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. 2022 Jun 7;41(14):e109958. doi: 10.15252/embj.2021109958

Figure EV3. HSBP represses HEI10 transcription by inhibiting HSFs.

Figure EV3

  1. Heatmap representation of transcript levels in Col‐0 and hsbp‐3 for Arabidopsis class A HSF genes in seedlings and buds from RNA‐seq data.
  2. RT–qPCR analysis of relative HEI10 transcript levels following transient transfection of HSFA1a and HSBP effector constructs in Arabidopsis protoplasts and after heat treatment (40°C for 1 h). Immunoblots of HSF1a‐HA and HSBP‐Myc in transfected protoplasts. Coomassie‐stained membrane served as a loading control.
  3. As in B, for HSFA7a and HSBP.
  4. DAP‐seq data of HSFs at HEI10 regulatory regions. Candidate HSEs (HEI10_1 to 5) around HEI10 are shown.
  5. As in (B), but showing ChIP–qPCR analysis of HSF7a at HEI10 regulatory regions in protoplasts expressing HSF7a‐HA. IgG was used as a negative control.
  6. As in (B), but showing HSBP ChIP–qPCR analysis in heat‐treated seedlings using anti‐HSBP antibody. Seedlings (10‐day) were incubated at 37°C for 3 h and used for ChIP analysis.
  7. RT–qPCR analysis of relative HEI10 transcript levels in Col and hsbp‐3 after heat treatment. Seedlings (10‐day‐old) were incubated at 37°C for 4 h. Data points (color) indicate values of replicates. Black dots and horizontal lines mean ± s.d. values (one‐sided Welch’s t‐test). n ≥ 6 two or three technical duplicates of three biological replicates.
  8. Co‐localization of the fluorescent fusion proteins HSBP‐RFP and HSFA1a‐GFP in protoplasts. Heat indicates incubation of transfected protoplasts at 40°C for 1 h. Scale bars: 5 μm.
  9. As in (H), but showing HSBP‐CFP and HSFA1a‐GFP (left), and HSBP‐GFP and nucleus marker (ARR2‐mRFP) (right) after H2O2 treatment. Scale bars: 5 μm.
  10. Nuclear location of HSBP in male meiocytes of HSBPpro:HSBP‐YFP plants. Nuclei spreads were stained with DAPI. Scale bars: 10 μm.
  11. Immunoblot analysis of HSBP and histone H3 levels using total proteins and nuclear extracts of Col seedlings. Seedlings (10 days old) were incubated at 37°C for 3 h. The Coomassie‐stained membrane was used as a loading control. Experiments were performed at least three times.
  12. Co‐immunoprecipitation analysis of Myc‐HSBP with HSF7a‐HA. IB, immunoblot; IP, immunoprecipitation. Ponceau S staining of the membrane, a loading control. Experiments were performed at least three times.

Data information: (B, C, E, F) Experiments were performed three times. Data points (black) indicate three technical duplicates of three biological replicates. Red dots and horizontal lines indicate mean ± s.d. values (one‐sided Welch’s t‐test).

Source data are available online for this figure.

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