<i>ISG15</i> expression and protein ISGylation are induced during <i>in vivo</i> erythroid differentiation.

<p>(<b>A</b>) Sorting procedure of the different erythroblasts populations from bone marrow cells of WT Bl6/J mice. (<b>B</b>) RT-PCR analyses of <i>ISG15, UbcM8, Herc6, Ube1L, Bcl-X_L</i> and <i>ß-Major-Globin</i> mRNA in sorted Pro-erythroblasts (Pro-E: large cells, CD71^high, Ter119^med), basophilic erythroblasts (EryA: large cells, CD71^high, Ter119^high), polychromatic (EryB: small cells, CD71^med, ter119^high) and orthochromatic erythroblasts/reticulocytes (EryC: small cells, CD71^low, ter119^high) as described in Mat & Met. Experiments were normalized to 18S Ribosomal RNA expression. (<b>C</b>) Statistical analysis of the induction of the expression of <i>ß-Maj-Globin, Bcl-X_L, ISG15</i>, <i>Ube1L, UbcM8</i> and <i>Herc6 m</i>RNA. Quantification was performed as described in Mat & Met. Note the two-fold decreased expression of a second housekeeping gene, ß-actin during differentiation. au = arbitrary unit. (<b>D</b>) Western blot analyses of whole cell extracts of the indicated erythroid subpopulations using anti-ISG15 (top panels) antibody. Middle panels show GAPDH levels as loading control. Bottom panels show globin accumulation as detected with Ponceau staining of the membranes. Whole cell extracts were prepared from sorted wild-type bone marrow cells as in A or from RBC of WT and <i>ISG15^-/-</i> mice. (<b>E</b>) Statistical analysis of the induction of ISG15 at the protein level during <i>in vivo</i> erythroid differentiation normalized to ß-Actin. Quantification was performed as described in Mat & Met. au = arbitrary unit.</p

<i>ISG15</i> expression during erythroid differentiation is independent of IFN signaling and partially dependent upon Epo signaling.

<p>(<b>A</b>) semi-quantitative RT-PCR analyses of <i>ISG15, Ube1L, UbcM8, Herc6, Irf7, Bcl-x_L</i> and <i>ß-Major-Globin</i> mRNA expression in WT or <i>IFNAR^-/-</i> differentiating primary erythroblasts. Bone marrow erythroblasts of each genotype were maintained in proliferation conditions (SCF, Epo and Dex) for one week and next induced to differentiate in response to Epo alone for three days. Cells were collected every 24 hours as indicated and RNA extracted. The experiment was normalized to <i>ß-Actin</i> mRNA expression. (<b>B</b>) Statistical analysis of the induction of the expression of <i>ß-Maj-Globin, Bcl-X_L</i>, <i>ISG15</i>, <i>Ube1L</i>, <i>UbcM8</i> and <i>Herc6</i> mRNA in differentiating WT and IFNAR^-/- erythroblasts. Quantification was performed as described in Mat & Met. Note the unchanged expression of a second housekeeping gene <i>HPRT</i>. au = arbitrary unit. (<b>C</b>) Whole cell protein extracts were prepared from WT or <i>IFNAR^-/-</i> erythroblasts maintained as in A and analyzed on a 10% acrylamide gel for <i>ISG15</i> expression using an anti-ISG15 antibody. Anti-ß-Actin and anti-GAPDH were used as loading controls. (<b>D</b>) Statistical analysis of the induction of ISG15 during erythroid differentiation as normalized to ß-Actin. Quantification was performed as described in Mat & Met. au = arbitrary unit. (<b>E and F</b>) A p53^-/- erythroid cell line expressing exogenous hBcl-X_L was switched from proliferation conditions (Epo, SCF, Dex) to differentiating medium in the presence or absence of Epo. Cells were collected every 24 hours as indicated and analyzed for their ability to (e) survive as measured by propidium iodide staining in flow cytometry analyses; (f) differentiate as shown by analysis of their morphology after benzidine/May-Grunwald staining. Note the significant induction of cell death in control cells maintained in absence of Epo; in contrast, hBCL-xL erythroblasts are strongly protected from apoptosis. (<b>G</b>) Cells were lyzed according to cell number and volume. ISG15 expression was analyzed on a 15% acrylamide gel using anti-ISG15 antibody, activation of the EpoR/STAT5 signaling pathway was monitored using anti-P-STAT5 antibody, differentiation was monitored using anti-Globin antibody and loading control was performed using anti-SAM68 antibody. (<b>H</b>) Mock and mscv-puro-STAT5^S710F transduced p53^-/- erythroid cell line maintained under proliferation conditions were lyzed and analyzed for ISG15 expression using anti-ISG15 antibody on a 10% acrylamide gel (Top panel). P-STAT5 was detected at a higher level in mscv-puro-STAT5^S710F transduced cells while only a modest increase in the total amount of STAT5 can be noted. Anti-β -Actin was used as a loading control.</p

<i>ISG15</i> deficiency impairs <i>in vitro</i>-induced erythroid differentiation.

<p>(<b>A</b>) Erythroblasts from the BM of either wild-type or <i>ISG15^-/-</i> mice were maintained under proliferation conditions (SCF, Epo, Dex) and switched to Epo alone to induce differentiation. Cumulative cell number of three independent wild-type (black symbols) and <i>ISG15^-/-</i> erythroblasts cultures (open symbols) are shown both under proliferation and differentiation conditions. Cells were counted with an electronic counter (CASY Scharfe). (<b>B</b>) Quantitative determination of hemoglobin contents of differentiating WT and <i>ISG15^-/-</i> erythroblasts 24, 48 and 72 hours after differentiation induction. Normalized values (hemoglobin level per 10⁶ live cells) are shown. (<b>C</b>) Cytocentrifugation analyses followed by Benzidine-May-Grunwald staining of cells maintained either under proliferation conditions (day 0) or induced to differentiate in response to Epo. Differentiating cells are stained in brown by Benzidine (black arrow), immature eryhroblasts stain in blue. Note the presence of a significant proportion of immature cells in <i>ISG15^-/-</i> culture (open arrow) as compared to WT culture. Representative fields are shown. (<b>D</b>) Quantification of cells of increasing maturity 24 h, 48 h and 72 h after the cells had been induced to differentitate. Cells (≥200) were counted per slide and mean values ±s.d. calculated from at least three independent experiments.</p

Altered distribution of erythroid progenitors in <i>ISG15^-/-</i> bone marrow and splenic cells.

<p>(<b>A</b>) Blood was collected from mice at 8–10 weeks of age. Hematologic measurements were performed on a MS9 Hematology Analyzer (MELET SCHLOESING Laboratoires). The data are mean +/- SEM (N = 14). RBC indicates red blood cells; HGB, hemoglobin; MCH, mean corpuscular hemoglobin; MCHC, MCH concentration (calculated); HCT, hematocrit; MCV, mean corpuscular volume; RDW, RBC distribution width, Retic, reticulocytes; PLT, platelets; WBC, white blood cells; MONO, monocytes, LYMP, lymphocytes and GR, granuloctes. (<b>B</b>) Quantitative analysis of the distribution of the different erythroblasts subsets in age-matched WT versus <i>ISG15^-/-</i> mice. Flow cytometry analyses using the cell surface markers CD71 and Ter119 of bone marrow and spleen cells isolated from WT or <i>ISG15^-/-</i> mice (as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0026068#pone-0026068-g001" target="_blank">Figure 1A</a>). Dead cells (7AAD⁺) were excluded from the analysis. The data are mean +/- SEM (n = 13). (<b>C</b>) 2.10⁵ BM and 2.10⁶ spleen cells from mice at 8–10 weeks of age were used to assay BFU-E and CFU-E numbers in MethoCult M3334 (StemCell Technologies). For CFU-E assays, colonies were counted at day 2 and for BFU-E assay, at day 4. The data are mean +/- SEM (n = 11).</p

Enforced ISG15 expression facilitates erythroid terminal differentiation.

<p>(<b>A</b>) Western blot analysis of whole cell extract obtained from proliferating p53^-/- erythroid cell line transduced with either the control MSCV-puro, or MSCV-puro-ISG15-flag retroviruses. An anti-ISG15 antibody was used to show ISG15 expression and induction of protein ISGylation on a 10% acrylamide gel while anti-STAT5 was used as a loading control. (<b>B</b>) Hemoglobin content quantification analyses 48 and 72 hours after differentiation induction. (<b>C</b>) Quantification of cytospin preparation of 48 h-differentiating control and ISG15-overexpressing erythroblasts. Cells (≥200) were counted per slide and mean values ±s.d. calculated from at least three independent experiments (<b>D</b>) Flow cytometry analysis of erythroid cell surface marker Ter119, 72 hours after differentiation induction. Grey line: isotypic control, grey dashed line: control proliferating erythroblasts (1,34% Ter119 positive cells), black dashed line: control differentiating erythroblasts (24,2% Ter119 positive cells); black line: ISG15-differentiating erythroblasts (34,4% Ter119 positive cells).</p

Identification of several ISGylation substrates in erythroid cells and increased ubiquitination in <i>ISG15^-/-</i> RBCs.

<p>(<b>A</b>) Proliferating erythroblasts were left either untreated (1), or stimulated with IFNß (100 U/ml) for 24 h (3), or induced to differentiate for 72 h (lane 4 to 8). Cells were lyzed according to size and number and ISG15/ISGylation level was compared by western blotting to the level of ISG15/ISGylation present in proliferating Flag-ISG15-expressing p53^-/- erythroid cell line. The arrow points to ISG15 band (<b>B</b>) Quantification of ISG15 level in independent western blot experiments. (<b>C</b>) Proliferating control and Flag-ISG15-expressing p53^-/- erythroid cell line were lysed and ISGylated proteins searched after a Flag immunoprecipitation. Anti-Flag detect ISG15-Flag and ISGylated proteins (arrow heads) in the crude lysates (dashed line) and are found enriched after the immunoprecipitation (plain line bar). Stars point to light and heavy chains of immunoglobulin. For the detection of ISGylated Globins, cells were induced to differentiate for 72 hours in order to induce globin expression. ISGylated proteins are indicated by arrowheads. Normal unmodified molecular weight of the proteins are: ERK2 (42kDa), PLCγ (150kDa), STAT5 (90kDa), Globin (13kDa). For ERK2 and STAT5 detection, cell lysates were run on a 10% acrylamide gel, for ISG15 and Globins detection on a 15% acrylamide gel and for PLCγ on a 8% gel. (<b>D</b>) Proliferating or 48 hours-differentiating control and STAT5-Flag-expressing p53^-/- cell line were lysed and analyzed for exogenous STAT5 expression either using a Flag (upper left panel) or a STAT5 antibody (upper right panel). Note the modest increase in the total amount of STAT5 induced by the expression of STAT5-Flag. ISGylated STAT5 was searched after a Flag immunoprecipitation followed by either a Flag (bottom left panel) or an ISG15 western blot analyses (bottom right panel). Extracts were run on a 7% acrylamide gel. (<b>C</b>) Western blot analysis of RBC extracts from WT and <i>ISG15^-/-</i> mice using a 15% acylamide gel. ISG15 expression and ISGylation were analysed using anti-ISG15 antibody (top panel), ubiquitination was monitored using anti-ubi antibody (intermediate panel) and anti-β-Actin was used as a loading control.</p

Long-term reconstitution with Lgl1^−/− bone marrow does not induce leukaemia.

<p>Unfractionated bone marrow was isolated from chimeric mice one-year post initial foetal liver reconstitution, and transplanted into Ly5.1/Ly5.2 heterozygote recipients. (<b>A–C</b>) Peripheral blood was analysed for the proportion of primary Ly5.2⁺ donor cells in peripheral blood at 5 weeks, 14 weeks, and 16 weeks post transplantation. (<b>D–F</b>) At 16 weeks, the lineage potential and existence of leukemic clones of transplanted bone marrow was assessed by measuring the proportion of primary donor Ly5.2+ B cells (B220⁺, CD19+), CD4 T cells (CD3⁺, CD4⁺) and CD8 T cells (CD3⁺, CD8⁺). Data shown are pooled results from 3 individual donors per genotype.</p

Lgl1 does not function as a tumour suppressor in conjunction with common oncogenes.

<p>Lg1^+/+ and Lgl1^−/− foetal liver cells were infected with either empty vector MigR1-GFP or ICN1-ΔRamΔP-GFP constructs and transplanted into lethally irradiated Ly5.1 recipient mice. (<b>A</b>) Kaplan-Meier analysis of survival of mice following reconstitution with infected foetal liver cells. Kaplan-Meir analysis of survival in lethally irradiated Ly5.1 mice reconstituted Ly5.2+ Lgl1^+/+,Lgl^+/− and Lgl^−/− foetal liver cells in the presence of either the TEL-JAK2 (<b>B</b>) or Eµ-Myc (<b>C</b>) transgenes. Splenocytes (<b>D</b>) or Eµ-Myc B cell lymphomas (<b>E</b>) were isolated from primary chimeric wildtype and Lgl deficient mice and assayed by Q-PCR for expression of both Lgl1 and Lgl2 in triplicate samples. Statistical differences between the means of two data groups was determined by using two-tailed unpaired Student's <i>t</i> test, and <i>p</i> values <0.05 were considered significant. For survival analysis, Kaplan-Meier survival curves were analyzed using Log-rank (Mantel-Cox) test. P<0.05 were considered significant.</p

Analysis of peripheral blood in primary reconstituted Lg1^−/− mice.

<p>Lethally irradiated Ly5.1 mice were reconstituted with 1×10⁶ foetal liver cells isolated from E14.5 embryos from Lg1^+/+, Lg1^+/− and Lgl1^−/− mice. (<b>A</b>) >6weeks post reconstitution, composition of the hematopoietic compartment was determined by ADVIA analysis of peripheral blood. (<b>B</b>) Donor derived Ly5.2⁺ lymphocyte populations in the blood of Lg1^+/+ and Lgl1^−/− chimeric mice were identified by flow cytometry and frequencies quantified in (<b>C</b>). Statistics represent mean ± SEM from one reconstitution. Lgl<i>^+/+ n = 4–11</i>, Lgl<i>^+/− n = 7–9</i> Lgl^{−<i>/</i>−}<i>n = 7–9</i>. One representative reconstitution is shown from 4 independent experiments</p