Fig. 6.

Tobacco smoke oxidant(s) act as the key trigger for Rtp801 activation and consequent oxido-nitrosative stress and apoptosis/necrosis of lung cells exposed to CS. (A) CSE was first directly incubated with a pure protein BSA (1 mg) for 15 min and then subjected to Oxyblot analysis or immunoblot analysis for nitrotyrosine formation in which prenitrated BSA was used as the positive control (+ve con). (B) CSE (1%), either freshly prepared or kept in an airtight flask for 15 h following preparation (for time-dependent deactivation of its oxidative potency) was added at a regular intervals of 2.5 h to cultured NL-20 cells followed by overnight incubation at 37 °C. Cell lysates prepared from the treated cells were then subjected to immunoblot analysis by using anti-Rtp801, anti-NF-κB–p65, anti-iNOS and anti-nitrotyrosine antibodies or oxyblot analysis using anti-DNP antibody and relevant secondary antibodies. Such treated NL-20 cells were also separately stained with a solution of AO and EtBr for assessing their apoptosis/necrosis levels. (C) Representative sections of NL-20 cells showing overall levels of apoptosis/necrosis (10 fields). (D) In a separate experiment, NL-20 cells pretreated with 200 μM ascorbate were incubated overnight with CSE (1%) and their lysate were probed for the same markers as in B. (D) A portion of the same NL-20 cells used in the above experiment were stained with a solution of AO and EtBr to evaluate their apoptosis/necrosis levels. (E) Representative sections of NL-20 cells showing levels of apoptosis/necrosis (10 fields). Normal cells appeared green, apoptotic cells showed condensed/fragmented orange chromatin (red arrow, Inset), and necrotic cells showed complete orange nucleus (yellow arrow, Inset). Data shown are representative of three independent experiments done under similar conditions. (Scale bars: C and E, 100 μm; E, Inset, 50 µm.)