Evaluation of prosaposin- and progranulin-depleted cells. (A) Immunoblot evaluation of prosaposin protein levels in cells transfected with control and prosaposin siRNAs. Vinculin was used as a loading control. (B) Quantification of prosaposin levels normalized to vinculin (n = 3 independent experiments; mean ± SEM; unpaired t test; ****, P < 0.0001). (C) Confocal immunofluorescence images showing progranulin and GM130 (cis-Golgi) localization in control and prosaposin siRNA–treated cells. Scale bar, 10 µm. (D) Manders colocalization coefficients for progranulin and GM130 in control and prosaposin KD cells. Data were collected from n = 20 cells. Error bars show mean ± SEM. Mann–Whitney U test. (E) Confocal immunofluorescence images show cathepsin D and LAMP1 localization in control and prosaposin siRNA–treated cells. Scale bar, 10 µm; inset, 5 µm wide. (F) Manders colocalization coefficients for cathepsin D with LAMP1 in control and prosaposin KD cells (20 cells per condition). Error bars show mean ± SEM. Unpaired t test. (G) Immunoblot evaluation of progranulin and spliced XBP1 protein levels in control and prosaposin KD cells treated for 7 h with the indicated concentrations of CB-5083, an ERAD inhibitor. Immunoblot quantification shows that progranulin levels are not significantly increased in prosaposin KD cells treated with CB-5083 compared with DMSO control. Progranulin levels were normalized to vinculin. n = 3 independent experiments; mean ± SEM; one-way ANOVA with Bonferroni’s multiple comparisons test. (I) Confocal immunofluorescence images showing localization of prosaposin and LAMP1 (lysosomes) in control and progranulin KO cells. Scale bar, 10 µm; inset, 3.38 µm wide. (J) Manders colocalization coefficients for prosaposin with LAMP1 in control and progranulin KO cells. n = 20 cells. Error bars show mean ± SEM. Unpaired t test.