Figure 1.

A significant reduction in DRAM2 expression is observed in CORD21-ROs and RPE cells

(A) Schematic diagram showing the RO differentiation procedure. DF-differentiation and MM-maintenance media.

(B) DRAM2 co-localizes with mitochondrial marker TOMM20 and lysosomal marker LAMP2 in the ISs of wild-type ROs (white arrowheads). These are representative images from 15 ROs imaged from three different differentiation experiments. Scale bars represent 20μm and 10μm in the top and bottom magnified panels, respectively.

(C) WB shows a significant reduction in DRAM2 expression in day 220 CORD21-ROs. Data are presented as mean +SEM (n = 3 different differentiation experiments each consisting of 48 ROs/sample), ^∗p <0 .05.

(D) DRAM2 localizes to the ISs of wild-type and isogenic control (as indicated by white arrowheads) but is absent in CORD21-ROs. Scale bar, 20 μm. These are representative examples from 15 ROs imaged from three different differentiation experiments/sample.

(E) Schematic diagram of iPSC-directed differentiation to RPE cells.

(F) WB shows a significant reduction of DRAM2 protein abundance in CORD21-RPE cells. Conversely, DRAM2 is detected in the wild-type and the isogenic control ROs. Data are presented as mean +SEM (n = 3 different differentiation experiments each consisting of 2 wells of a 12-well plate of RPE cells/sample), ^∗∗∗p <0 .001.

(G) Co-localization of DRAM2 with TOMM20 and LAMP2 in RPE cells derived from WT iPSCs and absence of DRAM2 protein in CORD21-P2 RPE cells. A punctate pattern of protein expression can be seen in the CORD21-P2c isogenic control (white arrows). These are representative examples from 15 RPE transwells imaged from three different differentiation experiments. Scale bar, 20 μm.