Phosphoproteomics identifies microglial Siglec-F inflammatory response during neurodegeneration (2020)
Figure 6

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

(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 hours, then fluorophores are added to media. Cells with endocytosis substrates (Aβ, Dextran) were assayed after 3 hours, while phagocytosis substrates (FluoSpheres) were assayed after 24 hours. 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.

Acknowledgements

We thank members of the White and Tsai laboratories for numerous discussions and feedback. We also thank C. Whittaker for help with RNA-seq analysis, J. Cheah for help with BV-2 assay development and screening, and R. Ahn for discussions on signal transduction mechanisms. N.M. was partially supported by the NIH Biotechnology Training Grant T32GM008334. D.M. was supported by NIRG-15-321390, and Arizona Alzheimer's Consortium. This work was supported by the Center for Precision Cancer Medicine at MIT, NIH grants U54-CA210180, R37-NS051874, RF1-AG054321, a grant from the Simons Center for the Social Brain, the Glenn Foundation and NDC Belfer (The Neurodegeneration Consortium, The Robert A. and Renee E. Belfer Family Foundation, and the Oskar Fisher Project) to L.-H.T.

Author Contributions

N.M., L.-H.T., and F.W. designed the study. A.N., L.A.W., N.M., L.A.A., and P.-C. P. performed animal tissue dissections. D.M. collected human brain tissue samples. N.M. collected proteomics data sets. N.M. wrote proteomics data integration software. N.M. and B.A.J. designed enrichment algorithms. N.M. and L.A.A. performed histology analysis. N.M., W.T.R., and J.P. developed and ran iMGL assays. N.M, F.H.R., and L.L. cloned DNA constructs and performed BV-2 cell assays. N.M., F.W., and L.-H.T. wrote the manuscript.

Conflict of interest

The authors declare no competing interests.