Search

Additional file 5: Supplementary Table 1. Complete list of antibodies used in this study.

Additional file 3: Supplementary Figure 3. Che-1 phosphorylation is required for its pro-proliferative ability. A: WB analysis with the indicated antibodies showing the transfection efficiency of the experiment described in Fig. 3A. B, C and D: Cell proliferation analysis (left) and relative WB analysis (right) of KMS27, HeLa and 293 T cells transiently transfected with Myc-Che-1, Myc-Che-1 3S or control vector (pCS2MT). Bar plot shows the average number of cells observed in these experiments (n = 5). E: Cell number-normalized total RNA quantification of the indicated cell line transiently transfected with Myc-Che-1 wt, 3S mutant or control vector. Error bars represent the SD of triplicate experiments (n = 3). F: Nuclear extracts from HCT116 cells transiently transfected with Myc-Che-1 wt or 3S and subjected to IP with H3 antibody. Immunoprecipitated complexes were then analysed by WB with the indicated antibodies. Input corresponds to 10% of the nuclear extracts used for IP. G: Cell proliferation analysis of KMS27 cells transiently transfected with the indicated peptides. Bar plot shows the average number of cells observed in these experiments (n = 3). Statistical significance is indicated by asterisks as follow: *P

Additional file 4: Supplementary Figure 4. CK2 phosphorylates Che-1. A: Nuclear extracts from HCT116 cells were subjected to IP with CK2 antibody. Immunoprecipitated complexes were then analysed by WB with the indicated antibodies. Input corresponds to 10% of the nuclear extracts used for IP. B and C: Representative WB analyses of total cell extracts showing the transfection efficiency of the experiments described in Fig. 5B and C. D: The panel shows the MS/MS spectrum of the phosphopeptide YLVDGTKPNAGSEEISSEDDELVEEK identifying the phosphorylation of Che-1 residues S320 and S321. E: Representative WB analyses of total cell extracts showing the transfection efficiency of the experiment shown in Fig. 5D. F: Nuclear extracts from HCT116 cells treated or not with 80 μM TBB for 4 h and then subjected to IP with Che-1 antibody. Immunoprecipitated complexes were then analysed by WB with the indicated antibodies. Input corresponds to 10% of the nuclear extracts used for IP. G: WB of the IP experiments used for densitometryc analysis shown in Fig. 5E.

Additional file 2: Supplementary Figure 2. Che-1 is highly phosphorylated. A: Two different replicates of 2D- experiments of total cell extracts from HeLa cells treated or not with λ-PP related to Fig. 2B. B: Two different replicates of 2D-Gel electrophoresis (top) and representative WB (bottom) of HeLa cells transiently transfected with Myc-Che-1 or Myc-Che-1 3S, related to Fig. 2D.

Mammalian target of rapamycin (mTOR) is a key protein kinase that regulates cell growth, metabolism, and autophagy to maintain cellular homeostasis. Its activity is inhibited by adverse conditions, including nutrient limitation, hypoxia, and DNA damage. In this study, we demonstrate that Che-1, a RNA polymerase II-binding protein activated by the DNA damage response, inhibits mTOR activity in response to stress conditions. We found that, under stress, Che-1 induces the expression of two important mTOR inhibitors, Redd1 and Deptor, and that this activity is required for sustaining stress-induced autophagy. Strikingly, Che-1 expression correlates with the progression of multiple myeloma and is required for cell growth and survival, a malignancy characterized by high autophagy response.