SCLEM of whole uncoated cells.

<p>(a) FM image of three adenocarcinoma cells actin labeled with Alexa488. The three cells are connected via tentacles and larger extrusions. Scalebar 5 µm. (b) SEM image of the boxed area in (a), showing detailed information on the connections between the cells. A dense network of tentacles and lamellae stretches between the upper and the right cell. Scalebar 3 µm(c) FM image of an extension connecting another two adenocarcinoma cells. Clear variations in actin concentration along the extrusion can be observed. (b) BSE image of the extrusion in (a). Red arrows mark areas with increased concentration of tentacles that occur before and after the thinner parts of the extrusion. Scale bar is 10 µm. (d, e) SE and BSE high-magnification images of the boxed areas in (b) showing a region rich in tentacles and small lamellar extrusions. Scale bars are 2 µm. (f) Fluorescence intensity profiles, normalized on the maximum, taken along the red and blue lines in (a). (g) Normalized SE intensity profile taken at the corresponding locations marked in (c).</p

SCLEM on FM and EM stained tissue sections.

<p>(a) FM image of human skin tissue stained with DiIC18 fluorescence and uranyl acetate and osmium tetroxide for EM contrast. Scalebar 5 µm (b) BSE image of a selected region from (a), showing a cell nucleus not discernible in (a) (marked with a red arrow), and bundles of longitudinally and transversally cut collagen fibers. Scalebar is 5 µm (c, d) High-magnification images of the areas marked with (c) a red star, scalebar 1 µm, and (d) a yellow star, scalebar 2 µm.</p

Simultaneous Correlative Light and Electron Microscopy.

<p>(a) schematic lay-out for SCLEM, BSE: backscattered electrons, SE: secondary electrons, ETD: Everhard-Thornley detector, LED: light emitting diode, CCD: charge coupled device camera. (b) inside view of the integrated microscope for SCLEM showing optical objective lens in epi-configuration underneath sample holder and electron lens.</p

Antibodies used for immunohistochemical detection of PR.

<p>Antibodies used for immunohistochemical detection of PR.</p

The Progesterone receptor has no influence on intestinal polyposis.

<p>A–C) Development of spontaneous polyposis in the <i>Apc^Min/+</i> mouse is not altered by <i>PRKO</i> (10 female animals per genotype).</p

Lack of off-target effects from progestins on intestinal proliferation or development of acfs.

<p>A) Treatment of a panel of colon cancer cell lines with MPA or progesterone (P4) has no effect on viability at relevant concentrations. B) BrdU incorporation in small intestine or colon after challenging female animals with MPA or progesterone (P4) for four consecutive days. C–E) Acf count in the Azoxymethane treated rat shows acf number (C), localization of acfs throughout the colon (D) and multiplicity (E) (number of crypts per acf).</p

Progesterone Receptor expression in mesenchymal cells in the intestine, not in the epithelium.

<p>A–E) PR immunohistochemistry on the mouse colon (A) and small intestine (B,C) where rare cells express PR (arrowhead). And in an adenoma of an <i>Apc^Min/+</i> mouse (D). PR is widely expressed in the mouse uterus (E). F,G) <i>In situ</i> hybridization in mouse uterus (F) and small intestine (G). All murine tissue shown was taken from A female animal in diestrous stage, when progesterone is high <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0022620#pone.0022620-Wood1" target="_blank">[39]</a>. H–J) PR Expression in the human colon is located in mesenchymal cells (I) and the smooth muscle layer (J), similar to the mouse intestine. For <i>in situ</i> hybridization, thick (10 µm) sections were used, which makes identification of mesenchymal cells difficult. K) PR immunoblot on the mouse colon and small intestine. L) PR immunoblot on a panel of colon cancer cell lines shows no expression of either PR-A or PR-B isoform. The breast cancer cell line T47D is used as a positive control.</p