pRS/βIII
Spot # / Protein ID / Mass / Score / Match / MW / pI* / T-test / Ratio
130 / Vinculin / 123722 / 742 / 30 / 123668 / 5.51 / 0.00035 / 1.51
209 / Glycogen phosphorylase / 96635 / 231 / 9 / 96695 / 6.4 / 0.00035 / 1.51
642 / Serpin B5 precursor / 42111 / 204 / 6 / 42138 / 5.72 / 1.10E-08 / 1.51
679 / Tropomyosin beta chain / 32831 / 144 / 5 / 32850 / 4.66 / 4.80E-07 / 6.39
930 / Profilin-2 / 15036 / 70 / 1 / 14915 / 6.78 / 2.20E-06 / 1.59
834 / Peroxiredioxin 4 / 30521 / 182 / 7 / 30540 / 5.86 / 3.10E-06 / -1.84
705 / Peroxiredoxin 2 / 21878 / 213 / 6 / 21761 / 5.67 / 0.00013 / -1.94
162 / Heat shock protein-90 / 83212 / 885 / 20 / 83133 / 4.97 / 0.00074 / -1.53
665 / Glutathione S-transferase
Mu 3 / 26542 / 248 / 7 / 26428 / 5.37 / 2.60E-06 / -1.82
309 / Tubulin beta 3 / 50400 / 440 / 11 / 50433 / 4.83 / 0.0011 / -1.63
Differentially expressed proteins in the cytoplasmic fraction.
Table 1: Proteins that were identified to be differentially expressed in the cytoplasmic fraction of H460 βIII-tubulinshRNA expressing NSCLC cells.
Table 2: Proteins that were identified to be differentially expressed in the nuclear fraction of H460 βIII-tubulin shRNA expressing NSCLC cells.
MASCOT / ProtoNet / pRS/Ctrl vs.pRS/βIII
Spot # / Protein ID / Mass / Score / Match / MW / pI* / T-test / Ratio
929 / 60S acidic ribosomal
Protein P0 / 34252 / 73 / 2 / 34273 / 5.72 / 1.50E-05 / 2.49
1240 / Ferritin light chain / 20007 / 39 / 1 / 19888 / 5.51 / 4.00E-05 / 2.06
1029 / Microtubule associated
Protein RP/EB family
member / 29980 / 43 / 1 / 29999 / 5.02 / 0.003 / 1.77
848 / Guanine nucleotide-binding
Protein G / 44280 / 63 / 1 / 44308 / 6.23 / 0.013 / 1.71
901 / Eukaryotic translation
Intitation factor 3 subunit 1 / 36479 / 47 / 1 / 36502 / 5.38 / 0.0011 / 2.32
766 / Vimetin / 53619 / 183 / 7 / 53554 / 5.06 / 0.042 / -1.89
525 / Heterogeneous
Nuclear ribonucleoprotein L / 60149 / 74 / 4 / 60187 / 6.65 / 0.0058 / -1.94
479 / ATP-dependent DNA
Helicase 2, subunit 2 / 82652 / 68 / 1 / 82573 / 5.55 / 0.014 / -1.82
Supplementary Figure legends:
Supplementary Figure 1: 2D-DIGE experimental design. Schematic diagram demonstrating the experimental design for 2D-DIGE proteomics in NSCLC cells.
Supplementary Figure 2:Purity ofnuclear and cytoplasmic fractions isolated from βIII-tubulin shRNA and control shRNA NSCLC cells. Representative western blots of nuclear and cytoplasmic fractions isolated from control shRNA (pRS/CtrlSH1 and pRS/CtrlSH2) and βIII-tubulin shRNA (pRS/βIIISH4 and pRS/βIIISH59) NSCLC cells for 2D-DIGE experiments. Enrichment of nuclear fractions was confirmed with Lamin B1 staining, while the cytoplasmic fraction enrichment was confirmed by β-tubulin staining. Samples were separated on the same gel, transferred onto membrane and membranes cut for probing against respective proteins. Representative image of n=3 individual isolations used for 2D-DIGE study.
Supplementary Figure 3:Two-Dimensional Difference Gel Electrophoresis (2D-DIGE) of cytoplasmic and nuclear extracts of H460 NSCLC cells expressing βIII-tubulin or control shRNA. A and B) Representative 2D-DIGE overlay images (pRS/CtrlSH 1 & 2 in green and pRS/βIIISH4 and 59 extracts in red) and identified protein spots (numbers refer to identified proteins) for both broad and narrow range pI’s in both the cytoplasmic and nuclear fractions, n= 3 independent experiments.
Supplementary Figure 4: Validation of proteins found to be differentially expressed in H460 βIII-tubulin shRNA expressing cells. Representative 2D-DIGE densitometric volume of each protein is shown in a 3D-view generated by the DeCyder Image Analysis software package. Arrows indicate the 3D-densitometric peaks for profiln 2 and peroxiredoxin 4 (Prdx4) in both control (pRS/CtrlSH 1 & 2) and βIII-tubulin shRNA expressing (pRS/βIII SH4 and SH59) NSCLC cells. Western blots and densitometry graphs confirming the stable suppression of βIII-tubulin in two individual βIII-tubulin shRNA expressing (pRS/βIII SH4 and SH59) clones and the associated protein changes identified by 2D-DIGE in βIII-tubulin shRNA expressing H460 cells (pRS/βIII SH4 and SH59) vs. controls, (pRS/CtrlSH 1 & 2). A) and B)Profilin 2 and B) and C)peroxiredoxin 4 (Prdx4), n=3-5 independent experiments all samples were normalized to the housekeeping protein GAPDH (*p<0.001, pRS/βIIISH4 and SH59versus pRS/CtrlSH 1 & 2).
Supplementary Figure 5:βIII-tubulin shRNA NSCLC cells show increased maspin expression. A) Representative western blot showing an increase in maspin protein expression in the H460 βIII-tubulin shRNA expressing cell clone 60 (pRS/βIIISH60) vs. 2 individual control shRNA clones (pRS/CtrlSH1 and SH2) n=3 independent experiments all samples were normalized to the housekeeping protein GAPDH. B) Densitometry graph demonstrating a significant increase in maspin protein expression in the H460 βIII-tubulin shRNA expressing cell clone 60 (pRS/βIIISH60) vs. 2 individual control shRNA clones (pRS/CtrlSH1 and SH2) n=3 independent experiments (*p<0.001, pRS/βIIISH4 and SH59 versus pRS/CtrlSH1 and SH2). C) A graph demonstrating a significant increase in maspin mRNA levels in the H460 βIII-tubulin shRNA expressing cell clone 60 (pRS/βIIISH60) vs. 2 individual control shRNA (pRS/CtrlSH1 and SH2) n= 4 separate experiments, all samples were normalized to the housekeeping gene β2-microglobulin (*p<0.001, pRS/βIIISH4 and SH59 vs. pRS/CtrlSH1 and SH2).
Supplementary Figure 6: Suppression of βIII-tubulin increases cell adhesion and decreases cell migration in NSCLC cells. A)A graph showing βIII-tubulin shRNA expressing NSCLC cells (pRS/βIIISH4) with increased adhesion to the ECM protein fibronectin vs. control (pRS/CtrlSH2) cells, n = 3 experiments, *p<0.05. B) A graph showing βIII-tubulin shRNA expressing A549 NSCLC cells (pRS/βIIISH61) with a significant decrease in cell migration vs. control (pRS/CtrlSH27) cells, n = 3-5 experiments, *p<0.05.
Supplementary Figure 7: A) A graph showing a decrease in βIII-tubulin mRNA expression in A549 NSCLC cells stably expressing βIII-tubulin shRNA (pRS/βIIISH61) vs. cells stably expressing control shRNA (pRS/CtrlSH27). B) Representative western blot demonstrating potent knockdown of βIII-tubulin protein expression in A549 NSCLC cells stably expressing βIII-tubulin shRNA (pRS/βIIISH61) vs. cells stably expressing control shRNA (pRS/CtrlSH27). GAPDH was used a protein loading control, n = 3 experiments.
Supplementary Table 1:Proteins identified to be differentially expressed in the cytoplasmic fraction of βIII-tubulin shRNA expressing NSCLC cells. Table lists some of the proteins which were identified to be differentially expressed in the cytoplasmic fractions of 2 independent βIII-tubulin shRNA expressing NSCLC cell clones (pRS/βIIISH4 and SH59) compared to 2 independent control shRNA NSCLC cell clones (pRS/CtrlSH1 and SH2).
Supplementary Table 2:Proteins identified to be differentially expressed in the nuclear fraction of βIII-tubulin shRNA expressing NSCLC cells. Table lists some of the proteins which were identified to be differentially expressed in the nuclear fractions of 2 independent βIII-tubulin shRNA expressing NSCLC cell clones (pRS/βIIISH4 and SH59) compared to 2 independent control shRNA NSCLC cell clones (pRS/CtrlSH1 and SH2).
Supplementary Methods
Two-Dimensional Differential Gel Electrophoresis (2D-DIGE).
To identify differentially expressed proteins in NSCLC cells with stable suppression of βIII-tubulin, we performed 2-D DIGE on H460 βIII-tubulin shRNA (pRS/βIIISH4 and pRS/βIIISH59) or control shRNA (pRS/CtrlSH1 and pRS/CtrlSH2) cells (Supplementary Fig 1). 2D-DIGE was performed as we previously described with modifications (15). Refer to Supplementary Method for detailed methodology. Nuclear and cytoplasmic fractions of cells (three independent isolations of each sample) were prepared using the Pierce Nuclear and Cytoplasmic extraction kit as per the manufacturer’s instructions. Antibodies specific to the cytoplasm (total β-tubulin) and nucleus (Lamin B1) were used in western blotting to confirm the purity of the cellular fractions (Supplementary Figure 2). Nuclear and cytoplasmic fractions were processed through the 2-D clean-up kit (GE Healthcare, Australia), followed by sample lysis in DIGE lysis buffer (30mM Tris, 7M urea, 2M thiourea, 4% CHAPS) and pulse sonication on ice. Protein concentration was quantitated using the PlusOne 2-D Quant Kit (GE Healthcare) according to the manufacturer’s instructions. Samples were labeled with the CyDyefluors (GE Healthcare) as described (15). Fluor (200 pmol) was added to 50µg protein and reaction quenching was achieved by adding 1µL of lysine. An internal standard was created by pooling equal amounts of each protein sample and labeling with Cy2. 50µg of the pooled Cy2 labeled sample was run on each 2-D gel along with 50µg of each sample labeled with Cy3 and a separate sample labeled with Cy5. Samples were combined as outlined in supplementary table 1, before the first dimension focusing in order to verify correct experimental design. Combined samples were mixed with an equal volume of 2x sample buffer (7M urea, 2M thiourea, 2% pH 3-10 carrier ampholytes, 2% DDT, 4% CHAPS) prior to cup loading onto either pH 4.5-5.5 or pH 4-7 24 cm Immobiline pH gradient strips and isoelectric focusing on a Multiphor II apparatus. Second dimension separation and gel scanning and Image analysis was performed as described (15). Analysis of the images was performed using the Batch Processor and Biological Variation Analysis (BVA) Module of the DeCyder software version 5.0. Within the analysis, each sample is normalized to its respective in-gel Cy2 internal standard, followed by matching all controls and samples between the different gels. For analysis of the effects of βIII-tubulin knockdown, H460 pRS/βIIISH4 and pRS/βIIISH59 cells were grouped and compared to controls, H460 pRS/CtrlSH1 and pRS/CtrlSH2 cells. These comparisons were performed for both nuclear and cytoplasmic fractions at the two pH ranges, pH 4.5-5.5 (narrow) or pH 4-7 (broad). The BVA module was used to compare each group and data is expressed as average expression ratios and student’s t-tests of individual protein spots.
Cell Migration Assay.
Cell migration was assessed by seeding A549 NSCLC cells stably expressing βIII-tubulin shRNA (pRS/βIIISH61) or control shRNA (pRS/CtrlSH27) (20X103 cells) into the wells of modified Boyden chambers. Culture inserts (Becton-Dickinson) containeda porous membrane at the bottom (8µm pores). 24h later, cell migration was assessed as previously described (16). Cell migration was expressed as a migration index (%): (number of cells on the undersurface of the membrane / total number of cells on both surfaces of the membrane) X100.