LKB1 mutant tumors show mislocalization of activated AMPK.

<p>A–C) Representative images of phospho-Thr172-AMPK (red) localization in mitotic cells from wild-type tissues. Images were not rotated, and not all cells had an obvious apical surface in the tissue section. The inset in C shows the apparent localization of phospho-AMPK to kinetochore regions. Microtubules are green and DNA is blue. D–I) Representative images of phospho-Thr172-AMPK (red) localization in mitotic cells from LKB1 mutant polyps. Images were not rotated, and not all cells had an obvious apical surface in the tissue section. Microtubules are green and DNA is blue. Scale bar, 10 µm.</p

AMPK inhibition by Compound C causes spindle misorientation and other mitotic defects in MDCK cell cysts.

<p>A) Representative spindles from vehicle treated control cysts. Images were rotated in three dimensions to show the cyst lumen, place the apical cell surface at the top of the cell, and place both spindle poles in a single plane. Spindle angle was measured relative to the apical surface. Microtubules are green, actin is red, and DNA is blue. B–D) Representative spindles from cysts treated with 20 µM Compound C for 4 hours prior to fixation. Images in B are displayed as in A. Images in C and D are taken from single lumen cysts, although the lumen is not visible in every image. Insets in D show magnified cells with misattached chromosomes marked by white arrows. Scale bar, 10 µm. E) Quantification of spindle angles. Each dot represents a single spindle angle measurement. Blue bars represent means and standard error of the mean. See text for numbers. P<0.0001 for the difference. F) Data from (E) presented as the % of angles that range from 0° to 30°, 30° to 60°, and 60° to 90°.</p

ZO-1 and E-cadherin localization are unaffected by LKB1 RNAi in MDCK cell cysts.

<p>Representative images of ZO-1 (red) and E-cadherin (green) immunofluorescence in control MDCK cell cysts and in cysts with LKB1 RNAi are shown. Sections were taken through the midpoint of the cyst to show the hollow lumen as well as the apical and lateral surfaces of cells at the widest part of the cyst structure. Bars, 10 µm.</p

<i>STK11</i> mutation causes spindle misorientation in vivo.

<p>Tissue immunofluorescence was done on upper gastrointestinal tumors from <i>STK11</i>/LKB1 heterozygous mutant mice and corresponding gastrointestinal tissue from wild-type littermates. A) Representative images from <i>STK11</i>/LKB1 wild-type tissues and <i>STK11</i>/LKB1 mutant polyps. The brush border at the apical cell surface is at the top of each panel (arrowhead at left and arrow at right) and the spindles were rotated in three dimensions to place both spindle poles in a single plane. Microtubules are green, actin is red, and DNA is blue. Spindle angle was measured as the angle between the spindle axis and the apical surface. The wild-type spindle is oriented parallel to the apical surface, and the three <i>STK11</i>/LKB1 mutant spindles lack this planar orientation. B) Quantification of spindle angles. Each dot represents a single spindle angle measurement. Blue bars represent means and standard error of the mean. See text for numbers. P<0.0001 for the difference. C) Data from (B) presented as the % of angles that range from 0° to 30°, 30° to 60°, and 60° to 90°. D) Spindles from <i>STK11</i> mutant cells contain astral microtubules. White arrows show astral microtubules of misoriented spindles in <i>STK11</i>/LKB1 mutant polyps. These spindles are from the original data set used to calculate spindle angle, but the confocal stacks were adjusted, rotated, and processed differently from (A) to show astral microtubules optimally, and spindle angle cannot be appreciated from them. Scale bars, 10 µm.</p

KIT activation & up-regulation, concomitant parallel induction of ET3, KIT⁺Melan-A^–- progenitor cells, and melanocyte regeneration in proportion to sun-exposure.

<p>(<b><i>A</i></b>), IHC of KIT and ET3 on serial sections of human skin specimen obtained from a lower extremity-amputation. Sole represents active suppression of melanogenesis (<i>a</i> and <i>d</i>), dorsum of big toe represents intermediate sun-exposure (<i>b</i> and <i>e</i>), and lateral lower leg represents heavy sun-exposure (<i>c</i> and <i>f</i>). (<b><i>B</i></b>), IHC of KIT, Melan-A, and ET3 on serial sections of human skin punch biopsy specimens obtained from sun-protected axilla (<i>g</i>, <i>i</i>, <i>k</i>) and chronic heavy sun-exposed forearm (<i>h</i>, <i>j</i>, <i>l</i>) from the same individual. Lymphocytes serve as internal negative control for KIT, ET3 and Melan-A; mast cells serve as internal positive control for KIT. Together, these images demonstrate that human skin exhibits sun-exposure-dependent up-regulation of KIT (<i>a-c</i>) and concomitant parallel sun-exposure-induced increasing induction of ET3 (<i>d-f</i>). Chronic sun-exposure induces intense dendritic pattern of KIT expression as well as a large increase in the number of KIT-expressing-cells in the basal layer (<i>h</i>) consisting of KIT⁺Melan-A⁺ mature melanocytes (<i>j</i>) and KIT⁺Melan-A^–melanocyte progenitor cells as evidenced by the difference between (<i>h</i>) and (<i>j</i>).</p

Microarray analysis comparing a highly aggressive GIST with an indolent GIST: A selective list of target genes whose expression was up- or down-regulated by KIT signaling.

<p>Microarray analysis comparing a highly aggressive GIST with an indolent GIST: A selective list of target genes whose expression was up- or down-regulated by KIT signaling.</p

Immunohistochemical studies on human colon myenteric plexus demonstrate that 48 hours fasting results in activation of SCF-KIT signaling and concomitant parallel induction of endothelin-3.

<p>Human colon specimens post 48 hours fasting demonstrate intense KIT staining in ICCs within the myenteric plexus (<i>a</i>), and intense ET3 staining within the myenteric plexus (<i>b</i>). In sharp contrast, the surrounding longitudinal and circular smooth muscle cells (SM) show negative ET3 staining.</p

Autophosphorylation, internalization, and nuclear localization of activated KIT with tyrosine phosphorylation at 568/570 (pY568/pY570KIT).

<p>(<b><i>A</i></b>), IHC of frozen sections of an aggressive GIST (<i>a-c</i>) and a normal human adult testis as external control (<i>d-f</i>) using pan-KIT antibody (<i>a</i> and <i>d</i>), pY568/pY570KIT antibody (b and <i>e</i>, red arrow indicates nuclear localization), and pY703KIT antibody (<i>c</i> and <i>f</i>) respectively. (<b><i>B</i></b>), <i>In situ</i> IHC to assess kinetics of SCF-induced nuclear translocation of pY568/pY570KIT using WM793 melanoma cells cultured in 4-well chamber tissue culture treated glass slides. Control (<i>g)</i> without SCF stimulation, after addition of SCF to culture media, the nuclear localization of pY568/pY570KIT increases progressively (<i>h-j</i>) in more than 90% of WM793 cells, reaches a plateau about 40–60 minutes (<i>i</i> and <i>j</i>), begins to decrease at 90 minutes (<i>k</i>), and is completely absent in nucleus with relocation back to the cytoplasm at 4 hours, some residual cytoplasmic staining remains visible (<i>l</i>).</p