Fig. 8
From: Copper-enriched automotive brake wear particles perturb human alveolar cellular homeostasis
NAO BWPM induces pseudohypoxic HIF pathway activation in a copper-dependent manner. GSVA was used to interrogate a 15-gene signature indicative of HIF transcription factor signalling to generate a HIF score A GSVA of a 15-gene set HIF score. B Heatmap of genes within the HIF Score. C Correlation between the HIF score and Oxidative Stress Score. D Correlation between [Cu] and Oxidative Stress Score. E–G: ATII cells were transfected with either a hypoxia response element (HRE) reporter firefly luciferase plasmid, or hypoxia-inducible factor α C-activation domain (HIFα-CAD) firefly plasmid, and both with a Renilla luciferase control plasmid using lipofectamine LTX. Cells were then exposed to 8 µg/cm2 of NAO BWPM for 24 h and luciferase activity was determined using a Promega Dual-Luciferase® Reporter Assay System. E. The mechanism of the HIF-CAD reporter assay. F. FIH inhibition following NAO BWPM exposure was determined using a HIFα-CAD reporter. n = 3. G. HRE binding following NAO BWPM exposure was determined using an HRE reporter. The plasmid contained an HRE binding region, and increased HIF transcription factor complex binding to this HRE binding site causes increased luciferase activity. PHD inhibitor DMOG was used as a positive control. n = 3. H and I: ATII cells were exposed to NAO brake-wear PM (8 µg/cm2), TEPA (50 µM), NAC (10 mM), or Ascorbate (5 mM) alone for 24 h. The cells were also exposed to NAO and TEPA together for 24 h, with no pre-treatment, NAO + NAC and NAO + Ascorbate refer to 24 h NAO exposure after a one-hour pre-treatment with respective agents. H. Representative HIF1α western blot showing the impact of NAO BWPM on HIF1α stabilisation, as well as the impact of copper chelator TEPA. I. Densitometric analysis of the western blots. n = 3. In F, G, and I data was represented as mean + SEM. In A: Box contains median, upper, and lower quartiles, with whiskers representing the range. In A and I, a RM one-way ANOVA test was used with a Dunnett’s post-hoc test. In C and D, a Pearson’s correlation was used. In F and G, and two-tailed paired t-test was used