Inhibition of HSP90 function in zebrafish embryos by radicicol. (A) Zebrafish embryos were treated with 5 μM radicicol (radicicol group) or DSMO (control group) for indicated times, then the mRNA levels of BAG3 and HSPB1 were detected using qRT-PCR. (B,C) Zebrafish embryos were treated with 5 μM radicicol for 24 h, then HSP90 mRNA (B) and protein (C) levels were determined by qRT-PCR and immunoblot. n = 3, two tailed t-test, ***P < 0.001.

Impact of embryonic HSP90 inhibition on cold tolerance of zebrafish. (A) Schematic diagram of the cooling time course. The temperature decreased from 28 to 18°C at a rate of 1°C/h, remained at 18°C for 12 h (overnight), then decreased at a rate of 1°C/h until reaching a final temperature of 8°C. (B) Zebrafish from control and radicicol group were exposed to a temperature of 8°C according to the protocol in (A), then survival time and the median lethal time (LT50) were measured. Dotted blue line (bottom) indicates the starting time at 8°C. Broken orange lines indicate the range of survival time of zebrafish of control group. Red box indicates zebrafish with increased survival time (cold tolerant zebrafish). (C,D) Body weight and length of fishes with different survival time for both groups.

HSP90 inhibition enhanced cold tolerance is inheritable. (A) Three-month-old F1 zebrafish of cold tolerant parental fish (Radicicol-F1) and control F1 zebrafish (control) were exposed to cold pressure as in Figure 2A, then survival time and the median lethal time (LT50) were observed. (B) Three-month-old F1 zebrafish by breeding cold tolerant and control parental fish (Radicicol-con-F1) and control F1 zebrafish (control) were exposed to cold pressure as in Figure 2A, then survival time and the median lethal time (LT50) were observed.

GO and KEGG pathway enrichment analyses of DEGs by RNA-seq. The total RNAs from the muscle tissues of three cold tolerant zebrafish from radicicol group (CT) and three control zebrafish (CON) were subjected to RNA-seq and subsequent analyses. (A) The Volcano plots of DEGs between cold tolerant (CT) and control (CON) fish. Abscissa represents log2 (fold-change), and ordinate represents –log10. Red dots denote the significantly up-regulated genes. Green dots denote the significantly down-regulated genes. Gray dots denote the non-differentially expressed genes. (B,C) GO and KEGG pathway enrichment analyses of up-regulated and down-regulated genes, respectively. Red bars represent up-regulated genes. Green bars represent down-regulated genes. Abscissa represents fold enrichment. (D) Motif enrichment analysis of up-regulated genes.

Roles of atg9b and psmd12 in cold tolerance in zebrafish cells. (A,D) zebrafish ZF4 cells were treated with 5 μM radicicol for indicated times, then the mRNA levels of atg9b and psmd12 were examined using qRT-PCR. (B,E) Zebrafish ZF4 cells were infected with shRNA lentivirus targeting atg9b or psmd12 for 24 h, then the mRNA levels of atg9b and psmd12 were examined using qRT-PCR. (C,F) Zebrafish ZF4 cells were infected with shRNA lentivirus targeting atg9b or psmd12 for 24 h, then the cells were moved to an incubator at 10°C for 24 h, then cell viability was examined using trypan blue staining. All experiments were performed in triplicate. n = 3, two tailed t-test, ^∗p < 0.05, ^∗∗p < 0.01, ^∗∗∗p < 0.001.

The impact of embryonic low-conductivity stress on cold tolerance of zebrafish. (A) Zebrafish embryos were incubated in low-conductivity water or regular embryo water for indicated times, the mRNA levels of BAG3 and HSPB1 were determined by qRT-PCR (n = 3, two tailed t-test, ^∗∗∗P < 0.001). (B) Three-month-old zebrafish were subjected to cold pressure as in Figure 2A, then survival time was measured. (C) Three-month-old F1 zebrafish of cold tolerant parental fish (low-conductivity-F1) and control F1 zebrafish (control) were exposed to cold pressure as in Figure 2A, then survival time was observed. (D) Three-month-old F1 zebrafish by breeding cold tolerant and control parental fish (low-conductivity-con-F1) and control F1 zebrafish (control) were exposed to cold pressure as in Figure 2A, then survival time was observed.