Flow cytometry evaluation of MDRPs.

<p>LRP and MRP1 were not detected by means of flow cytometry in any cell line. ABCG2 was expressed on only 9% of the Hcc-1 cells, and was almost absent on the surface of the other cell lines. ABCB1 (PGP) was highly expressed on the plasma membrane of HepG2 (58%), Hcc-1 (18%) and HuH7 cells (13%), but expressed on only about 3% of PLC/PRF/5 cells. Left column negative controls; middle column ABCG2 expression; right column PGP expression.</p

Immunophenotyping.

<p>The table shows the mean percentage of cells positive for each marker.</p><p>Immunophenotyping.</p

Hypothesised mechanism of the enhanced efficacy of drug pre-treatment before verapamil administration and PGP blockade.

<p>A) HCC cells expressing active PGP can expel a drug (e.g. sunitinib) from the cytoplasm or store it in lysosomes. B) Blocking PGP with verapamil before the co-administration sunitinib and verapamil allows the drugs to enter the cell and diffuse into cytoplasm/nucleus. C) If sunitinib is used for pre-treatment, it is stored in giant lysosomes and, after the co-administration of sunitinib and verapamil and subsequent PGP blockade, the drugs can enter the cytoplasm/nucleus from both extra-cellular space and the lysosomes.</p

Cytoplasmic vesicle localisation.

<p>Fluorescence microscopy images confirming intra-cellular vesicle localisation. (A) Hcc-1, (B) HepG2, (C) Hep3B, and (D) SNU475 cells. Sunitinib autofluorescence (green), CD29 staining (red) and Hoechst 33342 nuclear staining (blue). Original magnification 40x.</p

Immunofluorescence staining of MDRPs.

<p>Representative pictures of MDRP staining in HCC cell lines. The expression and localisation of MDRPs was different in the various HCC cell lines: ABCG2, LRP and MRP1 were localised on the membrane of the few groups of cells on which they were expressed, whereas PGP was mainly found in the cell cytoplasm, particularly on the membrane of intra-cellular vesicles. Nuclei stained with DAPI (blue). Original magnification 20x.</p

Time-lapse experiments.

<p>Over a period of seven hours it is possible to see that some lysosomes released their content outside the cells (top row, white circles), whereas others started to accumulate new sunitinib in their lumens (bottom row, white arrowheads).</p

Sunitinib accumulation in cytoplasmic vesicles of HCC cell lines.

<p>Representative pictures of cell lines cultured without (left) or with (right) sunitinib 12 µM. Cytoplasmic vesicles are visible in the Hep3B and SNU475 cells only after sunitinib incubation. Original magnification 20x.</p

Chemoresistance assays.

<p>A) When treated with sorafenib, the cell lines with giant PGP-positive lysosomes (Hcc-1, HepG2, PLC/PRF/5 and HuH7) showed higher IC₅₀ values than those with normal lysosomes (Hep3B and SNU475) (p<0.01). B) The HCC cell lines were incubated with different concentrations of sorafenib in order to verify their chemosensitivity (green lines). The cells with larger cytoplasmic vesicles were characterised by a curve with a sort of plateau of viability at sorafenib concentrations of between 5 and 20 µmol. One hour of verapamil pre-treatment used to inhibit ABC proteins before co-incubation with sorafenib and sunitinib increased the chemosensitivity of all of the cell lines (black curves). *p<0.01 green vs. black curves. One hour of sorafenib pre-treatment (red lines) before co-incubation with sorafenib and sunitinib enhanced treatment efficacy in comparison with verapamil pre-treatment in the cell lines carrying giant lysosomes. ^§p<0.05 red vs. black curves.</p

CD133+ cells were isolated from the peripheral blood of 70 DMD patients and 20 age-matched control subjects and analyzed by flow-cytometry.

<p>Representative panels show the CD133+CXCR4+CD34+ subpopulation in healthy subjects (mean percentage±SD, 1.58±2.39 of total CD133+ cells) (upper right panel in A) and in DMD patients (3.87±0.63)(lower right panel in B). A subpopulation of CD133+CXCR4+CD34-cells was significantly increased in DMD patients (lower right panel in B) compared with healthy controls (lower right panel in A) (mean percentage±SD, 17.38±1.38 vs. 11.0±1.70 of total CD133+ cells). (C) Histogram showing the percentages of CD133+CXCR4+CD34+ cells of healthy controls compared to DMD patients. (D) Histogram demonstrating the percentages of CD133+CXCR4+CD34- cells in healthy controls compared with to DMD patients. *Significance (<i>P</i> < 0.05).</p

Representation of 2 different disease courses in 19 DMD patients stratified into 2 groups, in which a 24-month follow-up was available according to the results obtained from linear regression analysis between age and levels of the CD133+CXCR4+CD34- cells.

<p>MRC% scores are shown over time in 8 patients with a percentage of the cells localized above the threshold level for the corresponding age (A and C) and in 11 patients with values below the regression line during a total follow-up period of 24 months (B and D). At the end of the observation period, in the first group of DMD patients, the mean MRC% decreased by approximately 14%, and the total clinical score approximately 2.25 points. In the second group, the MRC% decreased by approximately 25%. In a subgroup including 4 DMD patients of the first group and in 5 DMD patients of the second group, we measured CD133+CXCR4+CD34- cells levels. Differences between the baseline values of CD133+CXCR4+CD34- cells and those at 2, 4, 6, 12 and 24 months are shown for individual patients in E and F. Despite the significant increase of CD133+CXCR4+CD34- cells compared to the patients of the second group (mean value±SD: 31.36%±14.67% vs. 11.12%±4.9%; P<0.0001), the 4 patients in group 1 displayed major intra-individual variability between successive measurements.</p