Figure 4

Evaluation of the functional effect of four rare ADAMTSL4 variants identified in childhood glaucoma patients, in HEK-293T cells. Subcellular distribution of the wild type protein (A) and variants R98W (E), S719L (I), R774W (M), and R1083H (Q) in HEK293T Cells (green signals). The cells were transfected with cDNA constructs encoding the different variants, fused to GFP at their C-terminal as a reporter protein and analyzed 48 h after transfection. (U) Non-transfected cells were used as a negative control. (B,F,J,N,R,V) Immunocytochemistry was performed to detect PDI, a marker of ER stress (orange signals). (C,G,K,O,S,W) DAPI nuclear staining (blue signals). (D,H,L,P,T,X) Merged signals. The scale bars in the panels correspond to 20 μm. The white arrowheads indicate increased PDI signal colocalized with the granular expression pattern of the R98W and R774W variants. The inserts in panels (E,M) show granular deposits of recombinant protein, and dotted squares indicate the magnified areas. The images are representative of five fields per variant. Additional representative photographs are shown in Figure S3. R98W: p.Arg98Trp. S719L: p.Ser719Leu. R774W: p.Arg774Trp. R1083H: p.Arg1083His.