A: Flowchart depicting cold and hot temperature treatments in zebrafish for sampling. Three-month-old zebrafish reared at 28 °C were subjected to cold treatment by exposure to 18 °C for 12 h and 8 °C for 12 h. Hot temperature treatment was carried out by exposing fish to 38 °C for 4 h, with controls maintained at 28 °C. Samples for qRT-PCR and immunoblot analysis were collected at three temperature-time points (28 °C, 38 °C 4 h, 8 °C 12 h). Sample size: n=30. B: Dusp1 expression under cold and hot treatment measured using qRT-PCR compared with normal temperature controls. Data are mean±SD. ^**: P<0.01; ^***: P<0.001. C: Western blot analysis of DUSP1 protein levels in gills under thermal stress, with ACTB as the loading control. D: Schematic of targeted dusp1 gene editing by CRISPR/Cas9 and disruption of dusp1 at exon 1. E: Western blot validation of loss of dusp1 in dusp1-KO fish indicated by two tissues examined.

Thermal challenges were the same as described above. Sample size was 30 for each thermal challenge and at least three biological replicates were performed. A: Kaplan-Meier curves of three genotypes (dusp1^-/-, dusp1^+/-, and WT) treated by 8 °C exposure. B: Quantified apoptotic signal in gills under cold exposure analyzed by ImageJ software. Student’s t-test, ^*: P<0.05; ^**: P<0.01. C: TUNEL assay showing apoptotic signal in gills under 8 °C treatment. Scale bar: 100 µm. D: Kaplan-Meier curves of three genotypes (dusp1^-/-, dusp1^+/-, and WT) under 38 °C exposure. E: Quantified apoptotic signal in gills under hot exposure analyzed by ImageJ software. Student’s t-test, ^**: P<0.01. F: TUNEL assay of apoptosis in gills under hot (38 °C for 4 h) treatment. Scale bar: 100 µm. G: Differences in upper and lower lethal temperature tolerance between WT and dusp1^-/- zebrafish. LOE indicates loss of equilibrium. Half survival rates (i.e., LOE rate) of dusp1^-/- fish occurred at 10 °C and 36 °C, respectively, under cold and heat challenges, while WT fish exhibited half survival rates at 8 °C and 38 °C, respectively. One-way ANOVA, ^*: P<0.05; ^**: P<0.01; ^***: P<0.001. H: Rescue of dusp1^-/- mutant by zebrafish dusp1 mRNA (Ad-dusp1). Adding dusp1 mRNA restored the survival rate of embryos under cold (8 °C) and hot (38 °C) treatment. The same amount of GFP mRNA (Ad-ctrl) and dusp1^-/- embryos without injection were used as controls. One-way ANOVA, ^***: P<0.001.

A: KEGG enrichment analysis of DEGs between dusp1^-/- and WT treated at different temperatures. B: Markedly reduced expression of mitochondrial-related genes in KEGG “oxidative phosphorylation” pathway identified in dusp1^-/- versus WT zebrafish at three temperatures. Log₂ transformation of gene fold-change is indicated by the color-coded scale. C: DCFH-DA probe for ROS in the gills of dusp1^-/- and WT zebrafish under different temperature treatments. Scale bar: 50 µm. D: Statistics of ROS fluorescence intensity detected in the gills of temperature-treated dusp1^-/- and WT fish with ImageJ software. Statistically significant results between dusp1^-/- and WT zebrafish are indicated by asterisks. Level of expression in WT (28 °C) was used as a normalizing factor and set to 1. Sample size: n=6 for each measurement, one-way ANOVA, ^**: P<0.01. E: Western blot assays of activated caspase-3, reflecting severity of mitochondrial-dependent apoptosis. ACTB was used as the loading control.

A: Scarlet (red) and brilliant green (blue) staining of gill sections of dusp1^-/- and WT zebrafish under different temperature treatments. Mitochondria are in dark red and gill filaments are in green. Scale bar: 50 µm. Sample size=6 for each temperature-time treatment. B: Relative intensity of red fluorescence of the gill reflecting average number of mitochondria present in the gill section. Student’s t test, ^*: P<0.05; ^**: P<0.01. Sample size: n=6. C: Transmission electron microscopy of mitochondrial structure in the gills of dusp1^-/- and WT zebrafish under different temperature treatments. Dark circular structures are typical mitochondria. Mitochondria with abnormal shapes are indicated by arrows. Scale bar: 1 µm. D: ATP production measured by ATP Assay Kit (Beyotime, China) in dusp1^-/- and WT zebrafish gills under different temperature treatments. Student’s t test, ^*: P<0.05; ^**: P<0.01. Sample size: n=6 for each temperature-time point. E: Oxygen consumption rate measured using a respiratory oxygen consumption meter in dusp1^-/- and WT fish under different temperature treatments. Sample size: n=6 for each curve, one-way ANOVA, ^*: P<0.05, ns: No significant difference. F–I: Western blot analyses of p-P38, p-ERK, and p53 in dusp1^-/- and WT zebrafish exposed to different temperatures. Sample size n=6 for each measurement.

A: Flow cytometry measurement of ROS content at three temperature-time points (37 °C, 43 °C 5 h, 13 °C 15 h) using a ROS Assay Kit (Beyotime, China) in DUSP1^-/- and control HEK293T cells. B: Statistics of ROS fluorescence intensity in samples measured in (A) analyzed using FlowJo software. One-way ANOVA, ^*: P<0.05; ^***: P<0.001. C, D: PI staining to detect dead cells in DUSP1^-/- and control HEK293T cells exposed to high and low temperatures. Scale bar: 100 µm. Student’s t-test, ^*: P<0.05; ^***: P<0.001. E: OD450 values of DUSP1^-/- and control cells measured by CCK8 assay. One-way ANOVA, ^*: P<0.05; ^**: P<0.01. F: Western blot analysis of activated caspase-3 to detect mitochondrial dependent apoptosis in DUSP1^-/- and control cells. ACTB was used as the loading control. G: MitoTracker staining of DUSP1^-/- and control cells to reveal mitochondrial morphology at different temperatures. Scale bar: 20 µm. H: Transmission electron microscopy of mitochondrial structure in DUSP1^-/- and WT HEK293T cells exposed to three temperatures. Arrows in panels of DUSP1^-/- cells indicate fragmented mitochondria. Scale bar: 2 µm. I, J: JC-1 probe to measure mitochondrial membrane potential in DUSP1^-/- and WT cells examined under fluorescence microscopy. CCCP was used as a positive control to induce a decrease in mitochondrial membrane potential. Scale bar: 20 µm. One-way ANOVA, ^**: P<0.01; ^***: P<0.001. K: Relative levels of ATP production in DUSP1^-/- and control cells exposed to three temperatures. One-way ANOVA, ^*: P<0.05.

A–C: Western blotting of p-P38 and P53 in DUSP1^-/- and WT 293T cells at three temperature time points. D–F: Western blot analyses of p-P38 and p53 in presence of Y27632. G, H: Western blot analysis of apoptosis-related protein BAX. I, J: Western blotting showing increased phosphorylation of DRP1 at S616 in DUSP1^-/- cells. ACTB was used as the loading control.

Red arrows indicate elevation in proteins when dusp1 is deleted (indicated by red cross).