Int. J. Environ. Res. Public Health 2012, 9 479 for 2-D gel analysis as described [25]. Isoelectric focusing gels (3% acrylamide) with gradients of approximately pH 4.5–7.5 were obtained with 0.44% pH 3.5–10 Ampholines and 1.76% pH 6–8 Ampholines (LKB). The pH gradient was determined by measuring the pH value of 1-cm slices of parallel isoelectric focusing gels following overnight-incubation in 0.025 M KCl. The second dimension gels using 7.5% acrylamide were run with molecular markers (Bio-Rad) and stained with 0.25% Coomassie Brilliant Blue. Gels were treated with EN3HANCE (New England Nuclear Co., Boston, MA, USA), dried and exposed to prefogged Kodak X-Omat AR film at –80 °C. Protein spots were quantitated by the 1 D Scan EX software (Scananalytics) as described [23]. 2.12. Reverse Transcription-Polymerase Chain Reaction (RT-PCR) Total RNA (500 ng) extracted from cultured cells or from lung tissues were converted to the cDNA which was then amplified by using the SuperScript One-Step RT-PCR with Platinum Taq Kit (Invitrogen) under the following conditions: reverse-transcript at 50 °C for 30 min and pre-denaturation at 94 °C for 2 min, denaturation at 94 °C for 30 s, annealing at 58 °C for 45 s, and extension at 72 °C for 1 min for a total of 35 repetitive cycles. Final extension was performed at 72 °C for 10 min. The primer pair used to amplify I-tubulin DNA is: forward, 5’-ATGAGGGAAATCGTG CACATCCAG-3’ and reverse, 5’-TTAGGCCTCTTCTTCTGCCTCC-3’. The primer pair used to amplify glyceradehyde 3-phosphate dehydrogenase (GAPDH) DNA, an internal control, is: forward, 5’-GACTCTACCCACGGCAA-3’ and reverse, 5’-GGATGACCTTGCCCACA-3’. PCR products were respectively separated on agarose gels, stained with ethidium bromide and visualized on a UV transilluminator. Bands of cDNA were quantitated with 1 D Scan analysis [23]. 3. Results To elucidate As3+ effects on cellular MT organization, growth arrested RFL6 cells on coverslips were incubated with or without freshly prepared As3+ solution at final concentrations as indicated. Cellular MT distribution was revealed by immunocytochemistry [21]. As shown in Figure 1(A), growth-arrested RFL6 cells contain an intricate, highly organized network of MTs that radiate outward from the MT organizing center (MTOC) in the perinuclear region to the cell periphery. Exposure of cells to 5 µM As3+ for 24 h induced partial loss of MTs and no-organized tubulins with brightly staining accumulated in the perinuclear area where the MTOC is generally located [Figure 1(B)]. Treatment of cells with 20 µM As3+ for 24 h resulted in severe loss of MTs. Instead, mast cells exhibited high-density of depolymerized tubulin mass concentrated in the central area of cells [Figure 1(C)]. Notably, the maximum doses of As3+ (20 µM) that disrupted the MT organization in the cell model did not affect cell viability seriously (about 10% decrease) as assayed by trypan blue staining (data not shown). Morphological alterations in MTs were reversible after removal of As3+. These studies indicated that the MT cytoskeleton is an important cellular target for As3+ insult.