Journal: Molecular Cancer

Article Title: EGFR/Src/Akt signaling modulates Sox2 expression and self-renewal of stem-like side-population cells in non-small cell lung cancer

doi: 10.1186/1476-4598-11-73

Figure Lengend Snippet: SP cells express stem cell-like markers. ( A ) Western blot analysis on SP and MP cells from A549, H1650 and H1975 cell lines for the indicated proteins. β-Actin was used as the loading control. ( B , C , D ) SP and MP cells were analyzed by real time qRT-PCR method for the expression of Twist, Slug and Snail genes. Average (±SD) fold change between MP and SP cells were plotted. ( E , F , G ) SP and MP cells were analyzed by real time qPCR method for the expression of ABCG2, Oct4, Sox2 and Nanog genes. Average (±SD) fold change between MP and SP cells were plotted. ( H ) The spheres formed in self-renewal assays of H1650 SP cells were stained for the expression of ABCG2, Oct4, Sox2 and Nanog. Confocal image of one of the z-stacks for a sphere is presented. DAPI was used to stain the nucleus.

Article Snippet: In the present study, Gefitinib or erlotinib is used at 500 nM, dasatinib or BIBW2992 is used at 200 nM and PP2 is used at 1 μM dose. siRNA against EGFR, Src family kinases, Akt and Sox2, Oct4 and Nanog was purchased from Santa Cruz Biotechnology or OriGene Technology Inc. Primary antibodies against Sox2 (#3579), Oct4 (#2750), Nanog (#4903), Phos-Src-pY 416 (#2101), pERK1/2 (#4376) and phospho-AKT-pS 473 (#4058) were purchased from Cell Signaling Technology; Phos-EGFR-pY 1068 (#44788G) from Invitrogen; EGFR neutralizing antibody (#05-101) from Milipore and isotype matched mouse IgG were purchased from Biolegend.

Techniques: Western Blot, Control, Quantitative RT-PCR, Expressing, Staining

Journal: Molecular Cancer

Article Title: EGFR/Src/Akt signaling modulates Sox2 expression and self-renewal of stem-like side-population cells in non-small cell lung cancer

doi: 10.1186/1476-4598-11-73

Figure Lengend Snippet: Establishment and characterization of H1650-SPAdh cells. ( A ) SP cells from H1650 cell line was plated on normal tissue culture plate (i) or poly-D-lysine-laminin (PDL-Laminin) coated surface (ii) in serum free medium containing N2-supplement, EGF and bFGF. H1650-SPAdh cells growing in self-renewing condition was cultured in serum for 5 days to induce differentiation, and reanalyzed for SP frequency (iii). ( B ) Serum induces differentiation of SPAdh cells as seen by ABCG2 expression and ( C ) real time qPCR analysis for stem cell markers, ABCG2, Oct4, Sox2, Nanog . ( D ) Cell cycle analysis for parental-H1650 or H1650-SPAdh and serum differentiated H1650-SPAdh cells grown on PDL-Laminin coated surface. Histograms were plotted using ModFit program. ( E ) The average number of spheres generated from 1000 H1650-SPAdh cells is plotted (mean ± SD). Phase contrast microscopy images of the spheres in presence or absence of indicated drugs.

Article Snippet: In the present study, Gefitinib or erlotinib is used at 500 nM, dasatinib or BIBW2992 is used at 200 nM and PP2 is used at 1 μM dose. siRNA against EGFR, Src family kinases, Akt and Sox2, Oct4 and Nanog was purchased from Santa Cruz Biotechnology or OriGene Technology Inc. Primary antibodies against Sox2 (#3579), Oct4 (#2750), Nanog (#4903), Phos-Src-pY 416 (#2101), pERK1/2 (#4376) and phospho-AKT-pS 473 (#4058) were purchased from Cell Signaling Technology; Phos-EGFR-pY 1068 (#44788G) from Invitrogen; EGFR neutralizing antibody (#05-101) from Milipore and isotype matched mouse IgG were purchased from Biolegend.

Techniques: Cell Culture, Expressing, Cell Cycle Assay, Generated, Microscopy

Journal: Molecular Cancer

Article Title: EGFR/Src/Akt signaling modulates Sox2 expression and self-renewal of stem-like side-population cells in non-small cell lung cancer

doi: 10.1186/1476-4598-11-73

Figure Lengend Snippet: Inhibition of EGFR signaling downregulates Sox2 expression (A) H1650-SPAdh cells were treated with EGFR or Src inhibitors for 4 days and western blot analysis was performed for the indicated proteins. ( B ) H1650-SPAdh cells were treated with MEK or PI3K inhibitor for 4 days and levels of Sox2 and activation of Akt or ERK was evaluated by western blot. ( C ) A549 and H1975 cells were treated with MEK or PI3K inhibitor for 5 days and the frequency of SP cells were examined by SP analysis. Data represents the fold change in mean (± SD) of SP frequency. ( D ) ABCG2 expression upon inhibitor treatment as detected by western blotting. ( E ) SP cell frequency in NSCLC cell lines transfected with specific siRNAs against Src or AKT. ( F ) ABCG2 expression in siRNA transfected cells as detected by western blotting. ( G and H ) SP cells were sorted and plated for self-renewal assay in the presence or absence of indicated drugs. Average number of spheres generated from 1000 cells is plotted (mean ± SD). Phase contrast microscopy images of the spheres in presence or absence of drugs are presented. * represent the p value of <0.05; ** represent the p value of <0.01; *** represent the p value of <0.001.

Article Snippet: In the present study, Gefitinib or erlotinib is used at 500 nM, dasatinib or BIBW2992 is used at 200 nM and PP2 is used at 1 μM dose. siRNA against EGFR, Src family kinases, Akt and Sox2, Oct4 and Nanog was purchased from Santa Cruz Biotechnology or OriGene Technology Inc. Primary antibodies against Sox2 (#3579), Oct4 (#2750), Nanog (#4903), Phos-Src-pY 416 (#2101), pERK1/2 (#4376) and phospho-AKT-pS 473 (#4058) were purchased from Cell Signaling Technology; Phos-EGFR-pY 1068 (#44788G) from Invitrogen; EGFR neutralizing antibody (#05-101) from Milipore and isotype matched mouse IgG were purchased from Biolegend.

Techniques: Inhibition, Expressing, Western Blot, Activation Assay, Transfection, Generated, Microscopy

Journal: Molecular Cancer

Article Title: EGFR/Src/Akt signaling modulates Sox2 expression and self-renewal of stem-like side-population cells in non-small cell lung cancer

doi: 10.1186/1476-4598-11-73

Figure Lengend Snippet: Sox2 is necessary for maintaining the self-renewal of SP cells. ( A ) H1650-SPAdh cells were transfected with non-targeting control siRNA, or siRNA against EGFR, Src, or Sox2 and western blot analysis was performed for Sox2, Oct4, EGFR and Src expression. ( B ) siRNA transfected cells were plated for self-renewal assay and analyzed after 5 days of plating. Average number of spheres generated per well from 1000 cells is plotted (mean ± SD). ( C ) NSCLC cell lines were transfected with control or Sox2 siRNAs and SP frequency was evaluated. Average fold change in SP-frequency (± SD) is plotted. ( D ) Decrease in ABCG2 expression in Sox2 siRNA transfected cells was detected by western blotting. The Sox2 expression was evaluated by qRT-PCR and results are presented as bar diagram.

Article Snippet: In the present study, Gefitinib or erlotinib is used at 500 nM, dasatinib or BIBW2992 is used at 200 nM and PP2 is used at 1 μM dose. siRNA against EGFR, Src family kinases, Akt and Sox2, Oct4 and Nanog was purchased from Santa Cruz Biotechnology or OriGene Technology Inc. Primary antibodies against Sox2 (#3579), Oct4 (#2750), Nanog (#4903), Phos-Src-pY 416 (#2101), pERK1/2 (#4376) and phospho-AKT-pS 473 (#4058) were purchased from Cell Signaling Technology; Phos-EGFR-pY 1068 (#44788G) from Invitrogen; EGFR neutralizing antibody (#05-101) from Milipore and isotype matched mouse IgG were purchased from Biolegend.

Techniques: Transfection, Control, Western Blot, Expressing, Generated, Quantitative RT-PCR

Journal: Molecular Cancer

Article Title: EGFR/Src/Akt signaling modulates Sox2 expression and self-renewal of stem-like side-population cells in non-small cell lung cancer

doi: 10.1186/1476-4598-11-73

Figure Lengend Snippet: Sox2 expression correlates with metastatic progression of adenocarcinoma. ( A ) Sox2 expression in Lung squamous cells carcinoma, adenocarcinoma and metastatic adenocarcinoma was analyzed from stage I/II or stage IV patients samples by immunohistochemistry. ( B ) Semi-quantitative scoring was performed for adenocarcinoma samples and the tumors with score of one or more was considered positive for Sox2 expression and listed. ( C ) Median score for Sox2 expression was calculated and plotted for different stages of NSCLC progression. The mean (± SD) of score is mentioned in parenthesis. Metastatic tumors showed significantly ( p = 0.01 ) higher expression of Sox2.

Article Snippet: In the present study, Gefitinib or erlotinib is used at 500 nM, dasatinib or BIBW2992 is used at 200 nM and PP2 is used at 1 μM dose. siRNA against EGFR, Src family kinases, Akt and Sox2, Oct4 and Nanog was purchased from Santa Cruz Biotechnology or OriGene Technology Inc. Primary antibodies against Sox2 (#3579), Oct4 (#2750), Nanog (#4903), Phos-Src-pY 416 (#2101), pERK1/2 (#4376) and phospho-AKT-pS 473 (#4058) were purchased from Cell Signaling Technology; Phos-EGFR-pY 1068 (#44788G) from Invitrogen; EGFR neutralizing antibody (#05-101) from Milipore and isotype matched mouse IgG were purchased from Biolegend.

Techniques: Expressing, Immunohistochemistry

Journal: Oncology Letters

Article Title: Macrophages activate mesenchymal stem cells to acquire cancer-associated fibroblast-like features resulting in gastric epithelial cell lesions and malignant transformation in vitro

doi: 10.3892/ol.2018.9703

Figure Lengend Snippet: Gastric epithelial cells incubated with CM from macrophage-hucMSCs exhibit several properties of stem cells. (A) GES-1 cells were incubated with CM from hucMSCs or macrophage-hucMSCs for 48 h, and then seeded in non-adherent culture conditions for spheroid formation. Depicted are representative images of spheroid colonies after 15 days. Magnification, ×400; scale bar, 50 µm. (B) Quantification of the soft agar colonies shown in (A). (C) Western blot analysis of Nanog, SOX2, and BMI-1 in GES-1 cells (the controls) and GES-1 cells co-cultured with CM from hucMSCs and macrophage-hucMSCs after 48 h. The expression of all analyzed proteins is significantly enhanced in the GES-1 cells cultured with CM from macrophage-hucMSCs, compared with the control. (D) Representative image of single-colony formations of GES-1 cells incubated with CM from hucMSCs or CM from macrophage-hucMSCs. (E) The number of colonies is depicted as mean ± standard deviation. *P<0.05, # P<0.01; GES-1 cells treated with medium only served as the control (magnification, ×100). Macrophage-hucMSC, human umbilical cord-derived mesenchymal stem cells pre-cultured with macrophages for 48 h; CM, conditioned medium; SOX2, SRY-box 2; BMI-1, polycomb complex protein BMI-1.

Article Snippet: The membranes were incubated with 5% skimmed milk to block non-specific protein at room temperature for 1 h. Membranes were incubated overnight at 4°C with primary antibodies at a dilution of 1:800 for rabbit polyclonal anti-N-cadherin (cat. no. BS2224; Bioworld Technology, Inc., St. Louis Park, MN, USA), 1:1,000 for rabbit polyclonal anti-E-cadherin (cat. no. BS1098; Bioworld Technology, Inc.), 1:500 for rabbit polyclonal anti-vimentin (cat. no. BS1855; Bioworld Technology, Inc.), 1:500 for rabbit polyclonal anti-α-SMA (cat. no. BS8796; Bioworld Technology, Inc.), 1:800 for rabbit polyclonal anti-B-cell lymphoma-2 (Bcl-2) (cat. no. BS70205; Bioworld Technology, Inc.), 1:500 for rabbit polyclonal anti-Bcl-2-associated X (Bax) (cat. no. BS1030; Bioworld Technology, Inc.), 1:500 for rabbit polyclonal anti-SOX2 (cat. no. BS6161; Bioworld Technology, Inc.), 1:1,000 for rabbit polyclonal anti-Bmi-1 (cat. no. BS6015; Bioworld Technology, Inc.), 1:10,000 for rabbit polyclonal anti-β-actin (cat. no. AP0060; Bioworld Technology, Inc.), 1:1,000 for mouse monoclonal anti-FAP (cat. no. sc-71094; Santa Cruz Biotechnology, Inc.,) and 1:1,000 for rabbit polyclonal anti-Nanog (cat. no. sc-33759; Santa Cruz Biotechnology, Inc.).

Techniques: Incubation, Western Blot, Cell Culture, Expressing, Standard Deviation, Derivative Assay