Journal: Cancer Research

Article Title: A Novel Small Molecule Inhibitor of Signal Transducers and Activators of Transcription 3 Reverses Immune Tolerance in Malignant Glioma Patients

doi: 10.1158/0008-5472.can-07-1243

Figure Lengend Snippet: Figure 4. WP1066 enhances phosphorylation of intracellular signaling molecules. Monocytes and Tcells isolated from the PBMCs of GBM patients were incubated with either the medium or medium supplemented with 5 Amol/L WP1066. After 2 h, monocytes and T cells were stimulated for 5 min with 2 Ag/mL LPS and 5 Ag/mL anti-CD3 antibody, respectively. Subsequently, cells were lysed, electrophoretically fractionated in 8% SDS-polyacrylamide gels, transferred to nitrocellulose membranes, and immunoblotted with anti-phosphotyrosine monoclonal antibody 4G10. A, for monocyte membranes, subsequently the same membrane was stripped and reblotted with antibodies to phosphorylated p72Syk (Tyr352), Syk, phosphorylated Hck, Hck, and Lyn. B, for the T-cell membranes, after stripping, the membrane was reblotted with antibodies to phosphorylated ZAP-70 (Tyr319), ZAP-70, p56-Lck, and h-actin.

Article Snippet: T cells from the same patients were isolated with human CD3 microbeads and a magnetic separation column (LS MACS column, Miltenyi Biotec) with the exception of one patient who lacked sufficient PBMCs for both monocyte and T-cell isolations.

Techniques: Phospho-proteomics, Isolation, Incubation, Membrane, Stripping Membranes

Journal: Leukemia

Article Title: Spred1 deficit promotes treatment resistance and transformation of chronic phase CML

doi: 10.1038/s41375-021-01423-x

Figure Lengend Snippet: A Expression of SPRED1 in BM CD34+ cells from patients with BC CML and CP CML by Q-RT-PCR (n=8 samples for BC CML and n=12 samples for CP CML) and western blot and in BM by immunohistochemistry staining (one of three independent experiments with similar results was shown) (left), and expression of miR-126 in CD34+ and CD34+CD38− cells from BC CML (n=6 samples) and CP CML (n=10 samples) patients by Q-RT-PCR (right). B SPRED1 mRNA expression by Q-RT-PCR and protein expression by western blot, miR-126 levels by Q-RT-PCR, cell cycling by Ki-67 and DAPi staining (top) or by cell trace violet staining (bottom) followed by flow cytometry analysis in CML CD34+ cells transduced with SPRED1 siRNA to knock-down (KD) SPRED1 or with a non-targeting control siRNA (Ctrl). UND: undivided cells, G0; DIV: division. C Representative colonies and quantification of colony forming cells (CFC) in CML CD34+ (left) and CD34+CD38− (right) cells transduced with Spred1 siRNA to KD SPRED1 or with ctrl siRNA (n=3). Results shown represent mean ± SEM. Significance values: *, p<0.05; **, p<0.01; ***, p<0.001.

Article Snippet: Human CD34 + cells were selected using the indirect CD34 microbead kit (Miltenyi Biotec, San Diego, CA) and CD34 + CD38 − cells were sorted after staining with human antibodies against CD34 and CD38 ( Supplementary Table 1 ) or selected using CD34+CD38- cell isolation kit (Miltenyi Biotec, San Diego, CA) according to the manufacturer’s protocol.

Techniques: Expressing, Reverse Transcription Polymerase Chain Reaction, Western Blot, Immunohistochemistry, Staining, Flow Cytometry, Transduction, Knockdown, Control

Journal: International journal of oncology

Article Title: CXCR4 is involved in CD133-induced EMT in non-small cell lung cancer.

doi: 10.3892/ijo.2016.3812

Figure Lengend Snippet: Figure 1. The expression of CD133 in lung cancer and normal lung tissues. Original values are presented as log2 ratios. Spots showing the level of CD133 mRNA in NSCLC and normal tissues. (A) GSE10072. (B) GSE40275. (C) GSE63459; *p<0.05, **p<0.01 and ***p<0.001. (D) Correlation between CD133 and CXCR4 in GSE30219.

Article Snippet: Cells were incubated at 4 ̊C with 100 μl FcR blocking reagent and 100 μl CD133 Micro Beads (130-097-049, Miltenyi Biotec, Germany) for 30 min.

Techniques: Expressing

Journal: International journal of oncology

Article Title: CXCR4 is involved in CD133-induced EMT in non-small cell lung cancer.

doi: 10.3892/ijo.2016.3812

Figure Lengend Snippet: Figure 2. CD133 and CXCR4 are highly expressed in NSCLC patients with metastasis. (A) Representative images of immunohistochemistry staining (mag- nification, x400) of NSCLC patients. (a) CD133 low expression; (b) CD133 high expression; (c) CXCR4 low expression; (d) CXCR4 high expression. Positive staining ratio of CD133 (B)/CXCR4 (C) in metastatic and non-metastatic NSCLC patients. Positive staining score of CD133 (D)/CXCR4 (E) in metastatic and non-metastatic NSCLC patients. (F) Correlation results of CD133 and CXCR4. (G) Correlation analysis between CD133/CXCR4 co-expression and disease- free survival (red and black curves indicate high and low CD133/CXCR4 co-expression groups of patient death, respectively; *p<0.05).

Article Snippet: Cells were incubated at 4 ̊C with 100 μl FcR blocking reagent and 100 μl CD133 Micro Beads (130-097-049, Miltenyi Biotec, Germany) for 30 min.

Techniques: Immunohistochemistry, Staining, Expressing

Journal: International journal of oncology

Article Title: CXCR4 is involved in CD133-induced EMT in non-small cell lung cancer.

doi: 10.3892/ijo.2016.3812

Figure Lengend Snippet: Figure 3. CD133 enhances A549 cells proliferation. (A) Effectiveness of magnetic cell sorting was verified by immunofluorescence assay (magnification, x400). Red and blue indicate CD133 expression and cell nucleus, respectively. (B) Formation of colonies by A549 cell lines after 2-week incubation. (C and D) Cell proliferation capacity was determined in A549 cells, CD133+ A549 cells and CD133-A549 cells by CCK8 assays.

Article Snippet: Cells were incubated at 4 ̊C with 100 μl FcR blocking reagent and 100 μl CD133 Micro Beads (130-097-049, Miltenyi Biotec, Germany) for 30 min.

Techniques: FACS, Immunofluorescence, Expressing, Incubation

Journal: International journal of oncology

Article Title: CXCR4 is involved in CD133-induced EMT in non-small cell lung cancer.

doi: 10.3892/ijo.2016.3812

Figure Lengend Snippet: Figure 4. CXCR4 is upregulated by CD133. Silencing effectiveness of CD133 siRNA was verified by qPCR (A) and western blot analysis (B). (C) Expression of CXCR4 after treatment with CD133 siRNA (50 nM, 48 h) by qPCR analysis. (D) Western blot analysis for the expression of CXCR4 after treatment with CD133 siRNA (50 nM, 72 h). β-actin as a loading control. (E) The statistics of (D). All experiments were performed in triplicate. ***p<0.001.

Article Snippet: Cells were incubated at 4 ̊C with 100 μl FcR blocking reagent and 100 μl CD133 Micro Beads (130-097-049, Miltenyi Biotec, Germany) for 30 min.

Techniques: Western Blot, Expressing, Control

Journal: International journal of oncology

Article Title: CXCR4 is involved in CD133-induced EMT in non-small cell lung cancer.

doi: 10.3892/ijo.2016.3812

Figure Lengend Snippet: Figure 5. CD133+CXCR4+ promotes EMT process in NSCLC cells. (A) Transwell assay for the invasion of CD133-CXCR4+, CD133+CXCR4- and CD133+CXCR4+ cells. (B) Western blot assay for the expression of E-cadherin and Vimentin in CD133-CXCR4+, CD133+CXCR4- and CD133+CXCR4+ cells. (C) qPCR for the expression of E-cadherin, Vimentin, Snail, Slug and Twist in A549/CD133+, A549/CD133+ shRNA-NC, A549/CD133+ shRNA-CXCR4 and A549/CD133+ with amd3100. (D and E) RT-PCR for the expression of E-cadherin, Vimentin, Snail, Slug and Twist in A549/CD133+, A549/CD133+ shRNA-NC and A549/CD133+

Article Snippet: Cells were incubated at 4 ̊C with 100 μl FcR blocking reagent and 100 μl CD133 Micro Beads (130-097-049, Miltenyi Biotec, Germany) for 30 min.

Techniques: Transwell Assay, Western Blot, Expressing, shRNA, Reverse Transcription Polymerase Chain Reaction

Journal: International journal of oncology

Article Title: CXCR4 is involved in CD133-induced EMT in non-small cell lung cancer.

doi: 10.3892/ijo.2016.3812

Figure Lengend Snippet: Figure 6. Vimentin is positively associated with CD133/CXCR4 co-expression. (A) Representative images of immunohistochemistry staining (magnification, x400) of NSCLC patients. (a) E-cadherin low expression; (b) E-cadherin high expression; (c) Vimentin low expression; (d) Vimentin high expression. Positive staining ratio of E-cadherin (B)/Vimentin (C) in metastatic and non-metastatic NSCLC patients. Correlation analysis for E-cadherin (D) or Vimentin (E) with CD133/CXCR4 co-expression in NSCLC patients.

Article Snippet: Cells were incubated at 4 ̊C with 100 μl FcR blocking reagent and 100 μl CD133 Micro Beads (130-097-049, Miltenyi Biotec, Germany) for 30 min.

Techniques: Expressing, Immunohistochemistry, Staining