GSK-3 acts downstream of PP2A and the PI 3-kinase-Akt pathway, and upstream of caspase-2 in ceramide-induced mitochondrial apoptosis

JCS03473 Supplementary Material

Files in this Data Supplement:

• Supplemental Figure S1 -

  Fig. S1. Ceramide activates GSK-3β in various cell types. 10I (A), SK-N-SH (B), and A549 (C) cells were treated with 25 μM of C₂-ceramide for various time periods as indicated with or without 10 mM of the GSK-3β inhibitor LiCl. We used western blotting to determine GSK-3β phosphorylation (P-GSK-3β) at serine 9. Total GSK-3β protein was the control. The relative ratios of P-GSK-3β to total GSK-3β are shown. β-actin expression was used as an internal control.

• Supplemental Figure S2 -

  Fig. S2. Inhibiting GSK-3β blocks ceramide-induced apoptosis in various cell types. (A) 10I cells were treated with 25 μM of C₂-ceramide for 6 hours with or without the GSK-3β inhibitors LiCl (10 mM) and SB216763 (10 μM). Cell apoptosis was detected using annexin V-FITC detection and TUNEL reaction, and then flow cytometric analysis. The percentages of apoptotic cells are marked in the histograms. (B) SK-N-SH and A549 cells were treated with 25 μM of C₂-ceramide for 24 hours with or without LiCl (10 mM) and SB216763 (10 μM). Cell apoptosis was detected using TUNEL reaction and then flow cytometric analysis. The percentages of apoptotic cells are shown (means ± s.d. of triplicate cultures). Cells treated with DMSO were used as the reagent control.

• Supplemental Figure S3 -

  Fig. S3. Ceramide inhibits PI 3-kinase activity. 10I cells were transferred to hybridoma serum-free medium and then treated with 25 μM of C₂-ceramide for various time periods as indicated. The membrane and cytosolic proteins were extracted, and the expression of PI 3-kinase p85 regulatory subunit was detected using western blot analysis. The ratios of membrane PI 3-kinase to cytosolic PI 3-kinase are shown.