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. Author manuscript; available in PMC: 2021 Mar 2.
Published in final edited form as: Neuron. 2020 May 5;107(1):65–81.e9. doi: 10.1016/j.neuron.2020.03.031

Figure 7. Galc-Deficient Macrophages Have a Defect in Myelin Degradation.

Figure 7.

(A) Western blot and quantification of Peripheral Myelin Protein 2 (PMP2) and Myelin Protein Zero (MPZ) after myelin phagocytosis assay.

(B) Western blot and quantification of PMP2 and MPZ when macrophages were co-incubated with myelin and either (i) the proteasome inhibitor bortezomib or (ii) the lysosome inhibitor chloroquine.

(C and D) Bright-field images (C) and quantification (D) of bone-marrow-derived macrophages in culture treated with either blank or C8-galactosylceramide for 24 h.

(E) Immunofluorescence of macrophages (anti-F4/80; red) cultured with C8-GalCer and stained for O1 (anti-GalCer; green) and DAPI (Blue).

(F and G) HPLC-MS measurement of C8-GalCer (F) and psychosine (G) from macrophages treated with 20 μM C8-GalCer.

(H) qPCR of BMDMs incubated with 20 μM DMSO (blank), 20 μM C8-GalCer, or 5 μM psychosine for markers related to the immunological phenotype and integrated stress response, normalized to β-Actin, and reported relative to average WT expression.

Scale bars, 60 μm (C) and 15 μm (E). Error bars represent mean ± SEM, n = 3 biological replicates and 3 technical replicates per experiment (n ≥ 5 for A and B). Statistical significance was calculated by Student’s t test (A), one-way ANOVA (D), or two-way ANOVA (B, F, G, and H).