Figure 2
FKBP12 reduces the amyloid property of mHTT in N2A-expressing cells.
(a) Whole-cell staining of Htt(Q)109-eYFP and Htt(Q)109-eYFP/FKBP12-V5 transfectants using ThT dye. Note that the inclusions of Htt(Q)109-eYFP and Htt(Q)109-eYFP/FKBP12-V5 were co-localized with ThT signal. Insets indicate the enlarged ThT signal. Both Htt(Q)109-eYFP and Htt(Q)109-eYFP/FKBP12-V5 were immunostained with anti-V5 followed by probing with Alexa Fluor 633 anti-mouse Ab. Scale bar: 20 μm. (b) Fold change in ThT fluorescence between the Htt(Q)109-eYFP and Htt(Q)109-eYFP/FKBP12 groups. (c) Representative diagram of the workflow using FACS to separate the lysates of cells co-expressing Htt(Q)n-eYFP (n = 25, 109) and FKBP12 into three fractions (P1, P2 and P3) based on their eYFP fluorescence intensity. (d) Sorting histograms of N2A cell lysate, Htt(Q)25-eYFP, Htt(Q)25-eYFP/FKBP12-V5, Htt(Q)109-eYFP and Htt(Q)109-eYFP/FKBP12-V5. (e,f) TEM micrographs of the P1 fraction from Htt(Q)109-eYFP and Htt(Q)109-eYFP/FKBP12-V5. Middle graphs are enlarged images of the black square in the corresponding left panel. Right graphs indicate the immunolabeling of the sorted particles in Htt(Q)109-eYFP and Htt(Q)109-eYFP/FKBP12 samples with EM48 antibody. White and black scale bars represent 500 and 100 nm, respectively. (g) Images of the sorted particles from the P2 fraction. (h) Images of the sorted materials from the P3 fraction. (i) Fold change in ThT of the collected aggregates from P1 between the Htt(Q)109-eYFP and Htt(Q)109-eYFP/FKBP12 groups. n = 3, *p < 0.05, as analyzed by Student’s t-test.
