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Fig. 4 VtgR-mediated heat stress response mechanism of vitellogenic oocyte formation in mud crabs. a H3K27ac signal intensity in the VtgR gene and VtgR expression level in different groups of mud crabs. A 4.99 kb deletion in the nineteenth intron of VtgR removes an enhancer crucial for both vitellogenic oocyte formation and heat stress responsiveness in “abnormal” crabs. Horizontal bars below the H3K27ac signals indicate enriched regions identified by MACS2. b Normalized read density for the differential peak within the deletion in normal stage II mud crabs reared at 25 °C and 33 °C. c Schematic representation of the heat shock factor 1 (HSF1) binding site on the VtgR promoter. d Relative induction of VtgR promoter activity in Drosophila S2 cells transfected with HSF1 in a dosage-dependent manner, as assessed by dual luciferase assays (n = 4 wells). Statistical significance was determined by one-way ANOVA with Tukey’s test. Representative results from three independent experiments are shown. e Electrophoretic mobility shift assay (EMSA) demonstrating HSF1 binding to the VtgR promoter. Representative results from three independent experiments are shown. f Relative induction of VtgR promoter activity, with or without the VtgR-Enhancer-25°C or VtgR-Enhancer-33°C, mediated by expressed HSF1 in Drosophila S2 cells, as measured by dual luciferase assays (n = 4 wells). Protein expression levels were detected by Western blotting, with actin as a protein loading control. Statistical significance was determined by two-sided Student’s t-test. g A proposed scheme for the VtgR-mediated heat stress protection mechanism of vitellogenic oocyte formation in mud crabs. Bars represent the mean ± SD. P values are indicated above the plots, with P < 0.05 considered statistically significant. Source data are provided as a Source Data file.