Slug controls mammosphere growth.

<p>A. Epithelial cells isolated from mammary glands were seeded individually in low-adherence culture wells. All wells were screened after 3 days. Wells containing 1–4 live cells (microsphere) were enumerated. After three weeks, Some of the microspheres evolved into mammospheres that were monitored. Values are reported as percentage compared to the number of live seeded cells or to the number of microspheres at 3 days. Bar = 50 µm. B. Cells from wild-type (WT) or Slug-deficient mouse (KO) were cloned to grow into primary microspheres and mammospheres. For secondary mammospheres, 10–20 primary mammospheres were pooled and enzymatically dissociated before individual cloning and further screening. Experiment was repeated three times. C. CommaDβ cells were transfected with control (siCt) or two distinct anti-Slug siRNA (siSlug1 and siSlug2) and seeded individually in 96 well low-adherence dishes. All wells were screened after 3 days. Microspheres and mammospheres were reported separately after 3 weeks. A significant decrease in viability was observed after 3 days in cells transfected with anti-Slug siRNA. This deficit was even stronger three weeks later (*Student test, p<0.05). CommaDβ cells were also transfected with control (Ct) or Slug full-length cDNA (Slug) expression vectors and seeded individually in 96 well dishes as previously. The number of mammospheres showed a significant and early increase in survival and growth for Slug-overexpressing mammospheres (*Student test, p<0.05).</p

Mammary glands from Slug-deficient mice exhibit several defects.

<p>A. Mammary glands from wild type (WT) or SlugKO (KO) mice were analyzed in wholemount preparations during tubulogenesis (6 weeks old mice). KO mammary gland displayed a growth delay visible (double arrows) downstream and upstream from the primary duct (arrow). More than 6 mice were examined with a similar phenotype. Size bar = 1 mm. B. At later stages (8 weeks), wholemount preparations show a recovery linked to overbranching visible at higher magnification (inserts). C–D. Orthotopic grafts combining explants from wild type (WT) or SlugKO (KO) were examined 15 weeks after transplantation during estrus cycle (C), including diestrus phase (D). A significant increase (*Student test, p<0.05) in the terminal branching pattern is found in explants from KO mammary glands at all stages. E. Mammary gland phenotype was quantified by several ways. Average length of 12 primary ducts was estimated based on wholemount preparations from three 6 weeks old-mammary glands. In addition, 5 SlugKO and 9 WT (10 weeks old) mammary glands were weighed and compared. We also determined the number of branching nodes, discriminating between primary and secondary branching, significantly more abundant in KO explants (*Student test, p<0.05). F. Finally, we quantified in the explants the increase in the number of terminal buds in homozygous (black, KO) as compared to wild-type (white, WT) transplants, as determined in 5 KO explants from mammary glands at diestrus stage or estrus phases. Transplanted mammary glands from Slug-deficient mice also express extraneous branching. Internodal segments were sorted into short segments (1–4 mm), medium segments (5–7 mm) and longer segments (8–15 mm). Short segments, resulting from an increase in branching nodes generation, were clearly predominant in KO transplants (*Student test, p<0,05).</p

FACS and immunofluorescence analysis show Slug restricted expression by basal-like cells.

<p>A. Using FACS, primary mouse mammary cells were sorted into CD24 Neg (stromal cells: S), CD24 High/CD49f Med (luminal-like cells: Lu) and CD24 Med/CD49 High (basal-like cells: Ba). Results of two experiments are averaged to show expression levels of CK14, CK18, Slug and Snail as quantified by RT-qPCR and normalized to GAPDH reference gene. B. Double immunofluorescence study of mouse mammary epithelial cell line CommaDβ cells show co-expression of Slug and CD49f by a defined sub-population (arrows). In contrast, Slug and CK8 are found mutually exclusive from each other (arrowheads). C. Double immunofluorescence study was quantified for CK5 (white bar), CK8 (white bar) and co-expressed Slug (black bar inside white bar). No CK8+ cell were found to coexpress Slug. CommaDβ were also sorted based on Sca-1 expression as previously described (Deugnier et al., 2006). Sca-1-high basal-like (Ba) and Sca-1-low luminal-like (Lu) cell populations were analyzed for CK14, CK18, Slug, Pcad and Snail. Expression levels obtained by RT-qPCR were normalized to GAPDH reference gene. Data are shown as the mean (+sd) of 3 independent immunobead sorting experiments.</p

Slug regulates mammary epithelial cell differentiation pathways.

<p>We quantified expression levels of various genes involved in mammary epithelial cell differentiation in WT and KO mammary glands from same litter mice. Two KO mice from the same litter were used for the 5 weeks study and the expression level averaged. A. EMT-master genes from Snail and Twist families were screened at both stages. B. Basal (CK14) and luminal (CK 8, 18, 19) cytokeratin genes were screened to evaluate the relative epithelial fraction and putative regulation at 5 weeks. Genes reflecting proliferation (PCNA1), stem/progenitor or luminal phenotype (Bmi1, CD133, Gata 3, Elf5, Muc1, ER, PR) were also screened at 5 weeks. Because CK average expression level was significantly increased, we related the expression levels from strictly epithelial genes (Gata3, Elf5, Muc1) to the mean CK expression level. Finally, members of the Sox family of transcription factors were also screened. Gray areas cover gene expression ratios considered as not significantly modified (ratio between 0.5 and 1.5). C. PCNA1 expression was quantified by calculating the percentage of cells expressing PCNA1 among 1–500 cells in growing ducts paraffin sections from 5–8 weeks old mice, showing a strong decrease in SlugKO cells (*Student test, p<0,05). D. PCNA1 and CK5 were visualized by double-immunofluorescence including DAPI co-staining on paraffin sections from wild-type (WT) or Slug-deficient mouse (KO) ducts, as indicated. E. ER was colocalized with CK5 and DAPI to show a relative overexpression in Slug-deficient mouse (KO) ducts. Arrows points to ER+ CK5- cells identified as luminal cells.</p

Slug, Twist and Zeb1 localization during mammary gland morphogenesis.

<p>A. At 3 weeks, Slug expression (blue X-Gal staining) is visible in the primary duct on a mammary gland wholemount (a, arrowhead), and section (b). Lower sectioning level (c) show Slug expression in polarizing basal epithelial cells during initial tubulogenesis (arrowhead). B. During mammary tubulogenesis at 6 weeks, Slug is found in growing tubules in wholemount (a) and tubule sections (b–c) involving epithelial cells (c, arrow) and peri-tubular mesenchymal cells (b, arrow and c, arrowhead). Cell location is better defined at higher magnification, including basal epithelial (arrow) and mesenchymal (arrowhead) cells (c). Tubule terminal end buds (TEB) sections also show expression by mostly basal epithelial cap cells (d–e, arrows). Co-labeling using DAPI (blue) and antibodies against Slug (red) and Pcad (P-cad, green) demonstrates a basal localization for Slug in 6 weeks (f) and adult (g) tubules. In addition, Twist (h) and Zeb1 (i) were located in tubules from 10 weeks-old mammary glands. Colabeling with CK5 show that only stromal cells express Twist (arrow). Conversely, CK8 colabeling show that Zeb1 is expressed by basal and luminal epithelial cells (arrows).</p

Slug controls cell proliferation, apoptosis, motility and cell lineage commitment.

<p>A-B. CommaDβ cells were transfected with control (Ct) and Slug expression vector (Slug), or with si control (siCt) and two distinct anti-Slug siRNA (siSlug1 and siSlug2). After fixation, DAPI staining and immulocalization (A), average percentage of cells expressing KI67 or caspase 3 were calculated and reported (B). C. Slug controls mammary epithelial cell motility. Cell motility was estimated using a wound healing assay in confluent CommaDβ cells. Ability to repopulate the wound area was estimated by measuring total uncovered substrate area after 48 h, reported to 0 h. Cells were transfected as indicated with Slug full-length cDNA (duplicate Slug1 and Slug2), control vector (duplicate Ct1 and Ct2), and with anti-Slug siRNA (siSlug1 and siSlug2), and two distinct controls siCt1 and siCt2. Experiment was repeated three times. D. Slug controls mammary epithelial cell commitment. Basal/Luminal differentiation phenotype was evaluated by immunofluorescence using cytokeratin expression pattern. CK5 (basal) was found to be significantly overexpressed in cells overexpressing Slug (Slug) as compared to control (Ct), when CK8 (luminal) was downregulated in cells overexpressing Slug.</p

CIP2A promotes mouse spermatogenesis.

<p>(A) Decreased sperm production in CIP2A^HOZ mice. (*** p<0.001; n(WT) = 6, n(HOZ) = 5). (B) Decreased epididymis weight in CIP2A^HOZ mice. (** p<0.01, n = 6). (C) Epididymal analyses showed decreased epididymis size in CIP2AHOZ mice.</p

CIP2A is co-expressed with PLZF in proliferatively active primordial germ cells and spermatogonia.

<p>(A) Most primitive male germ cells, spermatogonia (indicated by arrows), locate most basally in the seminiferous tubules and contain highest CIP2A, PLZF and ki-67 expression levels in the mouse testis. (B) Immunohistochemical staining of CIP2A, PLZF and ki-67 in adult mouse testis show that CIP2A is expressed in both PLZF and ki-67 positive spermatogonia (white arrow heads). (C) CIP2A gene promoter is exclusively active in basal spermatogonia. Enzymatic LacZ staining was used to detect expression of ß-galactosidase protein from the gene-trap cassette under CIP2A promoter. ß-galactosidase activity was observed only in the CIP2A^HOZ spermatogonia (arrowheads in larger magnification). (D) Human testicular gonocytes (Fetal) and pubertal spermatogonia (Pubertal) express CIP2A, PLZF and ki-67 (arrowheads) whereas juvenile testis samples (Juvenile) were devoid of expression of any of these markers. White bar represents 25 µm, black bar – 50 µm.</p

Characterization of hypomorphic CIP2A^HOZ mice.

<p>(A) Gene-trap strategy for inhibition of CIP2A expression. Murine CIP2A wild-type allele and the corresponding CIP2A locus with gene-trap vector insertion are presented in the upper panel (exon structure is represented with filled boxes, the gene-trap vector by clear boxes). The pGT0Lxf gene-trap vector contains a splice acceptor site (SA), β-galactosidase reporter gene (β-geo) and a SV40 polyadenylation site (pA) inserted in CIP2A locus intron 1. The lower panel indicates the position of the genotyping primers, a–b for the wild-type allele, c–d for the mutated allele. (B) CIP2A^HOZ mice grow and develop normally. There were no differences in morphology between adult WT or CIP2A^HOZ mice. (C) CIP2A mRNA expression in WT and CIP2A^HOZ adult mouse organs. Real-time PCR analysis for CIP2A mRNA in CIP2A expressing organs. Mouse beta-actin was used for normalization. (D) CIP2A protein expression in WT and HOZ testis tissue. (E) CIP2A immunoreactivity in WT and HOZ testis was evaluated by immunohistochemistry. No CIP2A protein expression was detected in CIP2A^HOZ tissues. Arrows indicate specific CIP2A staining in WT basal male germ cells.</p

CIP2A, PLZF and ki-67 expression during human ontogenesis [(Fetal (F), Juvenile (J), Pubertal (P)], <i>n</i> = 10.

<p>CIP2A, PLZF and ki-67 expression during human ontogenesis [(Fetal (F), Juvenile (J), Pubertal (P)], <i>n</i> = 10.</p