Additional file 3: Figure S3. of The polarity protein Scrib mediates epidermal development and exerts a tumor suppressive function during skin carcinogenesis

Analysis of Scrib-deficient DMBA/TPA-driven epidermal lesions. (A) Kaplan Meier survival plot for DMBA/TPA treated Scrib +/+ , Scrib +/fl and Scrib fl/fl mice. The average survival of Scrib fl/fl mice (23.5 weeks post-DMBA) was comparable to Scrib +/+ and Scrib +/fl mice (24.5 and 24.9 weeks post-DMBA respectively). No statistical difference in survival was observed between genotypes (χ2 = 0.029 – 2.15, df = 1, P ≥ 0.1426, Log-rank Mantel-Cox test, n = 12–16). (B) qRT-PCR for Scrib mRNA confirmed a significant reduction in Scrib +/fl and Scrib fl/fl volume-matched (40 – 80 mm3) early benign papillomas compared to Scrib +/+ lesions (*p < 0.0001, unpaired t-test, n = 3). (C) IF to detect Scrib (green) and DAPI (blue) in Scrib +/+ , Scrib +/fl and Scrib fl/fl DMBA/TPA-induced size-matched benign papillomas (n = 3, scale bar = 50 μm, insert 1–3 scale bar = 10 μm). Representative p-ERK IHC images (D) and quantitation (E) from Scrib +/+ , Scrib +/fl and Scrib fl/fl DMBA/TPA-induced size-matched benign papillomas (scale bar = 50 μm, P ≥ 0.5919, unpaired t-test, error bars = SD, n = 3). (F) IF to detect ZO-1 (green), E-cadherin (red) and DAPI (blue) in Scrib +/+ , Scrib +/fl and Scrib fl/fl DMBA/TPA-induced size-matched benign papillomas (n = 3, scale bar = 50 μm). (PPTX 1616 kb

Additional file 1: Figure S1. of The polarity protein Scrib mediates epidermal development and exerts a tumor suppressive function during skin carcinogenesis

Cell polarity is not deregulated in Scrib KO embryonic epidermis. (A) IF to detect pan-Dlg (green) and DAPI (blue) and (B) IF to detect E-cadherin (red) and DAPI (blue) in Scrib Wt, Het and KO embryonic epidermis at E17.5 (n = 3, scale bar = 50 μm, dashed line represents basement membrane). (PPTX 539 kb

The Asymmetric Cell Division Regulators Par3, Scribble and Pins/Gpsm2 Are Not Essential for Erythroid Development or Enucleation

<div><p>Erythroid enucleation is the process by which the future red blood cell disposes of its nucleus prior to entering the blood stream. This key event during red blood cell development has been likened to an asymmetric cell division (ACD), by which the enucleating erythroblast divides into two very different daughter cells of alternate molecular composition, a nucleated cell that will be removed by associated macrophages, and the reticulocyte that will mature to the definitive erythrocyte. Here we investigated gene expression of members of the Par, Scribble and Pins/Gpsm2 asymmetric cell division complexes in erythroid cells, and functionally tested their role in erythroid enucleation <i>in vivo</i> and <i>ex vivo</i>. Despite their roles in regulating ACD in other contexts, we found that these polarity regulators are not essential for erythroid enucleation, nor for erythroid development <i>in vivo</i>. Together our results put into question a role for cell polarity and asymmetric cell division in erythroid enucleation.</p></div

The ACD regulators Par3, Scribble and Gpsm2 are not required for stress erythropoiesis in the spleen.

<p><b>(A)</b> Graphs showing spleen weights relative to body weights determined from untreated and PHZ treated, age-matched mice of the indicated genotypes of the different mouse models. Data represent the means (+/- SD) of 3–5 independent experiments. <b>(B)</b> Graphs showing total cell numbers per spleen (on left) and percentages of erythroid cells (Ter119 positive) in spleen (on right) isolated from PHZ treated, age-matched mice of the indicated genotypes of the different mouse models. Data represent the means (+/- SD) of 3–6 independent experiments. <b>(C)</b> Bar graphs showing percentages of erythroid cells at the different developmental stages (I-V) during stress erythropoiesis in the spleen from PHZ treated mice of the indicated genotypes of the different mouse models. Data represent the means (+/- SD) of 3–5 independent experiments. *P< 0.05, **P< 0.01, ***P< 0.001, ****P< 0.0001 (unpaired student’s t-test).</p

The ACD regulators Par3, Scribble and Gpsm2 are not required for erythroid enucleation during stress erythropoiesis.

<p><b>(A)</b> Representative images of blood smears from peripheral blood derived from PHZ treated, age-matched mice of the indicated genotypes of the different mouse models. Scale bar = 10μm. <b>(B)</b> Whole blood analysis using the CELL-DYN Sapphire System was performed on peripheral blood derived from PHZ treated, age-matched mice of the indicated genotypes of the indicated mouse models. Data represent the mean (+/- SD) of 3–6 independent experiments. <b>(C)</b> Orthochromatic erythroblasts were enriched by FACS (Aria II) from spleen of PHZ treated mice of the indicated genotypes of the indicated mouse models and incubated in 96-well plates at 30000 cells per well. Graphs showing enucleation efficiencies 5h post sort quantified by LSR II. Data represent the mean (+/- SD) of 3 independent experiments. *P< 0.05, **P< 0.01, ***P< 0.001, ****P< 0.0001 (unpaired student’s t-test). <b>(D)</b> Whole blood derived from PHZ treated, aged-matched mice of the indicated genotypes of the different mouse models was exposed to decreasing concentrations of sodium chloride (NaCl) and the degree of hemolysis measured colorimetrically. Data represent the mean (+/- SD) of 3 independent experiments.</p

Sequences of primers used for QRTPCR.

<p>Sequences of primers used for QRTPCR.</p

The ACD regulators Par3, Scribble or Gpsm2 are not required for steady-state erythropoiesis in the bone marrow.

<p><b>(A)</b> Graphs showing total cell numbers per femur (on left) and percentages of erythroid cells (Ter119 positive) in femurs (on right) isolated from age-matched mice of the indicated genotypes of the different mouse models. Data are mean (+/- SD) of 3–6 independent experiments. <b>(B)</b> (i.) Diagram showing the distinct erythroid populations at the different maturation stages (I-V) that can be identified by FACS. (ii.) Representative FACS plot showing erythroid differentiation stages in the bone marrow. <b>(C)</b> Bar graphs showing percentages of erythroid cells at the different developmental stages (I-V) during homeostasis in the bone marrow harvested from age-matched mice of the indicated genotypes of the different mouse models. Data represent the mean (+/- SD) of 3–6 independent experiments. *P< 0.05, **P< 0.01, ***P< 0.001, ****P< 0.0001 (unpaired student’s t-test).</p