Fig. 6: Lysosomal perinuclear accumulation occurs in ALS-PFN1 iMGs.
From: Expression of ALS-PFN1 impairs vesicular degradation in iPSC-derived microglia
a, b De-quenched DQ-BSA fluorescence intensity measured over 4 h by live-cell imaging of PFN1 WT and C71G+/− iMGs (a ns P = 0.5740, F (1, 4) = 0.3737) and of PFN1 WT and M114T+/− iMGs (b ns P = 0.0717, F (1, 4) = 5.921). Data were normalized to total cell number. Statistics were obtained by two-way ANOVA for n = 3 independent differentiations. c The fluorescence intensity of Lysosensor DND-189 was quantified for WT (n = 6 for two different lines) and ALS-PFN1 (C71G+/− n = 3, solid squares and M114T+/− n = 3, open squares) iMGs at baseline or pre-treated with 200 nM Bafilomycin A (WT n = 5 from two different lines and ALS-PFN1 from C71G+/− n = 3, solid squares and M114T+/− n = 2, open squares, BafA; ***P = 0.0006 for WT untreated vs BafA or P = 0.0002 for ALS-PFN1 untreated vs BafA; ns P = 0.9840 for untreated WT vs ALS-PFN1 or P = 0.9938 for BafA WT vs ALS-PFN1). Data was normalized to WT iMGs for each independent differentiation. Statistics were determined by one-way ANOVA (F 0.1243 = (3, 18)) and Tukey’s multiple comparisons test. d Western blot analysis of LAMP1 and the mature form of Cathepsin D (mCTSD) from WT and C71G+/− (n = 3 independent differentiations) with GAPDH as a loading control. e, f Quantification of (d) for LAMP1 (e ns P = 0.9703, t = 0.03967, df = 4) and mCTSD (f ns P = 0.4999, t = 0.7409, df = 4). Levels of the target proteins were first normalized to GAPDH and then to the WT control lane corresponding to the same differentiation. Statistics were determined by unpaired two-tailed t-test. g Representative immunofluorescence images of LAMP1 in PFN1 WT (n = 7) and ALS-PFN1 (C71G+/− n = 4 and M114T+/− n = 3) iMGs. White asterisks indicate cells with perinuclear LAMP1 signal. Scale bar = 50 µm. h Percentage of iMGs showing LAMP1 perinuclear localization for WT (n = 5 for two iPSC lines) and ALS-PFN1 (C71G+/− n = 2, solid squares and M114T+/− n = 3, open squares) iMGs. Statistics were determined by unpaired two-tailed t-test (***P = 0.0004, t = 5.768, df = 8). All graphs show mean ± SEM and individual data points in the bar graphs represent independent differentiations. Source data are provided as a Source Data file.
