Overexpression of ST6GAL1 influences the invasive ability of Hca-P cells both in vitro and in vivo.

<p>(A) Hca-P cells were transfected with a pcDNA3.1 expression vector, and western blot analysis for ST6GAL1 was assessed. GAPDH was also examined and served as controls for sample loading. Relative signal intensities of ST6GAL1 protein levels were normalized against those of GAPDH by LabWorks (TM ver4.6, UVP, BioImaging systems) analysis, respectively (*P<0.05). (B) In vitro ECMatrix gel analysis is performed. The average number of cells that invaded through the filter was counted. Hca-P/ST6GAL1 cells were significantly more invasive (*P<0.05) than the Hca-P and Hca-P/mock cells. (C, fluorescence; ×100) The results of ST6GAL1-transfected CFSE⁺Hca-P cells invasion to lymph nodes were analyzed. The number of Hca-P/ST6GAL1 cells was increased, compared with the Hca-P/mock, Hca-P cells after 24 h. (D) The number of ST6GAL1-transfected CFSE⁺Hca-P cells invasion to lymph nodes was measured by flow cytometry. Surface labeling was expressed as the percentage of positive cells relative to the total number of analyzed cells (*P<0.05). (E) FITC-SNA binding profiles of Hca-P cells using flow cytometry. Histograms of fluorescence intensities of cells with specific carbohydrate expression as determined. Data are the average ± SD of triplicate determinants.</p

Differential FITC–lectin binding profiles of Hca-F and Hca-P cell lines using flow cytometry.

<p>(A) Histograms of fluorescence intensities of cells with specific carbohydrate expression as determined by flow cytometry using 7 different lectins. (B) The data are means ± SD of 3 independent assays of Hca-F and Hca-P cell lines, *P<0.05. (C) List of glycogenes responsible for lectin signals in Hca-F and Hca-P cell lines.</p

Differential expression of glycogenes in Hca-F and Hca-P cell lines.

<p>(A) The mRNA levels of glycogenes analyzed by real-time RT-PCR. The relative amount of glycogenes mRNA levels was normalized to GAPDH levels. Relative intensities ratio (>3-fold) of the glycogenes signals were observed. (B) Western blot analysis for enzyme was assessed. Data are the means ± SD of triplicate determinants.</p

Silence of ST6GAL1 effects on the invasive ability of Hca-F cells both in vitro and in vivo.

<p>(A) Silencing of ST6GAL1 in Hca-F cells was analyzed by RNAi approach. After Hca-F cells were transfected with ST6GAL1 siRNA for 30 h, western blot analysis for ST6GAL1 was assessed. GAPDH was also examined and served as controls for sample loading. Relative signal intensities of ST6GAL1 protein levels were normalized against those of GAPDH by LabWorks (TM ver4.6, UVP, BioImaging systems) analysis, respectively (*P<0.05). (B) In vitro ECMatrix gel analysis is performed. The average number of cells that invaded through the filter was counted. Hca-F-ST6GAL1 siRNA cells were significantly less invasive (*P<0.05) than the Hca-F and Hca-F-control siRNA cells. (C, fluorescence; ×100) The results of ST6GAL1 siRNA-transfected CFSE⁺Hca-F cells invasion to lymph nodes were analyzed. The number of Hca-F-ST6GAL1 siRNA cells was decreased, compared with the Hca-F-control siRNA, Hca-F cells after 24 h. (D) The number of ST6GAL1 siRNA-transfected CFSE⁺Hca-F cells invasion to lymph nodes was measured by flow cytometry. Surface labeling was expressed as the percentage of positive cells relative to the total number of analyzed cells (*P<0.05). (E) FITC-SNA binding profiles of Hca-F cells using flow cytometry. Histograms of fluorescence intensities of cells with specific carbohydrate expression as determined. Data are the average ± SD of triplicate determinants.</p

Summary of N-glycan in N-glycan in Hca-F and Hca-P cell lines identified by MALDI-TOF MS.

<p>The N-glycan were observed as [M+Na]+.</p><p>Hex, hexose; HexNAc, N-acetylhexosamine; Man, mannose; GlcNAc, N-actylglucosamine; NeuAc, N-acetylneuraminic acid.</p

N-glycans composition profiling of Hca-F and Hca-P cell lines using Mass spectrometry analysis.

<p>(A) MALDI-TOF MS spectra of N-glycans released from membrane protein of Hca-F and Hca-P cell lines. (B) Histograms of relative intensities of the N-glycan signals observed. Values are the mean ± S.D of three permethylated samples from N-glycan samples. The signal numbers correspond to those described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0065218#pone-0065218-t001" target="_blank">Table 1</a>.</p

Clinicopathologic characteristics of the leukemia patients.

<p>Clinicopathologic characteristics of the leukemia patients.</p

PI3K/Akt inhibition changes the chemosensitivity of K562/ADR cells both in vitro and in vivo.

<p>(A) The K562/ADR cells were pretreated LY294002 or Akt siRNA. Expressions of PI3K/Akt signaling molecules were then examined by western blot analysis. LY294002 or Akt siRNA treatment also alleviated chemoresistance of K562/ADR cells, revealed by <i>in vitro</i> (B) and <i>in vivo</i> (C). (D) Down-regulation of PI3K/Akt signaling molecules was also shown by IHC staining in xenograft tumors derived from LY294002 or Akt siRNA treatment cells (400×). (E) Flow cytometry analysis showed that inhibition of PI3K/Akt pathway resulted in reduced levels of P-gp and MRP1. The data are means ± SD of 3 independent assays. ^*P<0.05 vs DMSO treatment cells; ^#P<0.05 vs control siRNA treatment cells.</p

The differential expression of ST6GAL1 and ST6GAL2 between MDR and chemosensitive patients.

<p>P<0.05 vs. patients without MDR.</p

Overexpression of ST6GAL1 gene enhances the chemoresistance of K562 cells both in vitro and in vivo.

<p>After transfection, ST6GAL1 mRNA (A) and protein (B) were increased notably in K562 cells by real time PCR and western blot. (C) Cell chemosensitivity was assessed by cytotoxicity assays. The reported values are the IC₅₀ (Mean ± SD) of three independent experiments. IC₅₀ represents the drug concentration producing 50% decrease of cell growth. *P<0.05 vs K562/mock cells. (D) An increase of mean tumor in mice group with K562/ST6GAL1 was observed, as compared with that in K562 group and K562/mock group. Within K562/ST6GAL1 group, an increase of tumor growth was found in group without ADR, compared with that with ADR (*P<0.05). (E) Up-regulation of ST6GAL1 was also shown by IHC staining in xenograft tumors derived from K562/ST6GAL1 cells (400×). (F) Increased expression of ST6GAL1 was detected by flow cytometry analysis in K562/ST6GAL1 cells. The data are means ± SD of 3 independent assays (*P<0.05).</p