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. 2021 Oct 21;134(20):jcs258894. doi: 10.1242/jcs.258894

Fig. 7.

Fig. 7.

Levels and intracellular sorting of FABP7 are impacted by application of apoE4. (A,B) CHO cells stably co-expressing sortilin and FABP7 (CHO-S/F) were treated for 24 h with conditioned medium containing 5 µg/ml of apoE3 (+E3) or apoE4 (+E4; see Materials and Methods for details). Western blot analysis (A) and densitometric scanning of replicate blots (B) document reduced levels of FABP7 in CHO-S/F cells in the presence of apoE4 as compared with apoE3 (n=10 replicates for each condition from four independent experiments). Data are mean±s.e.m. given as percentage of FABP7 levels in apoE3-treated cells (set to 100%). *P<0.05 (unpaired, two-tailed Student's t-test). (C,D) Experiment as in A and B but using conditioned medium from primary astrocytes secreting apoE3 or apoE4 (n=3 replicates from one culture). Data are mean±s.e.m. given as percent of FABP7 levels in apoE3-treated cells (set to 100%). *P<0.05 (unpaired, two-tailed Student's t-test). (E) Proximity ligation assay (PLA) to visualize the intracellular localization of sortilin–FABP7 complexes (red signal) in CHO-S/F cells treated with apoE3- or apoE4-conditioned HEK293 medium for 24 h (left panel). For comparison, immunostaining of total sortilin (middle panels) and FABP7 (right panels) in treated cells are shown as well. Cell nuclei were counterstained with DAPI (blue). PLA signals for sortilin–FABP7 complexes change from a dispersed vesicular pattern with apoE3 to a perinuclear pattern with apoE4 (arrowheads). A similar change is seen for total sortilin, whereas the pattern for total FABP7 remains unaffected by apoE4. Images shown are representative of results from three independent experiments. (F,G) Colocalization studies in CHO-S/F cells documenting the presence of sortilin–FABP7 complexes (as deduced by PLA; red signal) in early endosomes, marked by antibodies against Rab5 (green signal; F) or recycling endosomes marked by Rab11 (green signal, G). (H,I) Extent of colocalization of PLA for sortilin and FABP7 with Rab5 (H) (n=34 cells for apoE3 and 33 cells for apoE4 treatment) and Rab11 (I) (n=38 cells for each condition) as documented by thresholded Manders’ coefficient tM1. This experiment was replicated three times. *P<0.05; **, P<0.01 (unpaired, two-tailed Student's t-test). Scale bars: 20 µm.