Bak and Bax mediate apoptosis in Ras/E1A-transformed MEF cells.

<p>(<b>A</b>) Expression of both anti-apoptotic and pro-apoptotic Bcl-2 proteins in the indicated cell lines were examined by western blot. (<b>B</b>) The indicated MEF cells were cultured in soft agar and representative images are shown. (<b>C</b>) The number of foci shown in (B). Data represent mean±S.D. of three independent measurements. Asterisks (*) indicate <i>P</i><0.05, Student’s unpaired t test. (<b>D</b>) Both untransformed and transformed Bak^−/−Bax^−/− MEF cells were resistant to apoptotic stimuli. The indicated cells were treated with 0.5 µM thapsigargin and 5 µM etoposide, and cell viability was measured 48 hours later. Mean±S.D. of three independent experiments are shown. Asterisks (*) indicate <i>P</i><0.05, “ns” indicates no significance (P>0.05), Student’s unpaired t test.</p

Intracellular nanoparticles colocalize with lysosomes.

<p>The indicated cell lines were first cultured with 0.1 mg/ml nanoparticles for 24 hours, then incubated with 2 µM acridine orange (A) or 50 nM LysoTracker Red (B). Bright field and fluorescence images were acquired through a fluorescence microscope. Representative images are shown. While lysosomes labeled with acridine orange or LysoTracker Red are shown in red (the middle panels), TiO₂ nanoparticles inside cells are observed as dark-colored aggregates (the left panels).</p

TiO₂ nanoparticles preferentially induce cell death in transformed cells.

<p>(<b>A</b>) The indicated cell lines were treated with various concentrations of P25 TiO₂ nanoparticles for 24 hours, and viability of cells was measured using TOTO-3 DNA dye exclusion method. Data represent mean±S.D. of three independent experiments. * p<0.05, Student’s unpaired t test. (<b>B</b>) Effects of TiO₂ nanoparticles on cellular metabolic activities were determined by measuring Alamar Blue fluorescence. The indicated MEF cells were treated with 0.5 mg/ml or 1 mg/ml TiO₂ nanoparticles for 24 hours. The cellular reducing activities of treated cells were normalized to that of corresponding untreated cell lines. Mean±S.D. of three independent experiments are shown. * p<0.05, Student’s unpaired t test. (<b>C</b>) Effects of TiO₂ nanoparticles on long-term cell viability were determined by clonogenicity assay. The normalized cell survival was calculated by dividing the number of wells with viable treated cells with that of untreated cells. Data represent mean±S.D. of three independent experiments. * p<0.05, Student’s unpaired t test.</p

Oncogenic transformation does not affect fluid phase endocytosis.

<p>(<b>A</b>) Untransformed and transformed Wild-type or Bak^−/−Bax^−/− MEF cells were incubated with 10 µg/ml or 30 µg/ml 10 kDa Dextran conjugated to Alexa Fluor 647 (Invitrogene). The uptake of Dextran into cells was measured by flow cytometry. (<b>B</b>) The intracellular levels of Dextran were measured as the intensities of Dextran fluorescence. Mean±S.D. of three independent experiments are shown. “ns” indicates no significance (P>0.05), Student’s unpaired t test.</p

Selective cytotoxicity of TiO₂ nanoparticles is related to an oncogenic transformation-induced increase of lysosomal activities.

<p>(<b>A</b>) The expression levels of lysosomal protein LAMP1 in the indicated cells were determined by western blot. (<b>B</b>) The expression levels of LAMP1 are higher in transformed cells than their untransformed counterparts. The intensities of LAMP1 and actin shown in (A) were quantified using ImageJ software (NIH). The relative LAMP1 levels were calculated with the intensity of LMAP1 normalized to that of actin in the same sample. Data represent mean±S.D. of three independent experiments. * p<0.05, Student’s unpaired t test. (<b>C</b>) The enzymatic activities of lysosomal acid phosphatase were measured. Mean±S.D. of three independent experiments are shown. “*” indicates P<0.05, Student’s unpaired t test. (<b>D</b>) Chloroquine alleviates death of transformed cells induced by TiO₂ nanoparticles. The indicated cells were cultured in the absence or presence of chloroquine or TiO₂ nanoparticles. Cell viability was measured 24 hour after the treatments using TOTO-3 DNA dye exclusion approach. Data represent mean±S.D. of three independent experiments. “*” indicates P<0.05; “ns” indicates no significance (P>0.05), Student’s unpaired t test. The viabilities of TiO₂ nanoparticle-treated transformed cell lines in the presence of chloroquine are significantly higher than those in the absence of chloroquine (p<0.05, Student’s unpaired t test), whereas the differences in the viabilities of untransformed cells with and without chloroquine exposure are not statistically significant (Student’s unpaired t test).</p

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

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 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

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