Fig 2: Netrin-1 as a PMN-dependent Hif1A target critical for HIF-dependent cardioprotection. (A,B) Isolated human PMNs from healthy donors underwent hypoxia for 0, 2 and 4 h followed by cell lysis and total protein harvest. (A) Immunoblot for NTN1. b-Actin (Actb) served as the loading control. One representative blot of three experiments is shown. (B) Quantitation by densitometry of the NTN1 immunoblot results relative to b-Actin (n = 2 per group; per group mean ± SD; compared by one-way ANOVA followed by Tukey’s multiple comparison test). (C–E) Ntn-1loxP/loxP mice were crossed with Cre recombinase-expressing mice under the control of the lysozyme 2 promoter (LysM Cre+) to generate mice deficient in myeloid-specific Ntn-1. Mice received vehicle or 1 mg DMOG 4 h prior to ischemia, then underwent 60 min of ischemia followed by 120 min of reperfusion and assessment of myocardial injury. (C) Infarct sizes were determined in vehicle or DMOG treated Ntn-1loxP/loxP LysM Cre+ (Ntn-1loxP/loxP LysM Cre+ with vehicle n = 5; Ntn-1loxP/loxP LysM Cre+ with DMOG n = 6). (D) Representative infarct staining from vehicle or DMOG treated Ntn-1loxP/loxP LysM Cre+. (E) Troponin serum levels of vehicle or DMOG treated Ntn-1loxP/loxP LysM Cre+ mice (Ntn-1loxP/loxP LysM Cre+ with vehicle n = 5; Ntn-1loxP/loxP LysM Cre+ with DMOG n = 6). All data are presented as mean ± SD. Statistical significance assessed by two-sided, unpaired Student’s t-test. DMOG-induced cardioprotection was absent in Ntn-1loxP/loxP LysM Cre+.