HSF1 is asymmetrically activated in KV of zebrafish embryos (A) Immunostaining of phosphorylated HSF1 at serine 326 as classified by the expression pattern of left/right ratio (>1: left, <1: right, ∼1: bilateral) in KV of zebrafish embryos at 8-somite stage. Cilia is indicated by acetylated α-tubulin. a: anterior, p: posterior, L: left, R: right. Scale bar: 20 μm. (B) Statistical analysis of the percentage for three groups in (A), n = 3 from 56 embryos. Data are expressed as mean ± SEM, p < 0.05. (C) Statistical analysis of the expression of pS326 HSF1 in the left and right part of KV in the case of left/right ratio >1, n = 5. Data are expressed as mean ± SEM, p < 0.05. (D) Immunostaining of wild-type zebrafish embryos (WT) or embryos treated with c21orf59 MO or control morpholino (cMO). Scale bar: 25 μm. (E) Statistical analysis of the expression level of pS326 HSF1 in the left and right part of KV in (D) (n = 4). Data are expressed as mean ± SEM, p < 0.05.

HSF1 is required for the LR asymmetry establishment of zebrafish embryos (A) The effect of hsf1 knockdown by morpholino (MO) injection on the LR asymmetry establishment in zebrafish embryos. (B) The effect of CRISPR/Cas9-mediated knockout of hsf1 on the LR asymmetry establishment in zebrafish embryos.

Immediate activation of HSF1 by fluid shear stress (A) The illustration of the flow chamber device for fluid shear stress experiments. (B) Western blot results of phosphorylated HSF1 at Serine 326 or 303 in MDCK cells after shear stress stimuli for different time. (C) Statistical analysis of the results in (B) (n = 3). The ratio of phosphorylated HSF1 at Serine 326 in nuclei and cytoplasm (N/C) in (B) (n = 10 ). Data are expressed as mean ± SEM, p<0.05. (D) Luciferase reporter assay for HSE activity in MDCK cells treated with shear stress for 1 h. (n = 3). Data are expressed as mean ± SEM, p < 0.05. (E) Fluorescent immunostaining of phosphorylated HSF1 at Serine 326 in MDCK cells 30 min after shear stress. Scale bar: 25 μm. (F) The ratio of phosphorylated HSF1 at Serine 326 in nuclei and cytoplasm (N/C) in (E). Data are expressed as mean ± SEM, p < 0.01.

HSF1 activity was regulated by substrate stiffness (A) The illustration of the experiment device for substrate stiffness stimuli to cultured cells. (B) Fluorescent immunostaining of phosphorylated HSF1 at Serine 326 in MDCK cells under substrate stiffness stimuli. Scale bar: 10 μm. (C) The ratio of phosphorylated HSF1 at Serine 326 in nuclei and cytoplasm (N/C) in (B) (n = 9). Data are expressed as mean ± SEM, ∗p < 0.05, ∗∗p < 0.01. (D–I) Fluorescence intensity of phosphorylated HSF1 at Serine 326 and DAPI along the lines in the magnified figures in (B).

Ca2+ and Akt signals were involved in HSF1 activation by shear stress (A) Western blot results of phosphorylated HSF1 at serine 326 and total HSF1 in MDCK cells under shear stress in the presence of inhibitors or DMSO. (B) Statistical analysis of the results in (A) (n = 3). Data are expressed as mean ± SEM, ∗p < 0.05, ∗∗p < 0.01,∗∗∗p < 0.001 (C) Western blot results of phosphorylated HSF1 at serine 326 and total HSF1 in MDCK cells under shear stress in the presence of Ca2+ antagonist or Ca2+. (D) Statistical analysis of the results in (B) (n = 3). Data are expressed as mean ± SEM, ∗p < 0.05, ∗∗p < 0.01. (E) The expression level of hsp70 mRNA in MDCK cells under shear stress in the presence of Ca2+ antagonist or Ca2+ (n = 3). Data are expressed as mean ± SEM, ∗p < 0.05, ∗∗p < 0.01.

Ca2+ and Akt signals were involved in HSF1 activation in vivo (A) Immunostaining of phosphorylated HSF1 at serine 326 in zebrafish embryos treated with DMSO or 1 μM thapsigargin (TG), scale bar: 25 μm. (B) The left/right ratio of phosphorylated HSF1 at serine 326 in (A) (n = 3). Data are expressed as mean ± SEM, ∗p < 0.05. (C) Immunostaining of phosphorylated HSF1 at Serine 326 in zebrafish embryos treated with DMSO or 5 μM MK-2206 (Akt inhibitor), scale bar: 25 μm. (D) The left/right ratio of phosphorylated HSF1 at serine 326 in (C) (n = 4). Data are expressed as mean ± SEM, ∗p < 0.05.