Analysis of <i>Eva1</i> expression.

<p><i>Eva1</i> real-time RT-PCR in fetal thymi (A), adult DN subpopulations (B, left panel) and thymocytes from mutant mice (B, right panel). mRNA from flow cytometrically purified TECs (CD45-) and intrathymic haematopoietic cells (CD45+) or DN1-3 subpopulations was reverse transcribed and used as the template for PCR with <i>Eva1</i>-specific primers. All samples were normalized to the geometric mean of the GAPDH housekeeping gene. NB, newborn; 2mth, two months; WT, wild type; ΔCAM, Tg-Calcineurin; <i>Prkdc</i>^scid, protein kinase, DNA-activated, catalytic polypeptide (C, upper panel) Eva1 interference in LSK cells by lentiviral vector was controlled by real time RT-PCR. LSK-CT cells shown a comparable expression of <i>Eva1</i> while LSK-EVAi cells shown a drastic decrease of <i>Eva1</i> expression, indicating that the interference has occurred. (C, lower panel), fluorescence microscopy analysis confirming <i>Eva1</i> interference in LSK-EVAi cells. LSK-WT, uninfected LSK; LSK-CT, LSK infected with a non-interfering lentiviral vector; LSK-EVAi, LSK infected with a <i>Eva1</i>-interfering lentiviral vector.</p

<i>In vivo</i> T cell development with LSK-EVAi cells.

<p>(A, left panel) <i>Rag2/γc</i> mice were transplanted with either LSK cells infected with a non-interfering (LSK-CT) or with an Eva1-interfering lentivirus (LSK-EVAi). After 8 weeks, thymi were excised and compared: thymus from mice transplanted with LSK-EVAi were comparable to untreated ones for dimension and total cellularity (A, right panel). Untr., untreated control animal. Scale bar, 2 mm. (B) Time course of T cell development of LSK-CT (white bars) or LSK-EVAi (black bars) cells respectively, in recostitution experiments in <i>Rag2/γc</i> mice. Flow cytometric analysis of CD25/CD44 and CD4/CD8 stainings revealed that LSK-EVAi reconstituted thymus had a delay of T cell differentiation with an accumulation of DN cells at 6 weeks after trasplant (left panel). Percentage of DN cells in LSK-EVAi reconstituted thymus persists higher than in LSK-CT reconstituted thymi at 8 weeks after transplant (right panel). Mean and SD of three independent experiments are shown (** p<0.01). (C) Counts of total DN cells generated in the three different mouse groups at six and eight weeks post-treatment.</p

A Small-Molecule Targeting the MicroRNA Binding Domain of Argonaute 2 improves the Retinoic Acid Differentiation Response of the Acute Promyelocytic Leukemia Cell Line NB4

Argonaute proteins are pivotal regulators of gene expression mediating miRNAs function. Modulating their activity would be extremely useful to elucidate the processes governing small-RNAs-guided gene silencing. We report the identification of a chemical compound able to compete with Argonaute 2 miRNAs binding, and we demonstrate that this functional inhibition determines effects similar to Argonaute 2 shRNA-mediated down-regulation, favoring granulocytic differentiation of the acute promyelocytic leukemia cell line NB4 in response to retinoic acid

Additional file 6: of Expression of ID4 protein in breast cancer cells induces reprogramming of tumour-associated macrophages

Figure S2. Predictive power of ID4 mRNA expression for overall survival (OS) was evaluated by Kaplan-Meier analysis on the TCGA cohort in BLBCs showing high or low CD68 (a and b) or macrophage signature (MacSig) (c and d) levels. Macrophage signature is composed of eight widely used markers for the mononuclear phagocyte system (CD14, CD105, CD11b, CD68, CD93, CD33, IL4R and CD163 [37]). e Evaluation of association between ID4 or CD68 and the pathological variables T, N, G and TP53 status in the BLBCs from the TCGA cohort. (PDF 4464 kb

Additional file 3: of Expression of ID4 protein in breast cancer cells induces reprogramming of tumour-associated macrophages

Figure S1. Comparison of ID4 mRNA expression in basal-like breast cancer (BLBC) and triple-negative breast cancer (TNBC) versus all other breast cancer subtypes (Others) in the indicated representative datasets [19, 22, 60]. (PDF 143 kb

Additional file 9: of Expression of ID4 protein in breast cancer cells induces reprogramming of tumour-associated macrophages

Figure S6 Differentiated U937 cells transfected with an empty vector (EV) or a granulin (GRN) expression vector and subsequently cultivated in the presence of CM from MDA-MB-468 cells were evaluated for their differentiation state (percentage of CD11b+ cells) (a) and for their viability (b) by, respectively, FACS analysis and ATPlite assay at the indicated time points after CM addition. c Overexpression of GRN evaluated by Western blotting. (PDF 141 kb

Additional file 5: of Expression of ID4 protein in breast cancer cells induces reprogramming of tumour-associated macrophages

Table S3 mRNAs modulated in an ID4-dependent manner in differentiated HL60 cells cultured with conditioned medium from control (CM EV) or ID4-overexpressing (CM ID4) MDA-MB-468 cells. The presence of HIF-1 consensus sequences on promoters was evaluated using the LASAGNA-Search web tool (http://biogrid-lasagna.engr.uconn.edu/lasagna_search/). The presence of putative binding sites for miR-107, miR-15b and miR-195 on 3′-UTR or coding (CDS) sequences of mRNAs was evaluated using the miRWalk analysis tool ( http://zmf.umm.uni-heidelberg.de/apps/zmf/mirwalk2/ ) by selecting the following databases: (1) 3′-UTR analysis = miRWalk, miRanda, miRDB, miRNAMap, Pictar2, RNA22, RNAhybrid, TargetScan; and (2) CDS analysis = miRWalk, miRanda, RNA22, RNAhybrid, TargetScan. (DOCX 22 kb

Additional file 2: of Expression of ID4 protein in breast cancer cells induces reprogramming of tumour-associated macrophages

Table S1 Characteristics of patients selected for the analysis of ID4 protein expression. (DOCX 17 kb

Additional file 8: of Expression of ID4 protein in breast cancer cells induces reprogramming of tumour-associated macrophages

Figure S5 a Expression of miR-107, miR-15b and miR-195 in differentiated HL60 cells cultured with CM from control (CM EV) or ID4-overexpressing (CM ID4) MDA-MB-468 cells. b–e Expression of miR-15b and miR-195 in HL60 and U937 cells cultured with CM from control (si-SCR) or ID4-depleted (si-ID4) BC cells. f miR-107, miR-15b and miR-195 expression evaluated by RT-qPCR in differentiated U937 cells cultured with CM from MDA-MB-468 cells depleted or not of VEGFA expression. VEGFA interference efficiency is shown in Fig. 3i. g Expression levels of miR-15b and miR-195 in differentiated U937 cells cultivated in RPMI medium (CTR) or CM from MDA-MB-468 cells for the indicated time points. h and i HIF1A mRNA (h) and protein (i) expression evaluated, respectively, by RT-qPCR and immunofluorescence in differentiated U937 cells transfected with control mimic or miR-107 mimic and cultured in the presence of CM from MDA-MB-468 cells for 48 hours. (PDF 2150 kb

Additional file 1: of Expression of ID4 protein in breast cancer cells induces reprogramming of tumour-associated macrophages

Figure S3 Growth curve of MDA-MB-468 cells depleted (si-ID4) or not (si-SCR) of ID4 expression by siRNA transfection (a). Cells were transfected for 16 hours, and then equal numbers of cells were plated and counted at the indicated time points. Efficiency of ID4 depletion at 48 hours and 72 hours was evaluated by Western blotting (b). (PDF 4554 kb