Article Figures & Data
Figures
- Fig. 1.
Organization of lung tumor tissue microarray (TMA). A, H&E staining of the entire TMA section. B, scheme of TMA. Tissues samples are represented by small rectangles that are arrayed in groups of 25 each. Tumor types are indicated as light gray (adenocarcinoma) or black (squamous cell carcinoma). The clear areas are control tissues including normal lung from patients with adenocarcinoma (#1), or squamous cell carcinoma (#2), and normal nonpulmonary organs (#3).
- Fig. 2.
Chromatin remodeling factors in lung cancer cell lines. A, immunoblotting of BRG1, BRM, BAF155, and Ini-1 among nine lung cancer cell lines. The EKVX cell line showed a band reactive to anti-BRM but nonimmunoreactive for anti-BRG1 antibody. The apparent sizes of BRG1 and BRM were a little larger than the known molecular size (180 kDa) but are the same as those shown in the data sheet from the supplier (http://www.scbt.com/). BAF155 and Ini-1 showed an immunoreactive band in eight cell lines. H358 appeared to be negative for Ini-1. B, anti-BRM immunohistochemical staining of the same lung cancer cells. Hop92 did not show positive staining by immunohistochemistry but showed a weak positive band on Western blot. Consistent with immunoblotting, immunohistochemical signals of BRM were lower than those of BRG1 in lung cancer cell lines. H520, EKVX, and H2170 showed membranous and cytoplasmic staining, whereas H358 and H226 showed weak and focal nuclear staining.
- Fig. 3.
Examples of immunohistochemical staining by BRG1 and BRM antibodies in primary lung tissues. A, BRG1 in normal lung. Most of the cells including pneumocytes and interstitial cells were positively stained in the nucleus. B, a lung tumor negative for BRG1 [TS0]. C, a tumor positive for BRG1 (TS3). D, BRM in normal lung. Most of the cells including pneumocytes and other interstitial cells are positive in the nucleus. E, tumor with negative BRM staining (TS0); the surrounding inflammatory cells were positive. F, tumor positive for BRM (TS3). G and H, membranous BRM staining in tumor cells. I, high magnifications (×400) of images shown in G and H. Diameter of tissue core = 0.6 mm.
- Fig. 4.
Association of staining between each pair of antibodies. A, P values of χ2 test between each antibody are shown for adenocarcinoma (gray area) and squamous cell carcinoma (white area). P ≤ 0.01 is considered significant and is indicated in the table. B, one-way clustering indicating the relatedness of expression patterns among all antibodies. Note that all nonnuclear signals clustered distantly from the nuclear signals of the same antibody. C, identification of two highly correlated sets of chromatin remodeling proteins in NSCLC.
- Fig. 5.
Immunohistochemistry scoring and Kaplan-Meier survival analyses for BRG1 and BRM in non-small cell lung cancer. A, immunohistochemical scoring for nuclear BRG1, nuclear BRM, and membranous BRM signals. Grids corresponded to tumor arrangements on the TMA as shown in Fig. 1B<$REFLINK> (gray and black blocks). Total scores (TSs) are shown, and the colors corresponded to the TS values. Red, TS3; yellow, TS2; white, TS1/TS0; black, excluded or missing tissues. B, Kaplan-Meier survival plots. The overall patient survival status for antibodies and selected cellular locations are shown. The number of patients in each arm and the associated P values are as indicated.
Tables
- Table 1
Clinical characteristics of all non-small cell lung cancer patients used in this study
Variable Category No. of patients (n = 230) Median survival (yrs) Gender Male 166 2.8 Female 64 4.6 Mean age ± SD (yrs) 64.6 ± 9.2 (36–86) Stage I 115 ] 4.2 II 29 III 30 ] 1.3 IV 7 Vital status Alive 52 7.3a Dead [ Tumor related 46 1.6 Other cause 20 1.7 Unknown 80 2.8 Median follow-up (yrs) 3.2 ± 3.2 a Median term of last follow-up.
- Table 2
Antibodies and protocols used for immunohistochemical staining
Antibody Species Source Clone Type Protocol Titration Retrieval HDAC1 Ra Santa Cruz H-51 IgG2a ABC 50 DC HDAC2 R Santa Cruz H-54 IgG ABC 200 MW BRG1 R Santa Cruz H-88 IgG ABC 200 DC BRM G Santa Cruz C-20 IgG ABC 100 DC BRM G Santa Cruz N-19 IgG ABC 100 DC BAF170 R Santa Cruz H-116 IgG ABC 100 MW BAF155 R Santa Cruz H-76 IgG ABC 100 MW RbAp48 G Santa Cruz K-15 IgG ABC 300 MW RB M Zymed RB-1 IgG1, κ ABC 100 DC Ini1 R Santa Cruz H-300 IgG ABC 50 DC mSin3A R Santa Cruz AK-11 IgG ABC 50 DC HAT1 G Santa Cruz C-20 IgG ABC 1000 DC Tip60 G Santa Cruz K-17 IgG ABC 100 DC AE1/AE3 M DAKO AE1/AE3 IgG1 ABC 100 None PGP9.5 R Biogenesis n/a n/a ABC 8000 DC a R, rabbit; DC, Decloaking Chamber; MW, microwave; G, goat; M, mouse; n/a, not available.
- Table 3
Immunohistochemical staining of chromatin remodeling factors using TMAa
The actual number of cases is shown for each scoring, whereas percentages are given for − and + cells. N, nuclear, C, cytoplasmic, M, membranous. Total scores 2 and 3 were designated as positive, whereas scores 0 and 1 were recognized as negative.
Total score BRG1 BRM BAF155 Ini-1 BAF170 HDAC1 HDAC2 RbAP48 RB mSin3A HAT1 Tip60 N C N M N C N C N N N N N N N C 0 74 221 156 222 122 162 135 148 43 73 110 55 71 72 27 84 1 44 4 45 16 51 21 17 19 25 20 28 37 45 9 38 36 2 77 7 38 15 44 69 73 53 60 75 46 78 78 60 54 64 3 50 13 29 15 30 21 25 28 122 70 63 71 68 100 127 54 − 48 92 74 89 70 67 58 47 27 36 54 35 44 29 37 37 + 52 8 26 11 30 33 42 33 73 64 46 65 56 71 63 63 a TMA, tissue microarray.
Volume 10, Issue 13
