Fig 2: LY2801653 treatment inhibits growth and invasion of SUM225 CCR2-H MIND lesions. NOD- SCID mice bearing SUM225 CCR2-H MIND lesions were treated with 12 mg/kg LY2801653 or vehicle control for 4 weeks (n = 8/group). Mammary tissues were measured for A. changes in mass, B. PCNA expression, C. cleaved caspase-3 expression or D. number of DCIS, DCIS+ microinvasive (DCIS+Mi) and invasive lesions through co-staining for a-sma (green) and CK/19 (red). Total number of lesions scored/group: n = 527 (vehicle), n = 948 (LY2801653). Statistical analysis was determined by Two Tailed t-test (A-C) or ?² test (D). Statistical significance was determined by p < 0.05. *p < 0.05, ***p < 0.001, n.s=not significant. Mean+SEM are shown. Scale bar= 400 microns.

Fig 3: NMDA-induced proliferative response at the central retina. (A) IHC detection of PCNA proliferation marker (red staining) in the central retina of saline-treated (saline) or NMDA-treated (NMDA) eyes at 3, 6, 14, 18, and 24 dpi. Cell nuclei are counterstained with DAPI (blue). The different retinal layers are indicated (ONL, outer nuclear layer; OPL, outer plexiform layer; INL, inner nuclear layer; oINL, outer INL; iINL, inner INL; IPL, inner plexiform layer; GCL, ganglion cell layer), and the dashed white line marks the transition between oINL and iINL layers. Arrowheads indicate PCNA-positive cells at the outer edge of oINL. Scale bar, 100 µm. (B) Quantification of the number of PCNA-positive proliferative cells per surface area, as identified by PCNA IHC, in all indicated morphologically distinct retinal layers (see fill pattern code in top left corner) from saline-treated (saline) or NMDA-treated (NMDA) eyes at 3, 6, 14, 18, and 24 dpi. Significant values based on Student’s t-test for both the whole-retina (top asterisks) and individual layers (asterisks between columns) are indicated (*p-value <0.05; **p-value <0.01).

Fig 4: Eye growth and developmental patterns of retinal cell proliferation in selected squamate models. (a) Scatter plot showing the increase in eye axial length (reflecting eye size) over embryonic (open circles) and postnatal (closed circles) development in P. vitticeps (blue), P. guttatus (green), and A. carolinensis (magenta). Head length rather than age was used as a proxy of developmental time because of considerable variations in growth rate at postnatal stages in a given species (see main text). The postnatal period covers the first two-thirds of posthatchling lifetime in all species. Colored lines represent species-specific regression lines for embryonic (dashed lines) or postnatal (solid lines) data points. (b–u) Change in embryonic eye size and retinal cell proliferation in P. vitticeps (b–i), P. guttatus (j–q), and A. carolinensis (r–u), as assessed by 3D-volume rendering and eye segmentation (green) of PTA-stained whole-embryos (b, j, r) or embryonic heads (i, q, u) as well as immunohistochemistry with PCNA proliferation marker at the peripheral dorsal retina (c–h, k–p, s, t). Regular developmental stages (every 10 days) covering the entire postoviposition embryonic period, from oviposition (b, c, j, k, r) to hatchling (i, q, u), are shown for each species. Solid arrowheads delimitate the retinal peripheral margin (RM), and open arrowheads indicate the boundary between the monolayered ciliary epithelium (CE) and the retinociliary junction (RCJ) at late embryonic stages. Scale bars, 100 µm (b, i, j, q, r, u) or 1 mm (c–h, k–p, s, t)

Fig 5: Slow-cycling label-retaining cells (LRCs) accumulate in two different regions of bearded dragon teeth.(A) Double IHC for PCNA (red staining) and BrdU (green) in pleurodont (left and middle panels) or acrodont (right) tooth sections collected from bearded dragons at 0 (d0), 28 (d28), and 58 (d58) days of chase after the first week of BrdU pulse. White arrowheads indicate the scattering of BrdU/PCNA-double positive cells throughout the dental epithelium at d28, and red arrowheads show the clustering of BrdU/PCNA-double positive LRCs in both OE and DL at d58. White asterisks indicate the position of taste buds in the OE, and white dashed lines delimitate the epithelium-mesenchyme junction. Scale bars: 100 µm. (B) Schematic drawing of the dental subdivisions used for cell quantification: I, OE, defined as a portion of epithelium directly lingual to the DL; II, external DL; III, deep DL; IV, SDL. (C, D) Total amount of BrdU/PCNA-double positive cells in the entire dental epithelium (C; dental zones I-IV) or relative distribution of BrdU/PCNA-double positive cells in the individual dental subdivisions (D; color-code as in (B)) of pleurodont and acrodont teeth at d0 and d58, n = 3 per group (ns, non-significant). (E, F) IHC for SOX2 (red staining) and BrdU (green; left, middle top, and right top panels) or double IHC/ISH for LGR5 (middle bottom) or IGFBP5 (right bottom) with BrdU in parallel sections of pleurodont (E) and acrodont (F) teeth at d58. Insets show high magnifications of OE (dental zone I as in (C)) or DDL (dental zone III) positive stainings. White arrowheads indicate co-expression of SOX2, LGR5, or IGFBP5 with BrdU. White or black dashed lines delimitate the epithelium-mesenchyme junction. Scale bars: 100 µm.10.7554/eLife.47702.006Figure 3—source data 1.Quantification of putative stem/progenitor cells in dental tissues.Absolute means of BrdU/PCNA-double positive cells in the entire dental epithelium (all regions) or relative amounts of BrdU/PCNA-double positive cells in the individual tooth regions (subdivisions I-IV, see Figure 3B) of pleurodont and acrodont teeth at d0 and d58, n = 3 biological replicates per group.