Inhibition of the Ubiquitin-Proteasome System Induces Stress Granule Formation -- Mazroui et al. 18 (7): 2603 Data Supplement - Supplemental Material -- Molecular Biology of the Cell

Inhibition of the Ubiquitin-Proteasome System Induces Stress Granule Formation
Mol. Biol. Cell Mazroui et al. 18: 2603

Supplemental Material

This article contains the following supporting material:

Supplemental 1 - Role of Hsp72 in SG disassembly in NIH-3T3. (A) Western blot analysis of the expression of Hsp72 during MG132 treatment. PARP was used as loading control. (B) NIH3T3 cells were treated for 3 or 6 h with 10 μM MG132 then fixed, permeabilized and used for immunofluorescence experiment to visualize SG formation using the anti-HuR and anti-G3BP antibodies. (C) NIH-3T3 cells were transfected with a pcDNA3 plasmid expressing the Flag-tagged Hsp72 protein. 48h later the cells were treated for 3h with10 μM MG132, then fixed, permeabilized, and used for the immunofluorescence experiment with anti-Flag and anti-HuR antibodies.
Supplemental 2 - GCN2-/- cells are able to form SGs in response to arsenite or Pateamine A. GCN2-/- cells were either untreated (Unt) or treated with 0.5 mM of arsenite (AS) for 30 min or with 0.5 μM Pateamine A (Pat) for 30 min, then analyzed by immunostaining with antibodies against HuR and FXR1. Shown are representative images from two independent experiments.
Supplemental 3 - While prolonged exposure of HeLa cells to MG132 prevented a significant increase in the phosphorylation of eIF2α by arsenite it did not prevent them from assembling SGs in response to Pateamine A or Hippuristanol. (A) Arsenite failes to significantly increases eIF2 phosphorylation in HeLa cells pretreated with MG132 for 7h. HeLa cells were either untreated or treated with 10 μM MG132 for various periods of time (0 to 8 hours) or with 0.5 mM arsenite (AS) for 30 min or 7h with MG132 followed by 30min exposure to 0.5 mM of arsenite (AS), then analyzed by western blot using the anti- phospho-eIF2α (P-eIF2α) or -eIF2α antibodies. (B) HeLa cells pretreated for 7h with MG132 are still able to form SGs in response to Pateamine A or Hippuristanol. HeLa cells were either treated with only 0.5 mM arsenite (AS) or 0.5 μM Pateamine (Pat) or Hippuristanol (Hipp) for 30 min or with 10 μM MG132 7h followed by 30min exposure to 0.5 mM AS or 0.5 μM Pat or 0.5 μM Hipp then analyzed by immunostaining with antibodies against HuR and Hsp72. HeLa cells treated with only 0.5 mM AS, 0.5 μM Pat or Hipp were used as a positive control for AS-, Pat-, Hipp-induced SGs. Shown are representative images from two independent experiments.
Supplemental 4 - MG132 treatment results in a three fold increase of G3BP and FMRP proteins in the insoluble fraction. Untreated HeLa cells, or cells that were incubated with MG132 for three hours, were lysed and total cell extracts (T), insoluble (Ins) and soluble (S) fractions were prepared. Proteins of each fraction were resolved on a 10% SDS-polyacrylamide gel, and analyzed by western blot. For C, D and E, representative of three independent experiments are shown.