Side population analysis.

<p>(A) Cytometric analyses of the side population. The CD133⁺ fraction includes a small subset (0.97%), expressing the characteristic profile of a side population at FACS. (B) ABCG2 expression in SAOS2 cell line, showing an evident positivity; the grey line indicates the isotype control.</p

Spheres assay.

<p>(A) Sphere clusters formed by CD133⁺ cells in semisolid medium after 24 hours (Original Magnification ×100); (B) CD133⁻ cells in semisolid medium after 7 days, do not form spheres. (Original Magnification ×100); (C) Sphere clusters formed by CD133⁺ cells after 48 hours (Original Magnification ×200); (D) Sphere clusters formed by CD133⁺ cells after a new sorting (Original Magnification ×400).</p

CD133 expression in adherent cells and floating spheres.

<p>(A) Immunohistochemical analyses on adherent cells and (B) floating spheres showing the presence of CD133 antigen (<u>arrows</u>). (Original Magnification. ×100); (C) Immunofluorescence analysis on SAOS-2 for CD133 PE, cytoskeleton is stained with phalloidin-FITC, nucleus with DAPI (Original Magnification. ×400); (D) Immunoflurescence analysis on SAOS-2 spheres for CD133 PE after 24 hours in adhesion. (Original Magnification ×200); (E) Confocal analyses on adherent cells and (F) floating spheres confirming the presence of the CD133 antigen. (Original Magnification. ×400).</p

Colony-forming efficiency of CD133⁺ cells versus CD133− population^a in SAOS2, MG63 and U2OS cell lines.

a<p>Results are the mean±standard devition of three experiments from different cases.</p>b<p>Represents the number of colonies with respect to the number of wells plated in experiments.</p>c<p>Ratio between the percentage of colonies formed by CD133+ versus CD133− cells.</p

Cytometric analyses for CD133 on SAOS2, MG63 and U2OS cell lines.

<p>A CD133⁺ cell population can be detected in all the three cell lines.</p

Cytometric analyses for CD133, CD44, CD90, CD34 and CD29 on SAOS2, MG63 and U2OS cell lines.

<p>All three cell lines are negative for CD34 antigen but they evidence equal strong positivity for CD29, CD44 and CD90.</p

CD133 intracellular expression in adherent cells.

<p>(A) Figure performed at FACS showing intracellular expression of CD133 antigen in SAOS-2, MG-63 and U2OS. (B) Figure showing at the Real time PCR the mRNA transcript expression in CD133⁺ and CD133⁻ cells. The levels are almost identical as detected by in both CD133 positive and negative cells.</p

Cytometric analyses for CD133 and OCT3/4 on sarcospheres in SAOS2, MG63 and U2OS.

<p>The blue line indicates isotype controls, red and green lines indicate the expression of CD133 at the 4^th and 6^th cell passage, respectively. In the histograms, OCT3/4 expression is analyzed at the 6^th cell passage; the green line indicates isotype controls. Sarcospheres both in CD133 and OCT3/4 are strongly positive.</p

Cell cycle, proliferation and growth analyses.

<p>(A) Figure of cell cycle analyses performed on SAOS2, MG63 and U2OS cell lines. A CD133⁺ cell population is mainly observable in the G2\M phase, whereas CD133⁻ cells were predominantly in G0\G1; (B) Figure showing Ki-67 reactivity. CD133⁺ cells resulted to be Ki-67⁺, whereas CD133⁻ were mainly negative for this marker; (C) Figure showing growth curves of CD133⁺ cells with respect CD133⁻ cells in SAOS2, MG63 and U2OS cell lines. CD133⁺ cells possess a high proliferative potential in all the three cell lines.</p

Cell cycle analysis performed using Hoechst 33342 and Ki67 both on CD34 negative and positive cells.

<p>(A) Hoechst 33342 analysis performed CD34⁻ cells: G₀G₁ phase (98%), S phase (0,65%) and G₂M phase (0,50%); (B) Ki67 analysis performed CD34- cells: Ki67 (10%); (C) Hoechst 33342 analysis performed CD34⁺ cells: G₀G₁ phase (84%), S phase (5%) and G₂M phase (10%); (D) Ki67 analysis performed CD34+ cells: Ki67 (85%).</p