Fig. 5.

Functional tests of TRIM17 for its role in hypoxia adaptation. A Coding sequence alignment of TRIM17 orthologous genes from mouse, NMR, BMR, and 7 other mammals. One stop codon in NMRs and one stop codon/four frameshifts in BMRs were observed that resulted in disrupted coding potential of TRIM17 gene. B–D Examples of inactivating mutations in toothed whales (B), baleen whales (C), and pangolins (D). The positions of all mutations shown in this figure are labelled under the corresponding alignment using mouse Trim17 coding sequence as reference. E–F Immunoblot analyses for Trim17 protein level changes during hypoxia treatments in primary cortical neurons (E) and N2a cell lines (F). “+” indicates hypoxia treatment and “−” indicates normoxia treatment. Quantification and relative fold change analysis of protein level showed a significant decrease of Trim17 protein induced by hypoxia in both primary cortical neurons and N2a cells. Data from three independent assays were used to perform paired two-tailed t-test, *P < 0.05. G–I Knockdown of Trim17 expression by shRNAs (shTrim17-1 and shTrim17-2) provided neuroprotection under hypoxia (1% O₂) compared to control (shCtrl). Apoptotic cells were detected by staining cells with PI and Annexin V-FITC followed by flowmetry analysis. Cells with Trim17 knockdown showed significantly lower apoptosis rate under hypoxia compared to control, while no significant changes were observed under nomorxia (G, H), paired two-tail t-test, *P < 0.05, **P < 0.01. Immunoblot analysis for corresponding cells showed elevated Mcl-1 protein level and decreased activated caspase-3 protein level under hypoxia in cells with Trim17 knockdown (I)