Figure 4. Modulation of chaperone activity regulates translation elongation pausing.

(A) Polysome profiles of a thermotolerance experiment. 3T3 cells were treated with mild (green), severe (red), or mild prior to severe (magenta) heat shock, or left untreated (blue). P:M ratios are 1.4 in Control, 0.33 in HS8M, 0.1 in HS2S, and 0.2 in HS8M2S. Profile is representative of two replicate experiments, where relative P:M ratios were 22.4% ± 1.3% of Control in HS8M, 5.8% ± 1.7% of Control in HS2S, and 12.2% ± 2.7% of Control in HS8M2S. (B) Normalized footprint density along mRNAs for a biological replicate experiment and for cells pre-treated with mild heat shock (8 h at 42° C) prior to severe heat shock (magenta). (C) Polysome profiles of cells treated with the Hsp70 inhibitor VER-155008 (Massey et al., 2010) at a 20 μM concentration (Hsp70 inhibitor, cyan line) or DMSO (Control, blue line) for 3hrs. P:M ratios are 1.52 in Control and 0.7 in Hsp70 inhibitor. Profile is representative of three replicate experiments, where relative P:M ratios for Hsp70 inhibitor were 46.5% ± 3.8% of Control. (D) Normalized footprint density along mRNAs for control cells after 3 hours of VER-155008 treatment (cyan). Control and severe heat shock plots (dotted lines) are identical to those in B. (E) Normalized footprint density along mRNAs for 293T cells overexpressing GFP or Hspa1a, with or without 2 hours of severe heat shock.

See also Figure S4.