Journal: Scientific Reports

Article Title: Prostate cancer-derived holoclones: a novel and effective model for evaluating cancer stemness

doi: 10.1038/s41598-020-68187-9

Figure Lengend Snippet: ( a ) Representative bright field images of parental cells and holoclones derived from DU145, PC-3, LNCaP and 22Rv1 prostate cancer cell lines at approximately 5–7 weeks following initial plating in high-salt agar. ( b – e ) Stemness gene expression profile in holoclones and parental cells derived from high-salt agar. ( b – d ) Relative quantification of change in expression of stem-associated genes (ALDH1, CD44, CD133, POU5F1, NANOG, CD24, c-Met, integrin α2, integrin β1 and integrin α6) in PC-3, 22Rv1 and LNCaP-derived holoclones, which were normalised to parental controls. ( e ) The associated heat map summarising changes in gene expression (holcolones versus parent) (Grey: undetected, green: downregulated, red: upregulated). Data represented as Mean ± SEM (*p < 0.05, unpaired Student’s two-tailed t test, n = 3).

Article Snippet: Appropriate antibodies, APC mouse anti-human CD133 (Miltenyi Biotec, Germany); PE-Cy7 mouse anti-human CD44, PE mouse anti-human CD29, FITC-mouse anti-human CD49b (BD Biosciences, San Jose, CA, USA) were added to each cell sample and incubated in the dark for 20 min. A further 100 μL PBA was added; cells were pelleted to remove unbound antibody and re-suspended in 200 μL PBA buffer.

Techniques: Derivative Assay, Gene Expression, Quantitative Proteomics, Expressing, Two Tailed Test

Journal: Scientific Reports

Article Title: Prostate cancer-derived holoclones: a novel and effective model for evaluating cancer stemness

doi: 10.1038/s41598-020-68187-9

Figure Lengend Snippet: qRT-PCR primer information.

Article Snippet: Appropriate antibodies, APC mouse anti-human CD133 (Miltenyi Biotec, Germany); PE-Cy7 mouse anti-human CD44, PE mouse anti-human CD29, FITC-mouse anti-human CD49b (BD Biosciences, San Jose, CA, USA) were added to each cell sample and incubated in the dark for 20 min. A further 100 μL PBA was added; cells were pelleted to remove unbound antibody and re-suspended in 200 μL PBA buffer.

Techniques: TaqMan Assay

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

Journal: Cancer research

Article Title: Microenvironment-driven dynamic chromatin changes in glioblastoma recapitulate early neural development at single-cell resolution

doi: 10.1158/0008-5472.CAN-22-2872

Figure Lengend Snippet: RESOURCES TABLE

Article Snippet: For inducible DLX5 expression, the same protocol was followed in GLICO. . Flow Cytometry analysis GSCs were stained with CD44 Antibody coupled to APC (#130–113-338, Miltenyi Biotec) followed manufacturer’s Cell surface flow cytometry staining protocol.

Techniques: Recombinant, Gene Expression, Software

Journal: Nature Communications

Article Title: KK-LC-1 as a therapeutic target to eliminate ALDH + stem cells in triple negative breast cancer

doi: 10.1038/s41467-023-38097-1

Figure Lengend Snippet: a Volcano plot showing up-regulated genes in docetaxel-resistant MDA-MB-231 cells (DTX_R) compared to their parental cells (Parental) (Log2 Fold change > 6, adj P value < 0.05, P values were determined using moderated t -statistic, adj P -value was calculated using Benjamini and Hochberg’s method and tails were two-sided). b Up-regulated genes in docetaxel-resistant MDA-MB-231 cells were intersected with the up-regulated genes in TNBC , . Expression levels of two genes were significantly higher in docetaxel-resistant MDA-MB-231 cells. c Heatmap showing expression of the two highly expressed genes. d Schematic showing the identification and isolation of primary ALDH + and ALDH - cells. e Among the two highly expressed genes, KK-LC-1 had the highest expression in ALDH + cells compared to ALDH − cells and was selected as a potential candidate that may regulate ALDH + cells in TNBC; data are presented as mean ± SD from three biologically independent experiments, statistical significance was determined using Student’s t -test and was two-sided. f, g : KK-LC-1 knockout significantly reduced the proportion of ALDH + cells and ALDH + CD44 high CD24 low cells in MDA-MB-231 cells; data are presented as mean ± SD from three biologically independent experiments and statistical significance was determined using Student’s t -test and was two-sided. h The proportion of ALDH + cells was significantly lower in ALDH + sh- KK-LC-1 #1 cells compared to ALDH + NC cells after 2 weeks in culture; data are presented as mean ± SD from three biologically independent experiments, statistical significance was determined using Student’s t -test and was two-sided. i A significantly higher proportion of ALDH + cells was derived from CD44 high CD24 low KK-LC-1 +/+ MDA-MB-231 compared to CD44 high CD24 low KK-LC-1 −/− MDA-MB-231 cells after 2 weeks in culture; data are presented as mean ± SD from three biologically independent experiments, statistical significance was determined using Student’s t -test and was two-sided. Source data are provided as a Source data file.

Article Snippet: MDA-MB-231 cells and MDA-MB-468 cells were resuspended in PBS and stained with phycoerythrin (PE)-conjugated anti-CD24 at a dilution of 1:100 (Cat: 130-112-656, Miltenyi Biotec) and allophycocyanin (APC)-conjugated anti-CD44 at a dilution of 1:500 (Cat: 12211-MM02-A, SinoBiological) for 30 min at 4 °C.

Techniques: Expressing, Isolation, Knock-Out, Derivative Assay

Journal: Journal for Immunotherapy of Cancer

Article Title: Molecular mechanisms underlying the modulation of T-cell proliferation and cytotoxicity by immobilized CCL21 and ICAM1

doi: 10.1136/jitc-2024-009011

Figure Lengend Snippet: CCL21+ICAM1 SIN stimulation is associated with a shift in T-cell differentiation toward effector/effector memory subtypes following antigen-specific and nonspecific activation. Flow cytometry analysis of DC-activated and bead-activated T cells (A–D, respectively) cultured for 3 or 4 days on uncoated or SIN-coated surfaces. The gating strategy is shown in . (A, B) Bar graph illustrating the percentage of viable DC-activated and bead-activated CD8 + T cells (mean±SEM) in three independent experiments. The calculated p values (using standard t-tests) are indicated in the figure. The upper pairs represent the differences between day 3 and day 4 for cells cultured on uncoated surfaces (left) and CCL21+ICAM1-coated SIN (right). (C, D) Distribution of subsets of differentiation markers measured on days 3 and 4. The percentages (mean±SEM) of effector T cells (TE, CD44 + /CD62L − /CD127 − ), effector memory T cells (TEM, CD44 + /CD62L − /CD127 + ), central memory T cells (TCM, CD44 + /CD62L + ) and naïve T cells (CD44 - /CD62L + ) in the DC-activated and bead-activated CD8 + T-cell populations are shown. The data shown here are representative of three independent experiments. The calculated p values (using standard t tests) for the proportions of effector, effector memory and central memory CD8 + T-cell subsets among the SIN-treated cells were compared with those among the untreated cells (p<0.001, p<0.001 and p<0.05, respectively). DCs, dendritic cells; SIN, synthetic immune niche.

Article Snippet: The supernatant was aspirated, and the cells were surface stained (at room temperature for 30 min) with LIVE/DEAD Fixable Blue dead cell stain (1:1000; Invitrogen, Paisley, UK) and with the following fluorescent monoclonal antibodies from the following sources: CD8a-Spark blue 550, CD25-PE-Fire 640, CD69-PerCP, FasL-APC, PD-1-BV421, LAG-3-BV785, and CD62L-BV570 (all from BioLegend); CD44-APC vio 770 (Miltenyi Biotec); and CD127-Alexa 594 (R&D Systems, Minneapolis, Minnesota, USA).

Techniques: Cell Differentiation, Activation Assay, Flow Cytometry, Cell Culture

Journal: Journal for Immunotherapy of Cancer

Article Title: Molecular mechanisms underlying the modulation of T-cell proliferation and cytotoxicity by immobilized CCL21 and ICAM1

doi: 10.1136/jitc-2024-009011

Figure Lengend Snippet: Prolonged stimulation of bead-activated T cells with CCL21+ICAM1 SIN increases T-cell survival and alters the prominence of subsets of differentiation markers. Flow cytometry staining was performed for T cells that were bead-activated and further cultured for 7 days with or without CCL21+ICAM1 SIN. The gating strategy is shown in . (A) Bar graph illustrating the percentage of viable CD8 + T cells as determined by flow cytometry analysis. The data are shown as the mean±SEM of three independent experiments. The calculated p values (using standard t tests) are indicated in the figure. (B) Distribution of differentiation subset markers in these cells on day 7. The percentages (mean±SEM) of effector T cells (TE, CD44 + /CD62L − /CD127 − ), effector memory T cells (TEM, CD44 + /CD62L − /CD127 + ), central memory T cells (TCM, CD44 + /CD62L + ) and naïveT cells (CD44 - /CD62L + ) among the CD8 + T cells are shown. The data shown here are representative of three independent experiments. The calculated p values (using standard t-tests) for the comparisons of the proportions of effector, effector memory and central memory CD8 + T-cell subsets among the SIN-treated cells and the untreated cells were calculated (p<0.01, ns and p<0.001, respectively). (C) Fold change in the MFI of activation, proliferation, killing and exhaustion markers in bead-activated CD8 + T cells in the presence or absence of SIN stimulation, measured on day 7. The data are presented as the mean fold change±SEM relative to nonactivated cells and were obtained from three independent experiments. The calculated p values (using standard t-tests) are indicated in the figure. The pairs of bars represent the MFI of cells cultured on uncoated (left) and SIN-coated (right) surfaces. MFI, mean fluorescence intensity; SIN, synthetic immune niche.

Article Snippet: The supernatant was aspirated, and the cells were surface stained (at room temperature for 30 min) with LIVE/DEAD Fixable Blue dead cell stain (1:1000; Invitrogen, Paisley, UK) and with the following fluorescent monoclonal antibodies from the following sources: CD8a-Spark blue 550, CD25-PE-Fire 640, CD69-PerCP, FasL-APC, PD-1-BV421, LAG-3-BV785, and CD62L-BV570 (all from BioLegend); CD44-APC vio 770 (Miltenyi Biotec); and CD127-Alexa 594 (R&D Systems, Minneapolis, Minnesota, USA).

Techniques: Flow Cytometry, Staining, Cell Culture, Activation Assay, Fluorescence