 cells.

Figure 6

  • A
    Schematic showing experimental setup to measure rates endocytosis in BV‐2 cells. Control (MIP empty vector) cells were plated together with cells stably expressing different Siglec constructs. Cells are allowed to adapt to the same media environment for 24 h, and then, fluorophores are added to media. Cells with endocytosis substrates (Aβ, Dextran) were assayed after 3 h, while phagocytosis substrates (FluoSpheres) were assayed after 24 h. Populations are separated, and relative uptake is measured using flow cytometry.
  • B, C
    Flow cytometry quantification of fluorescent monomeric Aβ uptake and Siglec‐F expression for (B) Siglec‐F and (C) Siglec‐F 2xY‐>F. Siglec‐expressing cells were plated with empty vector control cells in the same well alongside fluorescent substrates.
  • D–F
    Quantification of relative mean fluorescence intensities (MFI) between empty vector and Siglec‐expressing populations. Relative values are shown for each mouse and human Siglec construct for uptake of (D) monomeric Aβ, (E) 10,000 MW Dextran, and (F) 1 μm FluoSpheres. Bars indicate mean ± 95% CI; n = 6 replicates; *P < 5e‐2, **P < 1e‐2, ***P < 1e‐3, ****P < 1e‐4; ns: not significant, using unpaired Student’s t‐test, two‐sided. Dotted line indicates mean of 2xY‐>F group.
  • G
    pHrodo Dextran uptake in BV‐2 cells with stable expression of Siglec‐F estimated by Incucyte measurements. Bars indicate mean ± 95% CI; n = 6 replicates; *P < 5e‐2, **P < 1e‐2, ***P < 1e‐3, ****P < 1e‐4; ns: not significant, using unpaired Student’s t‐test, two‐sided.
  • H
    pHrodo dextran uptake in BV‐2 cells stably expressing Siglec‐F with 0 ‐ 2.5 μM SHP099 treatment. Data is from the same experiment as (G).
  • I
    Confluency doubling times for BV‐2 cells with stable expression of Siglec‐F with 0 ‐ 2.5 μM SHP099 treatment. Data is from the same experiment as (G). Bars indicate mean ± 95% CI; n = 4 replicates; *P < 5e‐2, **P < 1e‐2, ***P < 1e‐3, ****P < 1e‐4; ns: not significant, using unpaired Student’s t‐test, two‐sided. Dotted line indicates mean of untreated 2xY‐>F group.