Fig. 1
From: Neutral sphingomyelinase inhibition promotes local and network degeneration in vitro and in vivo
Inhibition of nSMase alters plasma membrane-associated lipids. A, B Example confocal images of (A) DMSO- and (B) GW4869-treated 3 DIV hRGCs expressing tdTomato (orange) immunolabeled against nSMase2 (green). (C) GW4869 reduced nSMase2 immunofluorescence in hRGC cultures (p = 0.0233). N = 4 independent samples of 112 DMSO-treated cells (95 images) and 118 GW4869-treated cells (88 images). D, E Images of fixed (D) DMSO- and (E) GW4869-treated hRGCs immunolabeled against GM1 ganglioside (green) and ceramide (magenta). Inset indicates points of overlapping signals from GM1 and ceramide (arrowheads). (F, left) GW4869 significantly increased GM1 ganglioside (DMSO: N = 125 cells across 110 images from 4 replicates; GW4869: N = 109 cells across 90 images from 5 replicates). (F, right) GW4869 decreased ceramide (Cer) localization to hRGCs (p < 0.001). DMSO: N = 48 cells across 26 images from 2 replicates; GW4869: 47 cells across 32 images from 2 replicates. G There is a positive relationship between GM1 and Cer immunolabeling intensity that is altered by GW4869 (DMSO: p = 0.0139, R2 = 0.12, GW4869: p < 0.001, R2 = 0.29). DMSO N = 48 cells from 2 replicates (26 images). GW4869 N = 47 cells across 2 replicates (32 images). H The percent of colocalization (Mander’s) between ceramide and GM1 immunolabeling is reduced by GW4869 (left, p < 0.001) but the percent of colocalization between GM1 and ceramide remained unaltered (right, p = 0.5908). DMSO N = 48 cells from 2 replicates. GW4869 N = 47 cells across 2 replicates. Statistics: C, H Mann–Whitney test, (F) t-test, (G) Linear regression. Data presented as mean ± sem