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1.
Fig. 2.

Fig. 2.K5-Ets1 BT skin demonstrates an expansion of the basal layer.. From: Aberrant epidermal differentiation and disrupted ΔNp63/Notch regulatory axis in Ets1 transgenic mice.

Immunofluorescent staining of E18.5 wild-type and K5-Ets1 BT skin for ΔNp63 (A–B), β4-integrin (C–D) and Ki67 (E–F) (green staining for all). Sections were also co-stained with antibodies to K5 or K14 (red) and with TOPRO-3 to mark the nuclei (blue). Scale bars are 37.5 µm in all cases.

Shu Shien Chin, et al. Biol Open. 2013 Dec 15;2(12):1336-1345.
2.
Fig. 7.

Fig. 7.Model of Ets1 regulation of Notch signaling.. From: Aberrant epidermal differentiation and disrupted ΔNp63/Notch regulatory axis in Ets1 transgenic mice.

When Ets1 is over-expressed it leads to upregulation of ΔNp63, which then inhibits Notch signaling and thereby prevents the transition of keratinocytes from basal to spinous cell fates. The dashed line indicates the possibility that Ets1 might also directly regulate expression of Notch genes in addition to its indirect control of Notch signaling via upregulation of ΔNp63.

Shu Shien Chin, et al. Biol Open. 2013 Dec 15;2(12):1336-1345.
3.
Fig. 3.

Fig. 3.Expression of spinous layer and granular layer markers is decreased in K5-Ets1 BT skin.. From: Aberrant epidermal differentiation and disrupted ΔNp63/Notch regulatory axis in Ets1 transgenic mice.

Immunofluorescent staining of wild-type and K5-Ets1 BT E18.5 skin using antibodies specific for spinous and granular layer markers (green). Involucrin (Inv, A–B), filaggrin (Fil, C–D), Blimp1 (E–F), K1 (G–H), K10 (I–J) and Irf6 (K–L). White arrows point to Blimp1+ or Irf6+ keratinocytes. Some panels were also co-stained with antibodies to K5 (red). All sections were stained with TOPRO-3 to mark the nuclei (blue). Scale bars in all cases are 37.5 µm.

Shu Shien Chin, et al. Biol Open. 2013 Dec 15;2(12):1336-1345.
4.
Fig. 5.

Fig. 5.Impaired Notch signaling in K5-Ets1 BT skin.. From: Aberrant epidermal differentiation and disrupted ΔNp63/Notch regulatory axis in Ets1 transgenic mice.

(A) Quantitative real-time PCR to measure mRNA levels of genes involved in the Notch signaling pathway. Data are represented as mean ± SEM. *P<0.05, Student's t-test. (B–E) Immunofluorescent staining for Notch1 in the skin of E14.5, E16.5 and E18.5 wild-type and E18.5 K5-Ets1 BT embryos (all green). (F–K) Immunofluorescent staining for Notch2, Notch3 and Hes1 in the skin of E18.5 wild-type and K5-Ets1 BT embryos (all green). White arrows point to membrane-associated staining of Notch2 and Notch3. Each section was co-stained with TOPRO-3 to mark nuclei (blue). Scale bars in all cases are 37.5 µm.

Shu Shien Chin, et al. Biol Open. 2013 Dec 15;2(12):1336-1345.
5.
Fig. 6.

Fig. 6.Ets1 regulates expression of ΔNp63.. From: Aberrant epidermal differentiation and disrupted ΔNp63/Notch regulatory axis in Ets1 transgenic mice.

(A) Diagram of the ΔNp63 proximal promoter with potential Ets1 binding sites indicated by black triangles. Arrows below indicate three primer sets used in the ChIP assay. (B) Normalized luciferase activity of the ΔNp63 promoter co-transfected with empty vector (pCMV-HA), a plasmid encoding wild-type Ets1 (pCMV-HA-Ets1) or a DNA-binding mutant of Ets1 (pCMV-HA-Ets1-R391D). (C) ChIP assay using chromatin derived from HA-Ets1 expressing keratinocytes and primer sets 1 (surrounds sites A and B), 2 (surrounds sites B, C and D) and 3 (lacks potential Ets binding sites).

Shu Shien Chin, et al. Biol Open. 2013 Dec 15;2(12):1336-1345.
6.
Fig. 4.

Fig. 4.K5-Ets1 BT adult animals suffer from a dramatic skin phenotype.. From: Aberrant epidermal differentiation and disrupted ΔNp63/Notch regulatory axis in Ets1 transgenic mice.

(A) Overview of the time course for induction of the Ets1 transgene in adult K5-Ets1 BT mice. (B) Adult BT mice induced at weaning develop non-healing sores and scabs on the skin after 3–4 months. (C) Hematoxylin and eosin (H&E) staining of adult wild-type and K5-Ets1 BT skin demonstrates hyper-proliferation, impaired differentiation, increased angiogenesis (black arrowheads) and infiltration of mononuclear cells (black arrows). Scale bars are 75 µm. (D–K) Immunofluorescent staining of wild-type and BT adult skin using antibodies specific for K10, loricrin (Lor), CD11b and Ki67 (all green). Each section was co-stained with either K5 or K6 (red) and with TOPRO-3 to mark nuclei (blue). Note overlap of green Ki67 staining with blue TOPRO-3 staining in parts J-K leads to a pale blue color in nuclei. Scale bars for D–G are 75 µm and for H–K are 37.5 µm.

Shu Shien Chin, et al. Biol Open. 2013 Dec 15;2(12):1336-1345.
7.
Fig. 1.

Fig. 1.Effects of over-expression of Ets1 in the basal layer of the skin.. From: Aberrant epidermal differentiation and disrupted ΔNp63/Notch regulatory axis in Ets1 transgenic mice.

(A) Immunofluorescent staining of E18.5 wild-type and K5-Ets1 BT skin for Ets1 (green staining). Sections were counterstained with TOPRO-3 to mark the nuclei (blue). Scale bars are 37.5 µm. (B) Embryonic E18.5 K5-Ets1 BT mice in which the Ets1 transgene was induced prenatally exhibit an eye-open-at-birth phenotype (arrows point to closed or open eyes in the wild-type and K5-Ets1 BT pups), but grossly normal skin. (C) K5-Ets1 BT embryos exhibit delayed skin barrier acquisition at E16.5 and E17.5. However, at E18.5, both wild-type and BT embryos show similar skin barrier function (note that the open-eye phenotype of BT embryos results in a blue-stained eye). (D) Hematoxylin and eosin (H&E) staining of E18.5 embryonic wild-type and BT skin demonstrates a second layer of basal-like epithelial cells (black bracket), impaired differentiation of suprabasal keratinocytes and increased angiogenesis (black arrows). Scale bars are 37.5 µm. Inset shows a higher magnification view of the skin.

Shu Shien Chin, et al. Biol Open. 2013 Dec 15;2(12):1336-1345.

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