Characterization of D. mawsoni lepa. (A) Coding sequence and encoded amino acid sequence. The box and the asterisk indicates the start codon, the stop codon, respectively. (B) Alignment of leptin-a homologs among the vertebrates. The signal peptide and conserved function domains are indicated with the boxes. Identical amino acid residues are shown in black and the amino acids with >50% similarity shown in gray. Two asterisks indicate the conserved cysteine residues, which form a single disulfide bridge. (C) Predicted tertiary structure in different species. Four α-helices are displayed in brown, blue, dark green, and green helices.

Expression patterns of DM-lepa in different tissues. The y axis indicates the relatively expression level with associated standard error bars, n = 3 (biological replicates), **P < 0.01, ***P < 0.001.

Positive selection analysis. Numbers at the branches indicate the bootstrap support. Amino acids marked in red are positive selection sites for D. mawsoni, *P < 0.05.

Over-expression of DM-lepa in ZFL cells. (A,B) DM-Lepa expression vector map. (C) Fluorescence observation after transfection of ZFL cells with empty plasmid and DM-lepa gene eukaryotic expression plasmid. Scale bar, 50 μm. (D) Western blot analysis of DM-LEPA in ZFL. (E) Assay of subcellular localization in ZFL cells. Green fluorescence show signal of DM-LEPA and blue nuclei stained with DAPI. Scale bar, 20 μm.

The effect of overexpression of DM-lepa on cells. (A,B) Cell proliferation was determined by assay of CCK8. (C) Apoptosis was evaluated with Hoechst/PI staining. PI stains dead cells or late-stage apoptotic cells with a red fluorescence, and Hochest stain the nuclei with a bright-blue fluorescence. (D) The percentage of apoptotic cells was determined using ImageJ software. (E) Protein levels of cleaved-caspase 3 within cells was detected by western blot analysis.β-actin as the loading control. (F) ROS was detected by flow cytometry and analyzed using FlowJo software. (G) The relative expression of ROS Data were analyzed and graphed using GraphPad Prism 8. The results are presented as mean ± SD (n = 3), *P < 0.05, **P < 0.01, ***P < 0.001.

Activation of stat3 signal pathway in DM-lepa-over-expressed cells. (A) Western blot analysis of the protein expression level of STAT3 in the cell lysates of the DM-lepa over-expression group and the control group. (B–F) A qRT-PCR assay of SOCS3/socs3a, c-myc/myca, Bcl-xl/bcl2l1, Bcl2/bcl2a, and Baxa/baxa mRNAs in control and DM-lepa transfected cells. The results are presented as mean ± SD (n = 3), **P < 0.01, ***P < 0.001.

Decreased p53 expression level and enhanced mdm2 expression in ZFL cells under cold temperature. (A) Western blot analysis of the protein expression level of P53 in the cell lysates of the DM-lepa over-expression group and the control group cells. (B) A qRT-PCR assay of MDM-2/mdm2 in control and DM-lepa transfected cells. The results are presented as mean ± SD (n = 3), ***P < 0.001.