(A) Representative 2-D Difference Gel Electrophoresis (DIGE) images from patient LA5. Protein extracts from normal lung parenchyma and lung adenocarcinoma were labeled with amine-reactive Cy3 (green) and Cy5 (red), respectively. Top left, top right, bottom left, and bottom right gel images depict total unfractionated protein extract, not heparin-binding fraction, weakly heparin-binding fraction, and strongly heparin-binding fraction, respectively. Numbers indicate 14 spots fulfilling the Boolean selection criteria (>2-fold change in at least 3 patients and >95% statistical significance of overall mean value difference). Note the increase of weak spots in heparin-binding fractions over unfractionated extracts, indicating the efficiency of low-abundance proteome enrichment by heparin fractionation. Importantly, 13 of the 14 differentially expressed spots (93%) remained entirely undetectable in unfractionated 2-D DIGE gels. (B) Heat map representation of protein expression level changes (spots 1–14). The columns correspond to patients LA1-5. Each cell is color-coded by fold increase (shades of red) or fold decrease (shades of blue) in cancer relative to matched normal parenchyma. Grey cells indicate that the spot intensities in normal and cancer specimens were approximately equal. When a specific protein spot was present exclusively in either normal or cancer tissue, it was represented as >8-fold decrease or >8-fold increase, respectively. (C) Bar graph representations of spot intensities for spots 1, 3, and 5. Blue and red bars denote normal lung and cancer tissue, respectively. Abscissae are labeled by patient number (LA1-5). The ordinate expresses the relative amount of protein in a given spot as parts per million (PPM) of the total integrated gel fluorescence at the respective excitation wavelength. (D) Detail views of spots 1, 3, and 5 for all 5 matched sample pairs (LA1-5; N, normal; C, cancer). Open arrowheads indicate the spot locations.

TAGLN (A), TAGLN2 (B), and PPIA (C) expression in unfractionated and weakly heparin-binding tissue protein extracts. The expression ratio of β-actin was used for normalization of each matched sample pair. TAGLN and TAGLN2 blots showed two or three specific bands and the sums of individual band intensities were used for comparisons (9, 10).

Photomicrographs of normal lung tissue (A) and a focus of AAH (B) stained with hematoxylin-eosin. Protein expression of TAGLN2 (C, D) in alveolar pneumocytes (left, normal; right, atypical adenomatous hyperplasia). Presence of the specific protein is indicated by the amount of brown staining. Nuclei were counterstained with hematoxylin (blue) for visualization purposes. Note the histopathologic features of AAH, such as slightly thickened alveolar walls and an increased number of alveolar lining cells with cytologic atypia. The tissue shown is from patient LA11. Original magnifications: 640×.

mRNA abundance of TAGLN, TAGLN2, and PPIA was compared between matched paired normal and lung adenocarcinoma tissues samples. The normalized expression ratios (cancer:normal) are shown relative to the internal control gene RPL13A (ribosomal protein L13A) transcript.

Photomicrographs of normal lung tissue (A) and lung adenocarcinoma (B) stained with hematoxylin-eosin. Protein expression of TAGLN (C, D), TAGLN2 (E, F), and PPIA (G, H) in lung tissue (left, normal; right, adenocarcinoma). Presence of the specific protein is indicated by the amount of brown staining. Nuclei were counterstained with hematoxylin (blue) for visualization purposes. The tissue shown is from patient LA13. Original magnifications: 400×.