HIF-1α protein accumulation is posttranscriptionally regulated by mTOR in LPS-stimulated human PMNs. (A) We assessed HIF-1α (top) and IL-8 (bottom) transcript expression with the use of RNA-seq in control and stimulated human PMNs isolated from a single healthy adult donor. (B) Using RNA-seq, we determined the sequence of the 5′-UTR for the transcript coding for HIF-1α and predicted both its secondary structure (image) and the energy required to resolve its secondary structure (−182.4 kcal/mole).²²  (C) qPCR was used to assess HIF-1α mRNA transcript expression in PMNs isolated from 3 different healthy adults at baseline and after LPS-stimulation over 1 hour. The results are presented as fold change over baseline ± SEM with baseline HIF-1α mRNA expression arbitrarily set at 1 (dashed line). One-way ANOVA analysis found no statistically significant differences. (D) We determined HIF-1α protein accumulation using In-Cell Western analysis for PMNs isolated from healthy adults at baseline and after LPS-stimulation for 1 hour with or without 1-hour pretreatment with rapamycin (200nM) or cycloheximide (500nM). The results are presented as fold change over baseline with baseline HIF-1α expression arbitrarily set at 1. The 1-way ANOVA with Tukey Multiple Comparisons posttesting was used. These results are representative of 5 separate experiments performed with human PMNs isolated from 5 different healthy adult donors. Statistical significance with *P < .05 and **P < .001. (E) We again assessed HIF-1α protein accumulation using In-Cell Western techniques in PMNs isolated from healthy adults at baseline and after LPS-stimulation for 1 hour with or without 1-hour pretreatment with rapamycin (200nM), torin 1 (250nM), or cycloheximide (500nM). The results are presented as absolute fluorescence normalized to cell number and are representative of 2 separate experiments performed with human PMNs isolated from 2 different healthy adult donors. (F) Human PMNs were stimulated with PAF (10nM) for 1 hour after a 1-hour pretreatment with or without rapamycin (200nM) or FK-506 (200nM). HIF-1α protein expression was quantified with semiquantitative immunocytochemistry, and the columns represent the mean fold change over baseline ± SEM. Control HIF-1α protein expression was arbitrarily set at 1 (dashed line). We used the single tailed Student t test for statistical analysis. *Significant difference (P < .05) between samples from PAF-stimulated PMNs and rapamycin-pretreated, PAF-stimulated PMNs. These experiments were performed with PMNs isolated from 3 different healthy adult donors.