Figure 1.
Bak and Bax mediate apoptosis in Ras/E1A-transformed MEF cells.
(A) Expression of both anti-apoptotic and pro-apoptotic Bcl-2 proteins in the indicated cell lines were examined by western blot. (B) The indicated MEF cells were cultured in soft agar and representative images are shown. (C) The number of foci shown in (B). Data represent mean±S.D. of three independent measurements. Asterisks (*) indicate P<0.05, Student’s unpaired t test. (D) 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 P<0.05, “ns” indicates no significance (P>0.05), Student’s unpaired t test.
Figure 2.
TiO2 nanoparticles preferentially induce cell death in transformed cells.
(A) 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) 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. (C) 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.
Figure 3.
Oncogenic transformation does not affect fluid phase endocytosis.
(A) 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) 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.
Figure 4.
The abundance of intracellular TiO2 nanoparticles is not influenced by oncogenic transformation.
The indicated MEF cells were cultured with or without 0.5 mg/ml TiO2 nanoparticles for 24 hours. The subcellular structures of control and treated cells were examined by transmission electron microscopy (TEM). (A) In the absence of nanoparticles, the indicated MEF cells exhibit comparable subcellular structures. Representative cross section images of the indicated cells are shown. The left panels are the enlargement of the corresponding inlets in the panels on the right. The large electron microscopic images were generated by stitching several images of smaller regions together. (B) Localization and abundance of intracellular TiO2 nanoparticles were determined by TEM. Representative TEM images of cross sections of the indicated cells show the intracellular presence of TiO2 nanoparticles. The large electron microscopic images were generated by stitching several images of smaller regions together. (C) The abundance of TiO2 nanoparticles in the treated cells was evaluated as the percentage of the total cellular area occupied by nanoparticles in cross sections of cells. Data represent mean±S.D. of at least 7 randomly selected independent cross sections.
Figure 5.
Intracellular nanoparticles colocalize with lysosomes.
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).
Figure 6.
Selective cytotoxicity of TiO2 nanoparticles is related to an oncogenic transformation-induced increase of lysosomal activities.
(A) The expression levels of lysosomal protein LAMP1 in the indicated cells were determined by western blot. (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. (C) 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. (D) 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).