Figure 5.

RPE proteome analysis of CORD21-RPE cells identifies changes in key proteins linked to vesicular-mediated transport

(A) Principal-component analysis (PCA) showed a distinct separation between CORD21-P1/P1c (blue) and CORD21-P2/P2c RPE cells (red).

(B) Venn diagram illustrates the 1,759 proteins that are commonly changed between P1/P1c and P2/P2c RPE cells (Tukey’s post hoc, FDR<0.05, n = 7 different differentiation experiments each consisting of 2 wells of a 12-well plate of RPE cells/sample).

(C) Dot plot revealed a total of 296 (red) and 375 (green) proteins that are up- and downregulated, respectively.

(D) Volcano plots show key targets involved in cellular transport in both comparison groups with logged FC > 2 that represent statistically significant changes (p < 0.05) (P1/P1c, n = 32) (P2/P2c, n = 20).

(E) GO enrichment analysis of RPE differentially expressed proteins conducted using Metascape identified respiratory chain complex I, carbohydrate metabolic process, carbon metabolism, generation of precursor metabolites and energy, transport of small molecules, and vesicle-mediated transport as affected biological processes.

(F and G) WB data showed PPT1 and NPC2 RPE intracellular deficiency is likely due to aberrant secretion to the extracellular media. Equal protein loading was visualized by the total protein stain, and data were normalized to the WT sample. Data are shown as mean +SEM (n = 3–4 different differentiation experiments each consisting of 6 wells of a 12-well plate of RPE cells/sample).

(H) Reduced CTSD enzymatic activity in CORD21-RPE lysates relative to isogenic controls. Endpoint activity assays for GBA and α-Mannosidase demonstrated a similar enzymatic reduction in CORD21-RPE cell lysates. Data are shown as mean +SEM (n = 3 different differentiation experiments each consisting of 2 wells of a 12-well plate of RPE cells/sample).