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. Author manuscript; available in PMC: 2015 Feb 1.
Published in final edited form as: Mol Genet Metab. 2013 Sep 21;111(2):172–183. doi: 10.1016/j.ymgme.2013.09.011

Figure 3. Caspase-3 activation, lysosomal compartment expansion and cytochrome C expression pattern in 145M-Twi from Twitcher.

Figure 3

(a) Culture 145M-Twi and 145C-Wt showed baseline caspase-3 activity, key component orchestrating apoptosis. Under 24h treatment with 15 and 30 μM of glucosylsphingosine (GlucoSP) and psychosine (PySP), the caspase-3 activation was significantly higher in the 145M-Twi cells. Triplicates were analyzed on each concentration tested. (b) Cleaved caspase-3 immunoblots (17 kD) from 145M-Twi and 145C-Wt cells treated at increasing concentrations of PySP are shown. α-tubulin (60 kD) was used as a loading control. The histogram shows the integrated density values (IDV) from the immunoblot for cleaved caspase-3. (c) Confocal immunocellfluorescence shows 145M-Twi and 145C-Wt stained with cytochrome C (Cyto C - green) and lysosomes labeled with lysosomal marker LysoTracker (LysoTr - red). Cell nuclei were stained with DAPI. The 145M-Twi and 145C-Wt were treated for 24h-period with 30 μM of glucosylsphingosine and psychosine. Increased lysosomal compartment (LysoTr –red) was noted in 145M-Twi cells. Under treatment with both glycosphingolipids, both 145M-Twi and 145C-Wt cells showed enhancement of lysosomal compartment, which was more evident in the 145M-Twi cells. Additionally, the 145M-Twi cells showed alterations in the Cyto C staining under glucosylphingosine and psychosine treatment. The long string pattern of Cyto C, representing intact mitochondria, became punctuated and dispersed, indicating the Cyto C translocation from the inner mitochondrial space into the cytosol. Higher magnification showing the Cyto C pattern is shown on the upper right corner of each panel of treated murine 145M-Twi and 145C-Wt cells.