Figure 8From: Target of rapamycin signaling regulates high mobility group protein association to chromatin, which functions to suppress necrotic cell deathDeregulation of HMG proteins induces necrosis. (A) H3WT cells were transformed with control vector or a vector expressing one of the five indicated yeast HMG genes from the GAL1 promoter. Equal numbers of cells were 5-fold serially diluted and spotted to SC-uracil plates containing glucose (repressive) or SC-uracil galactose (activating) media and incubated at 30°C for 4 days. (B-D) Cells containing control vector and either the galactose-inducible Hmo1 (B), Nhp10 (C) or Ixr1 (D) expression vectors were grown to log phase in raffinose media before serially diluting 5-fold and spotting to SC-uracil glucose plates. The remaining cultures were treated with 2% galactose for the indicated times before washing and spotting as described. Plates were incubated for 3 days at 30°C. (E) H3WT cells carrying control, GAL1-HMO1, GAL1-NHP10, or GAL1-IXR1 expression vectors were grown to log phase in SC-uracil raffinose media, induced with 2% galactose for 24 hours, stained with Annexin V and PI, and then analyzed by confocal microscopy. Scale bar indicates 10 μm. (F) The strains from (B-D) were induced with 2% galactose for 1 hour before preparing whole-cell extracts. 30 μg of extract was resolved by SDS-PAGE and immunoblotted with α-HA antibody to detect the HA-tagged Hmo1, Nhp10 and Ixr1. (G) Model for TORC1 signaling and regulation of HMG chromatin association. Details in the text. Gal, galactose; PI, propidium iodide; ura, uracil, vect, vector.Back to article page