Journal: Neuro-Oncology

Article Title: Dissecting inherent intratumor heterogeneity in patient-derived glioblastoma culture models

doi: 10.1093/neuonc/now253

Figure Lengend Snippet: Floating cells display enhanced mesenchymal properties. (A) MGG6 NS and FC mRNA were analyzed by qRT-PCR for different mesenchymal markers including CD44, vimentin, YKL-40, N-cad, and MMP2. (B) Cell lysates from different cultures were analyzed by western blotting for pSTAT3, STAT3, CD44, C/EBPβ, and β-actin. (C) FACS analysis showing proportion of CD44 positive cells in FC and their corresponding NS from different GSC cultures. (D) MGG29 NS were fractionated into CD44high and CD44low subpopulation by FACS and predominant cells were established and labeled with GFP and mCherry, respectively. Left: fluorescence analysis of a mixture of these 2 subpopulations cultured in serum; middle: FC collected and analyzed for both GFP and mCherry; right: flow cytometry of CD44high cells in both GFP and mCherry populations. (E) NS and FC were differentiated using StemPro Chondrogenesis Differentiation Kit and stained with fast green and safranin O (positive, #; negative, +). (F) NS and FC were cultured as a monolayer and their ability to invade/migrate was evaluated using scratch healing assay. Bright field micrographs are showing for different groups. Scale bar, 50 μm.

Article Snippet: CD44 expression was measured in 500000 cells dissociated with 2 mM EDTA and incubated in 80 μL PBS/0.1% bovine serum albumin at 4°C for 30 min in the dark with mouse monoclonal anti-human CD44 antibody conjugated to fluorescein isothiocyanate (FITC, 1:10; Miltenyi Biotec) or respective mouse immunoglobulin G1 control in the presence of FcR blocking reagent (1:5).

Techniques: Quantitative RT-PCR, Western Blot, Labeling, Fluorescence, Cell Culture, Flow Cytometry, Staining

Journal: Genes & Diseases

Article Title: ROS and Lipid Droplet accumulation induced by high glucose exposure in healthy colon and Colorectal Cancer Stem Cells

doi: 10.1016/j.gendis.2019.09.010

Figure Lengend Snippet: 72 hrs in HG cell culture medium is sufficient to increase CSC markers in CR-CSCs. a) Primary CR-CSC spheroids (#1, #4, #8 and #9) were cultured in ultra-low adhesion flasks and morphology was assessed by phase-contrast microscopy (scale bar: 1000 μm). b) Fold variation of protein expression in CR-CSCs treated with HG versus NG. Data are representative of 3 independent experiments performed with cells derived from 3 different CR-CSC lines. c) Representative Western blot of PI3K, phosphorylated (p-AKT) and total AKT in CR-CSC treated with HG or NG (#9 CR-CSC line is shown). β-Actin has been used as loading control. The uncropped blots are reported in Supplementary Fig. 1 . d) Fold variation of positivity for CD133 or CD44v6 in CR-CSCs treated as in ( b and c ), measured by FACS.

Article Snippet: Cells were dissociated, harvested, washed twice in Phosphate Buffer Solution (PBS) 1X (Thermo Fisher Scientific; #10010023) and stained with conjugated antibodies CD44v6-APC (2F10, mouse IgG1) (R&D systems; #FAB3660A), CD133/2-APC (293C3-APC) (Miltenyi Biotec; #130-090-854), or corresponding isotype-matched control (IMC) CD3e-APC (UCHT1, mouse IgG1) (R&D systems; #FAB100A).

Techniques: Cell Culture, Microscopy, Expressing, Derivative Assay, Western Blot, Control