Figure 1.
Validation of LKB1 rabbit monoclonal antibody D60C5 for immunohistochemistry in human and mouse paraffin-embedded, formalin-fixed samples.
A, Human cervical cancer cell lines. HeLa/Lkb1 = HeLa cells following transduction of lentivirus harboring a human LKB1 cDNA inducible expression construct. Relative expression levels of Lkb1 protein are indicated in parentheses. B, Mouse tissues (endometrium and lung) from animals harboring floxed alleles of Lkb1 following Cre-mediated recombination with Sprr2f-Cre (endometrium) or nasal-instillation of Adeno-Cre virus (lung). Distinct Lkb1-null clones are indicated by dashed lines (endometrium) or arrows (lung). Bars = 10 µm for each panel. Asterisks in the lung panels show invasive cancer cells subjacent to the dysplastic epithelium; these invasive cancer cells are also clearly Lkb1-null. C, Percent of Lkb1-null cells in Sprr2f-Cre; Lkb1L/L female mice by immunohistochemistry at 3, 6, 12, and 20 weeks of age. Error bar = S.E.M. D, Normal patterns of LKB1 protein in human lung and oviduct highlighting localization to the apical surface of ciliated cells (arrows). Note: in the oviduct, ciliated epithelial cells (arrows) are interspersed among nonciliated cells. Bars = 10 µm in both panels.
Figure 2.
Testing of another α-LKB1 monoclonal antibody (Ley 37D/G6).
Tissue sections are from the uterus of a 6-week old Sprr2f-Cre; Lkb1L/L female mouse. Rabbit monoclonal D60C5 readily distinguishes LKB1 positive from negative cells as shown previously. In contrast, the mouse monoclonal antibody Ley 37D/G6 shows a homogeneous pattern throughout the endometrial epithelium (serial step section) and fails to distinguish between LKB1 positive and negative cells. Size bars = 100 µ for each panel.
Figure 3.
Validation of rabbit monoclonal antibody D60C5 by Western blotting.
Positions of molecular weight standards (kilodaltons) are shown to the left of each blot. A, HeLa cells harboring Tet-On construct inducible with doxycycline. B, Comprehensive uterine cancer cell line panel (endometrial and cervical). Note: C4I harbors biallelic mutations of LKB1: a chromosomal deletion plus a point mutation that does not affect protein levels [1]. CaSki, C33, and ME180 do not harbor LKB1 mutations [1]. Endo was derived from normal endocervical epithelium immortalized with HPV E6/E7 [27].
Figure 4.
Scoring schema for LKB1 and pAMPKα (Thr172) expression in human lung cancer specimens.
Tissues were paraffin-embedded and fixed in formalin. Only staining in the malignant epithelial cells was scored. A, LKB1 immunohistochemistry and representative cases illustrating histologic scores. B, pAMPKα (Thr172) immunohistochemistry and representative cases illustrating histologic scores. The dynamic range was somewhat lower for pAMPKα (Thr172) vs. LKB1 but a wide range of staining intensities was also observed. Bar = 10 µm in all panels; all panels are at same magnification.
Table 1.
Summary of patient data.
Figure 5.
Validation of LKB1 antibody in situ assay in human lung tumor specimens.
A, LKB1 protein expression vs. gene expression scores. Researchers used the Agilent 44(p<0.01). B, Box plots showing comparison of gene expression scores in cases with confirmed LKB1 loss-of-function mutations vs. cases with no mutations. C, Protein expression by mutation status providing visual comparison of cases with mutation vs. no mutation. The x-axis shows the percentage of cases per LKB1 score (i.e. each side adds up to 1). The unsymmetrical shape indicates differences between the groups.
Figure 6.
Correlation between LKB1 and pAMPKα (Thr172) scores.
Heat map shows associations between LKB1 and pAMPKα (Thr172) protein expression scores. Kendall's tau provides a summary of the correlation (τκ = 0.49, p<0.001).
Table 2.
Coding mutations and LKB1 scores.