Fig. 7: ALS-PFN1 iMGs show deficits in autophagy-related proteins.
From: Expression of ALS-PFN1 impairs vesicular degradation in iPSC-derived microglia
a–c LC3I and LC3II expression in PFN1 iMGs by Western blot analysis from n = 5 independent differentiations. a Representative Western blot of LC3I, LC3II, and GAPDH as loading control. b, c Quantification of LC3I and LC3II in (a). b LC3II/LC3I ratio normalized to WT iMGs in each differentiation (ns P = 0.6978, t = 0.403, df = 8). c Total LC3 levels (LC3I + LC3II) normalized to GAPDH and reported as relative to WT iMGs (ns P = 0.7810, t = 0.288, df = 8). d–e p62 protein levels in PFN1 iMGs after bafilomycin A or rapamycin treatment compared to untreated cells. d Representative Western blots of p62 for PFN1 iMGs under untreated conditions or 0.1 µM rapamycin (n = 4 independent differentiations) and under untreated conditions or 100 nM bafilomycin A (n = 5 independent differentiations). e Quantification of (d), with p62 bands normalized to GAPDH and all data reported as relative to untreated WT iMGs. (Untreated WT vs C71G+/− *P = 0.0430, t = 2.594, df = 30; rapamycin WT vs C71G+/− ns P = 0.8917, t = 0.646, df = 30; bafilomycin A WT vs C71G+/− ns P = 0.3200, t = 1.597, df = 30). Statistics were determined by two-way ANOVA and Å Ãdák’s multiple comparisons test. f Representative immunofluorescence images of LC3 (green) and p62 (red) puncta with DAPI (blue) in PFN1 iMGs. Scale bar: 25 µm and 1 µm (inset). g, h Quantification of puncta per cell for LC3 (g ns P = 0.1689, t = 1.379, df = 289) and p62 (h ***P = 0.0002, t = 3.748, df = 289) from n = 147 WT and 144 C71G+/− cells across 4 independent differentiations. i, j Quantification of the puncta size for LC3 (i ns P = 0.1828, t = 1.332, df = 2434) and p62 (j ****P < 0.0001, t = 4.509, df = 3660) from n = 960 WT and 1476 C71G+/− puncta from the same cells as (g, h). k Pearson’s coefficient of the colocalization between LC3 and p62 puncta (**P = 0.0040, t = 2.898, df = 285) from n = 144 WT and 143 C71G+/− cells across 4 independent differentiations. All bar graphs show mean ± SEM and data points represent individual differentiations (b, c, e), cells (g, h, k), or puncta (i, j). Statistics for bar graphs other than e were determined by unpaired two-tailed t-test. Source data are provided as a Source Data file.
