Chemical and siRNA-mediated inhibition of catalase activity in A549 cells enhances the cytotoxic effects of heme.

<p><b>(A)</b> A549 cells were pre-treated with 5 mM 3-AT for 2 hours and then challenged with 100 μM heme for 36 hours. Cell viability was measured by alamar blue. <b>(B)</b> A549 cells were transfected with both 10 nM HO-1 and human catalase siRNAs for 24 hours and then challenged with 100 μM heme for 36 hours. Cell viability was measured by alamar blue. In both cases, data are means ± SEM of 3 independent experiments. *p≤0.05.</p

Heme exposure of HO-1KO A549 cells leads to lysosomal rupture and loss of viability which is prevented by pre-incubation with DFO.

<p>A549 cells were challenged with 100 μM heme for 24 hours and then stained with 5 μg/ml AO. <b>(A)</b> A549 cells deficient in intact lysosomes (‘pale’ cells) were measured by flow cytometry. <b>(B)</b> The summary of data shown in (A). Data are means ± SEM of 3 independent experiments. *p≤0.01. <b>(C)</b> A549 cells were pre-treated with 1 mM DFO for 2 hours and then challenged with 100 μM heme for 24 hours. Cell viability was measured using alamar blue. Data are means ± SEM of 3 independent experiments. *p≤0.05.</p

HO-1KO in A549 cells sensitizes the cells to heme-mediated cytotoxicity.

<p><b>(A)</b> A549 cells were transfected with the indicated HO-1 siRNA for 24 hours, then treated with or without 100 μM heme for 36 hours. Western blot shows substantial reduction in HO-1 mRNA expression in cells transfected with HO-1 siRNA. <b>(B)</b> Cells were exposed to 100 μM heme for 36 hours and viability was assessed with Alamar blue reduction. Data are means ± SEM of 6 independent experiments. *p≤0.01. <b>(C)</b> Human bronchial epithelial cells (HBEC) were transfected with the indicated HO-1 siRNA for 24 hours, and HO-1 expression was measured by western blot. <b>(D)</b> HBEC were transfected with the indicated HO-1 siRNA for 24 hours, then treated with or without 25 μM heme for 24 hours. Cell viability was measured by Alamar blue reduction.</p

TLR4 knockdown suppresses heme accumulation in HO-1KO A549 cells.

<p>Following 24 hour transfections with both 10 nM TLR4 and HO-1 siRNAs, the cells were challenged with 100 μM heme for 6 hours, detached with 0.05% trypsin, washed twice and lysed using snap freezing and thawing. Heme was measured spectrophotometrically. Data are means ± SEM of 3 independent experiments. *p≤0.01.</p

HO-1KO A549 cells accumulate increased amounts of “loose” and total intracellular iron.

<p>The cells were transfected with HO-1 siRNA for 24 hours and then challenged with 100 μM heme for a further 24 hours. The cells were harvested by scraping and extracted with cold 10% perchloric acid. <b>(A)</b> Concentration of “loose” iron in the extract supernatants. <b>(B)</b> “Bound” intracellular iron concentrations. Note that in a nitric acid digest, “bound” iron includes not just ferritin bound iron but also heme iron. Data are means ± SEM of 3 independent experiments. *p≤0.01.</p

HO-1KO blocks heme-mediated induction of ferritin and compensatory over-expression of ferritin H chain prevents heme-mediated cytotoxicity.

<p><b>(A)</b> A549 cells were transfected with 10 nM HO-1 siRNA and western blot indicates pronounced ferritin H chain induction in control cells and those transfected with scrambled siRNA but not HO-1 siRNA. <b>(B)</b> The summary of normalized ferritin H chain levels shown in (A). Quantification of ferritin H chain expression normalized to actin (n = 3 independent experiments). <b>(C)</b> Over-expression of ferritin H chain in HO-1KO A549 cells blocks heme-mediated cytotoxicity. A549 cells were transfected with 2 μg of FTH1 expressing pCMV6-ferritin for 24 hours (± HO-1 siRNA) and then challenged with100 μM heme for 36 hours. Cell viability was measured with alamar blue. Data are means ± SEM of 3 independent experiments. *p≤0.01.</p

siRNA-mediated knockdown of TLR4 suppresses heme-induced cytotoxicity in A549 cells.

<p><b>(A)</b> Western blot showing successful knockdown of TLR4 after 24 hour exposure to 10 nM TLR4 siRNA. <b>(B)</b> TLR4 knockdown prevents the cytotoxic effects of 36 hour exposure of A549 cells to 100 μM heme (viability assessed by alamar blue). Data are means ± SEM of 3 independent experiments.*p≤0.01.</p

Knockdown of TLR4 in A549 cells suppresses heme-induced intracellular ROS generation.

<p>Following 24 hour transfection with both HO-1 and TLR4 siRNAs the cells were challenged with 100 μM heme for a further 24 hours. The cells were then detached with 0.05% trypsin and stained with 10 μM DCFDA at 37°C for 30 minutes. ROS was measured using flow cytometry. Data are means ± SEM of 3 independent experiments. *p≤0.05.</p