Journal: Genes & Cancer

Article Title: CD133-positive cancer stem cells from Colo205 human colon adenocarcinoma cell line show resistance to chemotherapy and display a specific metabolomic profile

doi:

Figure Lengend Snippet: A-B. Cytometry analyses of Nestin, CK20, CD133, Oct3/4, CD44 and CD44v9 on 10% FBS Colo205 growing cells (control) and serum-free growing cultures (week 1 to week 5).

Article Snippet: The purity of sorted cells was evaluated by flow cytometry using FACSCalibur (BD Biosciences) after labeling with anti-human CD133/2 or CD44 antibody (Miltenyi Biotec).

Techniques: Cytometry, Control

Journal: Genes & Cancer

Article Title: CD133-positive cancer stem cells from Colo205 human colon adenocarcinoma cell line show resistance to chemotherapy and display a specific metabolomic profile

doi:

Figure Lengend Snippet: A. Tumorsphere evaluation of CD133+ and CD133- sorted cells. B. Invasiveness of different cell fractions measured using a collagen-based invasion kit (relative DO measured at 560 nm). C. Survival assay after chemotherapy treatment with cisplatin and 5-FU. D. Metabolite quantification after CE-TOF-MS experiments. Only metabolites of interest for Colo205 cells cultured in 10% FBS (control; green bar), CD133+ sorted cells (red bar) and CD44+ sorted cells (yellow bar) are reported here.

Article Snippet: The purity of sorted cells was evaluated by flow cytometry using FACSCalibur (BD Biosciences) after labeling with anti-human CD133/2 or CD44 antibody (Miltenyi Biotec).

Techniques: Clonogenic Cell Survival Assay, Cell Culture, Control

Journal: bioRxiv

Article Title: CLIC-dependent internalization of caveolin-1 to lysosomal vacuoles in response to osmotic regulation

doi: 10.1101/2025.08.26.672461

Figure Lengend Snippet: A) MDA-MB-231 cells were incubated in isotonic media, in hypotonic media for 5, 15, 30 and 60 minutes or under Recovery conditions in which hypotonic media was replaced after 60 minutes with isotonic media for 5, 15, 30 or 60 minutes. Cells were labeled for Cav1, fixed and imaged by TIRF widefield imaging. Average Cav1 intensity per cell was quantified and normalized to isotonic control. Representative images of select conditions are shown. (n=3 independent experiments with >30 cells for each condition; ANOVA with Dunnett’s post-test comparing to isotonic control; **p < 0.01; ***p < 0.001; Scale bar: 2µm). B) STED imaging of anti-Cav1 labeling MDA-MB-231 cells incubated in isotonic media or in hypotonic media for 15 or 60 minutes. Cav1 endocytic vacuoles were counted per cell. (n>28 from three independent experiments for isotonic and hypotonic 15 minutes, four independent experiments for other conditions; (ANOVA with Dunnett post-test comparing to isotonic control; *p < 0.05; ****p < 0.0001; Scale bar: 4µm; Inset scale bar: 1µm). C) MDA-MB-231 cells incubated with isotonic or hypotonic media for 60 minutes were fixed and labeled for Cav1, CD44 and β1-integrin (Scale bar: 4µm). The diameter of individual Cav1, CD44, and β1-integrin positive endosomes was measured from three independent experiments.

Article Snippet: Primary antibodies: rabbit anti-Cav1 mAb (3267) was purchased from Cell Signaling Technology, rat anti-CD44 mAb (22530) from Novus, and mouse anti-β1 integrin mAb (sc-53711) from Santa Cruz.

Techniques: Incubation, Labeling, Imaging, Control

Journal: bioRxiv

Article Title: CLIC-dependent internalization of caveolin-1 to lysosomal vacuoles in response to osmotic regulation

doi: 10.1101/2025.08.26.672461

Figure Lengend Snippet: A) MDA-MB-231 cells were incubated with anti-CD44 and anti-β1 integrin antibodies in isotonic media for 60 minutes at 4°C or 37°C or in hypotonic media for 5, 15, 30 or 60 minutes at 37°C. As indicated, an acid wash treatment was performed at 4°C to remove surface antibodies and cells were fixed and labeled with secondary antibodies to anti-CD44 and anti-β1 integrin as well as for endogenous Cav1. (n= >30 cells per condition from three independent experiments; ANOVA with Dunnett post-test comparing each condition to isotonic 4°C control; *p < 0.05; ***p < 0.001; ****p < 0.0001; Scale bars: 2 µm). B) MDA-MB-231 cells were transfected with wild-type CDC42-GFP or dominant negative (Dom Neg) CDC42-GFP, incubated with isotonic or hypotonic media for 60 minutes and then fixed and labeled for Cav1 and CD44. CDC42-GFP was also imaged and the number of Cav1 endocytic vacuoles counted per cell (n>30 cells from three independent experiments; ANOVA with Dunnett’s post-test comparing to isotonic control; ****p < 0.0001; Scale bar: 2µm).

Article Snippet: Primary antibodies: rabbit anti-Cav1 mAb (3267) was purchased from Cell Signaling Technology, rat anti-CD44 mAb (22530) from Novus, and mouse anti-β1 integrin mAb (sc-53711) from Santa Cruz.

Techniques: Incubation, Labeling, Control, Transfection, Dominant Negative Mutation

Journal: bioRxiv

Article Title: CLIC-dependent internalization of caveolin-1 to lysosomal vacuoles in response to osmotic regulation

doi: 10.1101/2025.08.26.672461

Figure Lengend Snippet: A) MDA-MB-231 cells were transfected with LAMP-GFP, incubated with isotonic or hypotonic media for 60 minutes and then fixed and labeled for Cav1. LAMP-GFP was also imaged and the number of Cav1 endocytic vacuoles counted per cell (n>30 from cells three independent experiments with >30 cells for each condition; ANOVA with Dunnett’s post-test comparing to isotonic control; ****p < 0.0001; Scale bar: 2 µm). B) Cav1 CRISPR knockout MDA-MB-231 cells were transfected with Cav1-WT-HA or Cav1-K*R-HA lysine mutant, incubated with isotonic or hypotonic media for 15 or 60 minutes and then fixed and labeled for Cav1 and CD44. (n>29 cells from three independent experiments; ANOVA with Tukey post-test comparing WT to K*R at the same isotonic or hypotonic conditions and the respective hypotonic condition to isotonic control; Scale bar: 2µm). C) MDA-MB-231 cells were transfected with LAMP1-mScarlet and Cav1-Halo, where indicated pre-treated with 100nM BafA for 2 hours, labeled with Halo-LIVE RED for 30 minutes and then incubated for 30 minutes with 1uM LysoSensor green and LIVE RED Halotag ligand. Cells were then placed in isotonic or hypotonic imaging media and multiple cells imaged over a period of 30 minutes in isotonic DMEM or after 30 minutes of hypotonic shock. LysoSensor and Cav1 intensity within LAMP1-positive lysosomes were quantified. (n>38 cells from three independent experiments; ANOVA with Tukey’s post-test; *p < 0.05; ****p < 0.0001; Scale bar: 2 µm).

Article Snippet: Primary antibodies: rabbit anti-Cav1 mAb (3267) was purchased from Cell Signaling Technology, rat anti-CD44 mAb (22530) from Novus, and mouse anti-β1 integrin mAb (sc-53711) from Santa Cruz.

Techniques: Transfection, Incubation, Labeling, Control, CRISPR, Knock-Out, Mutagenesis, Imaging

Journal: bioRxiv

Article Title: CLIC-dependent internalization of caveolin-1 to lysosomal vacuoles in response to osmotic regulation

doi: 10.1101/2025.08.26.672461

Figure Lengend Snippet: A) Cell volume of MDA-MB-231 and PC3 cells switched from isotonic to hypotonic media was measured by live phase imaging. Black line indicates addition of hypotonic media (n=3 independent experiments; each experiment includes 2-3 technical replicates and each technical replicate consists of 9 ROIs). B) MDA-MB-231 and PC3 cells were incubated in isotonic media, or hypotonic or hypertonic media for 5, 15, 30 and 60 minutes. Cells were labeled for Cav1 and CD44 and Cav1 endocytic vacuoles counted per cell. Representative images for select time points are shown. (n>28 cells from three independent experiments; ANOVA with Dunnett’s post-test comparing each condition to isotonic control; *p < 0.05; **p < 0.01; ****p < 0.0001; Scale bar: 2 µm). C) PC3 cells and PC3 cells stably transfected Cavin-1-GFP were incubated in isotonic, hypotonic and hypertonic shock media for 60 minutes and then fixed and labeled for Cav1. Cavin-1-GFP was imaged in the GFP channel and Cav1 endocytic vacuoles counted per cell. (n>28 cells from three independent experiments; ANOVA with Tukey’s post-test and comparing PC3 to PC3+Cavin-1 and each condition with the respective isotonic control; **p < 0.01; ***p < 0.001; ****p < 0.0001; Scale bar: 2 µm).

Article Snippet: Primary antibodies: rabbit anti-Cav1 mAb (3267) was purchased from Cell Signaling Technology, rat anti-CD44 mAb (22530) from Novus, and mouse anti-β1 integrin mAb (sc-53711) from Santa Cruz.

Techniques: Imaging, Incubation, Labeling, Control, Stable Transfection, Transfection

Journal: Materials Today Bio

Article Title: A bioactive composite scaffold enhances osteochondral repair by using thermosensitive chitosan hydrogel and endothelial lineage cell-derived chondrogenic cell

doi: 10.1016/j.mtbio.2024.101174

Figure Lengend Snippet: EndMT-derived tEPCs acquire an MSC-like phenotype. ( a ) Flow cytometry analysis of CD44, CD90, CD105 (mesenchymal stem cell markers), CD34 (hematopoietic and endothelial cell marker), and CD45 (leukocyte marker) expression in tEPCs. The empty areas show isotype control staining. The red-filled areas represent the expression of specific markers. ( b ) Representative immunofluorescence images of tEPC surface markers. tEPCs stain positive for MSC markers (CD44, CD90, and CD105) and negative for endothelial cell markers (CD31, eNOS, VE-cadherin, and vWF) (20 × magnification; scale bar: 50 μm). ( c , d ) CFU efficiency of EPCs and tEPCs, assessing self-renewal through the rate of colony formation in CFU assays. ( c ) Representative colonies of EPCs and tEPCs in 6-well plates (scale bar: 5 mm). ( d ) Columns illustrate the CFU efficiency. Values are reported as the mean ± standard deviation (SD) of six replicates. *** P < 0.001. ( e ) Multilineage differentiation potential of tEPCs induced to differentiate into ( i ) chondrogenic (10 × magnification; scale bar: 100 μm), ( ii ) osteogenic (10 × magnification; scale bar: 100 μm), or ( iii ) adipogenic (40 × magnification; scale bar: 10 μm) lineages. Abbreviations: CD, cluster of differentiation; CFU, colony-forming unit; EndMT, endothelial-to-mesenchymal transition; eNOS, endothelial nitric oxide synthase; EPCs, endothelial progenitor cells; MSCs, mesenchymal stem cells; tEPC, transdifferentiated EPCs; VE-cadherin, vascular endothelial cadherin; vWF, von Willebrand factor.

Article Snippet: Next, 1 × 10 6 tEPCs were suspended in 500 μL of PBS containing 20 μg/mL of fluorescein isothiocyanate (FITC)-conjugated antibodies against CD44 (Novus, Centennial, CO, USA), CD90 (Bioworld, Louis Park, MN, USA), CD105 (Bioss, Woburn, MA, USA), CD34 (Bioss), and CD45 (Bioss).

Techniques: Derivative Assay, Flow Cytometry, Marker, Expressing, Control, Staining, Immunofluorescence, Standard Deviation

Journal: International journal of molecular medicine

Article Title: Autophagy is essential for the endothelial differentiation of breast cancer stem‑like cells.

doi: 10.3892/ijmm.2019.4399

Figure Lengend Snippet: Figure 1. Flow cytometry and microsphere culture of BCSLCs. (A) CD24‑/low cells were isolated from MCF‑7 using immunomagnetic beads, and the expres- sion of CD24 in MCF‑7 and isolated CD24‑/low cells were assessed using flow cytometry. (a) Isotype control for (b); (b) MCF‑7 cells; (c) isotype control for (d); (d) isolated CD24‑/low cells. (B) CD44+ cells were further isolated from CD24‑/low cells and the expression of CD44 was assessed using flow cytometry. (a) Isotype control for (b); (b) CD44+ cells. (C) Expression of CD24 and CD44 in MCF‑7, BCSLCs and BCSLCs after eight passages. (a) isotype control for (b); (b) MCF‑7 cells; (c) CSLCs; (d) isotype control for (e); (e) BCSLCs after eight passages. (D) The isolated BCCSLCs were cultured in microspheres for 0 and 64 h in stem cell culture medium. BCSLC, breast cancer stem‑like cell; PE, phycoerythrin.

Article Snippet: The following primary antibodies were used: Fluorescein isothiocyanate (FITc)-conjugated anti-human cd44 (cat. no. 130-113-903; Miltenyi Biotec GmbH), phycoerythrin (PE)-conjugated anti-human cd24 (cat. no. 130-098-861; Miltenyi Biotec GmbH) PE-conjugated anti-human cd31 (cat. no. 130-110-807; Miltenyi Biotec GmbH) and FITc-conjugated anti-human cd105 (cat. no. 130-098-778; Miltenyi Biotec GmbH).

Techniques: Flow Cytometry, Isolation, Control, Expressing, Cell Culture, Stem Cell Culture

Journal: Journal of cell science

Article Title: Combined heterogeneity in cell size and deformability promotes cancer invasiveness.

doi: 10.1242/jcs.250225

Figure Lengend Snippet: Fig. 5. Small cells of varying deformabilities may correspond to CSCs. (A) Distribution of cell size and deformability for four distinct populations: small cells of varying deformabilities (A), soft cells of varying cell sizes (B), intermediately stiff cells of varying cell sizes (C), and a combination of populations A, B and C, i.e. population D. (B) Quantification (mean± s.e.m.) of t and d for populations A to D. (C) Dependence of δ on deformability for population A. (D) FACS expression profile of CD44 and CD24 in MDA-MB-231 cells. Q1, Q2, Q3 and Q4 represent quadrant gating of FACS. Inset boxes correspond to gating of FACS to get pure populations of mCSCs and hCSCs. (E) Representative phase contrast images of CD44hi CD24−mCSCs and CD44hi CD24+

Article Snippet: Cells were fixed and stained for nuclei, F-actin (Thermo Fisher Scientific, A22282) and CD44 (Novus, NBP1-47386) at day 0 (2 h after spheroids were embedded in collagen gels), day 1, day 3 and day 5.

Techniques: Expressing

Journal: Journal of cell science

Article Title: Combined heterogeneity in cell size and deformability promotes cancer invasiveness.

doi: 10.1242/jcs.250225

Figure Lengend Snippet: Fig. 6. Temporal enrichment of CSCs at the invasive front. (A) Schematic of the spheroid invasion assay. Single spheroids are implanted into three-dimensional collagen gels and their invasion tracked over a period of 6 days. (B) Representative cryo FEG-SEM image of 1.5 mg/ml collagen gel. (C) Representative image of a spheroid stained for CD44, phalloidin (for visualizing the F-actin cytoskeleton) and Hoechst 33342 for imaging nuclei. (D) Radial distribution of CD44 and F-actin intensity in cells within the spheroid at day 0. (E) Representative images of spheroids at days 1, 3 and 5, visualized by CD44 staining. (F) Segregation of invasive area of day 5 spheroids into seven zones. Zones 0, 1 and 2 were contained within the area encompassing day 0 spheroids. (G) Temporal zone-wise distribution of integrated CD44 intensity normalized with respect to CD44 intensity of the innermost zone (i.e. zone 0) (mean±s.e.m.; n>40 cells per zone per time point pooled from N=2 spheroids). ***P<0.001 (one-way ANOVA/Fisher test for parametric data and Mann–Whitney test for non-parametric data). a.u., arbitrary units.

Article Snippet: Cells were fixed and stained for nuclei, F-actin (Thermo Fisher Scientific, A22282) and CD44 (Novus, NBP1-47386) at day 0 (2 h after spheroids were embedded in collagen gels), day 1, day 3 and day 5.

Techniques: Invasion Assay, Staining, Imaging, MANN-WHITNEY