Additional file 1: Figures S1A, S1B, and S1C. of PML-RAR alpha induces the downmodulation of HHEX: a key event responsible for the induction of an angiogenetic response

Figure S1A Analysis of HHEX (top panel) and VEGF-A (middle panel) reported in 176 primary AMLs on the TCGA platform. The HHEX/VEGF-A ratio is reported in the bottom panel. Figure S1B Correlation between HHEX and VEGF-A levels observed in 18 primary APLs in the present study (p = 0.0484) and in 16 primary APLs in the TCGA data (p = 0.0284). Figure S1C Correlation between HHEX and VEGF-A levels observed in 18 primary APLs (p = 00484) and in 20 primary M5 AMLs in the TCGA data (p = 0.0174). (ZIP 689 kb

In hypoxia, HIF-1α is a direct transcriptional repressor of TM9SF4 in leukemic cells.

<p>(A) Performed under hypoxic condition (1% O₂), knockdown of HIF-1α in U937 and HL-60 cells by using HIF-1α small interfering RNAs (HIF-1α-siRNA), as compared to control siRNA (c-siRNA), was controlled at protein level by Western blot analysis in U937 cells (left panel) and at mRNA level by real time PCR analysis in HL-60 cells (right panel). (B-D) TM9SF4 expression in U937(HIF-1α-siRNA) and HL-60(HIF-1α-siRNA) cells, as compared to U937(c-siRNA) and HL-60(c-siRNA) control cells, cultured in hypoxia (1% O₂) compared to normoxia (N), was analyzed at mRNA level by real time PCR analysis (B) and at protein level by western blot (C) and flow cytometry (D) analysis in U937 and HL-60 cells. (A HL-60, B, D) The results of three independent experiments (mean ± SEM values) are shown; *, ** are p<0.05, p<0.01 respectively. (A U937, C) One representative experiment out of three is shown; nucleolin is used as an internal control of U937 nuclear protein extracts; actin is shown as internal control of total protein extracts (C).</p

TM9SF4 is expressed during Mo and G proliferation and differentiation of HPCs, but is overexpressed in AMLs.

<p>(A, B) Real time PCR analysis of TM9SF4 mRNA expression during selective Mo and G proliferation and differentiation of CD34⁺ HPCs (day 0). (C-F) Analysis of TM9SF4 protein expression is shown by flow cytometry (C, D) and Western blot (E, F) analysis in Mo and G differentiating HPCs. (G) Real time PCR analysis of TM9SF4 mRNA expression in primary leukemic cells of AMLs pertaining from M0 to M5 subtypes of FAB classification, as compared to normal CD34⁺ HPCs. (A—D) The results of three independent experiments (mean ± SEM values) are shown; n.s. is for not significant; significance *, **, *** are p<0.05, p<0.01, p<0.001 respectively and as compared to day 0; °, °° are p<0.05, p<0.01 respectively and between indicated days. (A, B, G) A.U. is for arbitrary units. (C, D) MFI is for Mean Fluorescence Intensity. (E, F) One representative experiment out of three is shown; actin is an internal control. (F) 293T is for protein extract prepared from 293T(pcDNA3.1/TM9SF4) overexpressing cells used as a positive control of TM9SF4 protein expression. (G) Results are presented as scatter plot. Mean is indicated; significance *, ⁺ and *** are p<0.05 and p<0.001 respectively.</p

TM9SF4 decreases during vitamin D3-induced Mo differentiation of U937 cells, but increases during ATRA-induced G differentiation of HL-60 cells.

<p>(A) Flow cytometry analysis of specific Mo markers CD11b and CD14 expression was performed from day 0 to 5 (d5) in U937 cells treated with vitamin D3 (VITD3) to induce Mo differentiation, as compared to untreated U937 control cells (d0). (B) Real time PCR analysis of TM9SF4 mRNA expression during VITD3- induced Mo differentiation of U937 cells. (C, D) Western blot (C) and flow cytometry analysis (D) of TM9SF4 protein expression in VITD3-treated U937 cells, as compared to untreated control U937 cells (d0). (E) Flow cytometry analysis of G markers CD11b and CD54 was performed from day 0 to 5 (d5) in HL-60 treated with all trans retinoic acid (ATRA) for G differentiation, as compared to untreated HL-60 control cells (d0). (F) Real time PCR analysis of TM9SF4 mRNA expression during ATRA- induced G differentiation of HL-60 cells. (G, H) Western blot (G) and flow cytometry analysis (H) of TM9SF4 protein expression in ATRA-treated HL-60 cells, as compared to untreated control HL-60 cells (d0). (A, B, D, E, F, H) The results of three independent experiments (mean ±SEM values) are shown; *, **, *** are p<0.05, p<0.01, p<0.001 respectively; n.s. is for not significant. (A, D, E, H) MFI is for Mean Fluorescence intensity. (B, F) A.U. is for arbitrary units. (C, G) One representative experiment out of three is shown; actin is shown as an internal control; (C) 293T is for 293T(pcDNA3.1/TM9SF4) overexpressing cells used as a positive control of TM9SF4 protein expression.</p

Hypoxia that activates HIF-1α, downmodulates TM9SF4 expression in leukemic cells.

<p>(A, B) Cell growth analysis of U937 (A) and HL-60 (B) cells grown 48 hours in hypoxia (1% O₂), as compared to normoxia (21% O₂). (C, D) Western blot analysis of HIF-1α nuclear protein expression in U937 (C) and HL-60 (D) cells cultured in hypoxia (1% O₂), as compared to normoxia (21% O₂). (E, F) Real time PCR analysis of TM9SF4 mRNA expression in U937 (E) and HL-60 (F) cells in hypoxia, as compared to normoxia. (G—J) TM9SF4 protein expression levels were analyzed by Western blot (G, H) and flow cytometry (I, J) analysis in U937 (G, I) and HL-60 (H, J) cells in hypoxia, as compared to normoxia. (A, B, E, F, I, J) The results of three independent experiments (mean ± SEM values) are shown; *, ** are p<0.05, p<0.01 respectively. (C, D, G, H) One representative experiment out of three is shown; nucleolin is an internal control of nuclear protein extracts (C, D); actin is an internal control of total protein extracts (G, H); 293T is for 293T(pcDNA3.1/TM9SF4) overexpressing cells, used as a positive control of TM9SF4 protein expression (G). (E, F) A.U. is for arbitrary units.</p

TM9SF4 is a new molecule involved in cell adhesion of leukemic cells.

<p>(A, B) Adhesion assays show that the number of adherent U937 cells to fibronectin (Fn)-coated plates under hypoxic condition (1% O₂) is significantly decreased compared to normoxic conditions (21% O₂). (C, D) In normoxia, adhesion assays show that the number of adherent TM9SF4-siRNAs transfected U937 cells to fibronectin (Fn)-coated plates (TM9SF4-siRNA) is significantly lower than the number of adherent control-siRNA transfected U937 cells (c-siRNA). (E; F) Knockdown of TM9SF4 expression in TM9SF4-siRNA transfected U937 cells (TM9SF4-siRNA) was controlled by Western blot (E) and flow cytometry (F) analysis of TM9SF4 protein expression compared to control-siRNAs transfected U937 cells (c-siRNA). (A, C) Pictures of one representative experiment of adhesion assays are shown. (B, D, F) Data are presented as the mean of six independent experiments (n = 6) ± SEM; **, *** are p<0.01, p<0.001 respectively. (E) One representative experiment out of three is shown; actin is shown as internal control.</p

In hypoxia, TM9SF4 is a direct target gene of HIF-1α in leukemic cells.

<p>(A) HIF-1α DNA binding site (HRE) identified on TM9SF4 partial promoter sequence is shown. (B) Chromatin immunoprecipitation (ChIP) experiments were performed by using protein extracts of U937 cells cultured in hypoxia (panels 1% O₂), as compared to normoxia (panels 21% O₂), immunoprecipitated with the anti(α)-HIF-1α or with an unrelated α-cAbl antibodies, or no antibody (No Ab), and analyzed by PCR amplification of the TM9SF4 region surrounding the HRE site (Prom-TM9SF4) or a GAPDH coding region used as an internal control. Input: PCR on 0.02% of total chromatin. One representative experiment of three is shown. (C) Promoter activity assays were performed first (<i>left panels</i>) by using TM9SF4 upstream DNA sequence subcloned in the pGL3Basic promoterless luciferase vector (Prom-TM9SF4) and transfected into 293T cells in the presence (white bars) or absence (black) of a HIF-1α expression vector, as compared to empty pGL3Basic vector (vector); then (<i>right panels</i>) by using Prom-TM9SF4, or the same region with a mutated HRE binding site (Prom- MutTM9SF4), cloned in the pGL3Promoter reporter vector containing a minimal promoter, and transfected with or without a HIF-1α expression vector. Data of luciferase activity detected are mean ± S.E.M. values of 3 independents experiments.</p

Effect of exogenous angiopoietins on the growth of UT7/mpl cells.

<p><i>Top Panels</i>: UT7/mpl cells have been grown either in the absence (C ) or in the presence of either GM-CSF, TPO, Ang-1, Ang-2, TPO+Ang-1 or TPO+Ang-2 and the number of living cells was determined at each day of culture. <i>Bottom Panel</i>, left: At day 4 and 7 of culture cell aliquots were harvested and analyzed for cell cycle by PI labeling and flow cytometry. The proportion of S phase cells (mean values ± SEM observed in three separate experiments) is reported. * p<0.05 <i>Bottom panel</i>, right: UT7/mpl cells have been plated in methylcellulose either in the absence (control) or in the presence of either TPO, TPO+Ang-1, TPO+Ang-2 or TPO+Ang-1+Ang-2 and after 10 days of <i>in vitro</i> culture the number of colonies was evaluated under an inverted microscope. Mean values ± SEM observed in three independent experiments are shown. ** p<0.01.</p

Tie-2 expression during Mk differentiation of cord blood or peripheral blood cord blood or peripheral blood CD34⁺ cells.

<p>Kinetics of Tie-2 expression during TPO-driven megakaryocytic differentiation of cord blood (CB, <i>Left Panels</i>) or cord blood (PB, <i>Right Panels</i>) CD34⁺ cells. Purified CB or PB CD34⁺ cells have been grown in serum-free medium in the presence of TPO and, at various days of culture, cell aliquots have been harvested and and analyzed for cell growth (<i>top panels</i>), for Tie-2 expression at mRNA and protein level (mean percentage of positive cells ± SEM observed in three separate experiments, <i>middle panels</i>) and for the expression of membrane differentiation markers CD34, CD41, CD61 and CD42b (<i>bottom panels</i>). In the <i>inset</i> the Tie-2 WB analysis on Mks grown from PB is reported. Mean values ± SEM observed in three separate experiments.</p

Angiopoietin and TPO-induced cell signaling in HUVEC cells.

<p>HUVEC cells, grown under standard endothelial cell culture conditions, have been starved overnight of growth factors and stimulated as indicated in panel B of <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0039796#pone-0039796-g010" target="_blank">Fig.10</a>. <i>A –</i> Flow cytometry and Western Blot analysis of Tie-2 expression in HUVEC cells starved of growth factors and then stimulated for 15 min either in the absence (C ) or in the presence of either TPO or Ang-1 or Ang-2. <i>B and C</i> - WB and densitometric quantification (<i>top</i> and <i>bottom</i> panels, respectively).</p