Figure 4.

Differential protein analysis identifies vesicle-mediated response as a key biological process in CORD21-ROs

(A) Principal-component analysis reveals clear separation between CORD21-P1/P1c (outlined in blue) and CORD21-P2/P2c ROs (red).

(B) Venn diagram shows an overlap of 101 commonly changed proteins following Tuckey’s post hoc test (false discovery rate [FDR]<0.05) (n = 3–4 different differentiation experiments each consisting of 48 ROs/sample).

(C) Dot plot highlighting commonly upregulated (red) and downregulated (green) proteins (n = 19).

(D) Volcano plots enable visual identification of tandemly changed proteins with logged fold change cutoff >2 that are also statistically significant (logged p < 0.05) (n = 19).

(E) Gene Ontology (GO) term enrichment of significantly changed proteins following the same trend by Metascape revealed major affected biological processes.

(F and G) PPT1 and NPC2 enzymes are severely deficient in the lysates of CORD21-P1 and CORD21-P2-ROs relative to controls. This deficiency associates with PPT1 and NPC2 hypersecretion to the extracellular media of matched CORD21-ROs. A total protein stain was used to confirm equal protein loading, and normalization was conducted to the WT sample. Data are presented as mean +SEM, n = 3 different differentiation experiments each consisting of 48 ROs/sample.

(H) Kinetic assay for the activity of CTSD shows reduced enzymatic activity in CORD21-ROs lysates relative to isogenic control (ANOVA). Endpoint enzymatic activity assays for GBA (ANOVA) and α-Mannosidase (ANOVA) demonstrated a similar reduction in enzymatic activities in day 220 CORD21-ROS. Data were shown as mean +SEM (n = 3 different differentiation experiments each consisting of 48 ROs/sample).