Fig. 1

GRN Knockout HEK-293 Cells Exhibit Autophagy-Lysosomal Abnormalities. We deleted the entire GRN coding sequence from HEK-293 cells using CRISPR-Cas9, resulting in complete loss of progranulin. (a, b), Lysates of GRN knockout (GRN KO) cells had elevated levels of mature cathepsin D (CatD, t test, p = 0.0013, n = 9 per group). (c, d), GRN KO cells also exhibited a deficit in autophagy, defined as a reduced LC3-II/LC3-I ratio after incubating for one hour with 50 µM chloroquine (CQ) (ANOVA effect of CQ, p < 0.0001, * = p = 0.0406 by Holm-Sidak post-hoc test, n = 9 per group). (e–g), Fluorescent imaging of cells transfected with GFP-LC3-RFP-LC3ΔG confirmed a reduction in autophagic flux in GRN KO cells. The ratio of GFP/RFP fluorescence was significantly increased in GRN KO cells versus controls (e, linear mixed effects model effect of GRN, p = 0.0002, *** = p < 0.001 by Tukey’s post-hoc test, n = 528–594 cells per group from 4 independent cultures). Additionally, incubation with 50 µM chloroquine for 3 h increased the GFP/RFP ratio in control cells (e, linear mixed effects model effect of CQ, p < 0.0001, *** = p < 0.001 by Tukey’s post-hoc test) but not in GRN KO cells (p = 0.534 by Tukey’s post-hoc test). Data in (e) are shown as the median fluorescent intensity normalized to control vehicle-treated cells from each culture, with violin plots showing the distribution of all cells measured and symbols showing the median value for each culture¹⁰⁸. (f), Similar results were obtained by analyzing the frequency distribution of the GFP/RFP ratio in cells transfected with GFP-LC3-RFP-LC3ΔG (f, Kolmogorov-Smirnov test, control vehicle vs. GRN KO vehicle, p = 0.0147, control vehicle vs. control CQ, p = 0.0472, GRN KO vehicle vs. GRN KO CQ, p = 0.7649). Scale bars in (g) represent 20 μm. (h, i), Restoration of progranulin (PGRN) to GRN KO cells reduced levels of mature CatD, though CatD still remained elevated compared to control cells (ANOVA effect of GRN KO, p < 0.0001, effect of PGRN expression, p < 0.0001, ** = p < 0.01, *** = p < 0.001, **** = p < 0.0001 by Holm-Sidak post-hoc test, n = 6 per group). (g, h), Restoration of PGRN to GRN KO cells also normalized the LC3-II/LC3-I ratio (ANOVA effect of PGRN expression, p = 0.0328, n = 7–9 per group), as GFP-transfected GRN KO cells had lower LC3-II/LC-I after chloroquine treatment than control cells (** = p = 0.0092 by Holm-Sidak test) while PGRN-transfected GRN KO cells did not differ from controls (p = 0.3058 by Holm-Sidak test). Full images of immunoblots are shown in Fig. S2.