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. 2019 Oct 28;38(24):e102566. doi: 10.15252/embj.2019102566

Figure 4. Phosphorylated HSF1 at Ser326 recruits SGO2 to the HSP70 promoter.

Figure 4

  1. Interaction of HSF1 with SGO2. Extracts in NP‐40 lysis buffer were prepared from control MEF cells (C) and cells treated with heat shock at 42°C for 30 min (HS). Complexes co‐immunoprecipitated using SGO2 antibody were blotted with HSF1 or SGO2 antibody. An arrowhead indicates SGO2 band.
  2. Schematic view of the mouse HSPA1A locus. Shaded boxes indicate DNA regions (1–9) amplified by RT–qPCR.
  3. Occupancy of HSF1 and SGO2 on the HSPA1A locus in control MEF cells (Cont.) and cells treated with heat shock at 42°C for 5, 10, and 30 min (HS). ChIP‐qPCR on each region shown in (B) was performed.
  4. HSF1‐dependent recruitment of SGO2 to HSP promoters. MEF cells, which were infected with Ad‐sh‐mSGO2‐KD1, Ad‐sh‐mHSF1‐KD2, or Ad‐sh‐SCR, were treated with heat shock at 42°C for 0, 5, and 30 min. ChIP‐qPCR analyses on promoters of HSP70.3 (HSPA1A), HSP40 (DNAJB1), and HSP25 (HSPB1) were performed (left). Extracts of cells were subjected to immunoblotting (right).
  5. HSF1 mutants lacking the d‐region did not interact with SGO2. HSF1‐null cells were co‐infected with adenovirus expressing hHSF1 mutants and HA‐tagged mSGO2, and treated with heat shock at 42°C for 30 min. Complexes co‐immunoprecipitated with hHSF1 were blotted with HA or HSF1 antibody.
  6. HSF1‐S326 mutants did not interact with SGO2. HSF1‐null cells were co‐infected with adenovirus expressing hHSF1‐S326 mutants and HA‐tagged mSGO2, and untreated (Cont.) or treated with heat shock at 42°C for 30 min (HS). Complexes co‐immunoprecipitated with hHSF1 were blotted with HA, HSF1, or HSF1‐phospho‐S326 (S326P) antibody.
  7. SGO2 occupancy in the presence of HSF1‐S326 mutants. Cells, in which endogenous HSF1 was substituted with each mutant, were untreated (Cont.) or treated with heat shock at 42°C for 5 min (HS). ChIP‐qPCR analyses of HSF1 and SGO2 in the pHSE were performed (left). Extracts of cells were subjected to immunoblotting (right). Red arrows indicate phosphorylated hHSF1 bands.
  8. HSP70 expression in the presence of hHSF1 mutants. Cells, in which endogenous HSF1 was substituted with each mutant, were untreated (Cont.) or treated with heat shock at 42°C for 30 min (HS). HSP70 mRNA levels were quantified by RT–qPCR.
Data information: Asterisks in (A), (F), and (G) indicate non‐specific bands. Error bars indicate SD (n = 3). Asterisks indicate *P < 0.05 or **P < 0.01 by Student's t‐test in (C, D, G, and H).