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: bioRxiv

Article Title: Suppression of Glucosylceramide Synthase Reverses Drug Resistance in Cancer Cells Harbor Homozygous p53 Mutants

doi: 10.1101/2025.11.02.686136

Figure Lengend Snippet: Cells were planted in 5% FBS EMEM medium containing 6 μM IRI alone or combined 4 μM Genz-161 for 6 days. A, Imaging flow cytometry analysis for colon CSCs (CD44v6 + /CD133 + ). BF, bright field. B, GCS inhibition decreased CSC population. *, p <0.001 compared to WiDr cells treated with vehicle; **, p <0.001 compared to WiDr cells treated with IRI alone. C, Representative CSC plots of cancer cells with various treatments.

Article Snippet: After treatments, suspended cells (10 6 cells/ml) were incubated with human CD44v6 Alexa Fluor ® 488-conjugated antibody (2F10; mouse IgG1; from R&D Systems, Minneapolis, MN, USA) and human CD133 APC-conjugated antibody (170411; mouse IgG2b; from R&D Systems) in 1% BSA-containing PBS at 4°C for 45 min. After washing, cells were resuspended in 1% BSA PBS (5 x 10 5 cells/150 μL) and analysed using an Amnis Imagestream Mark II Imagestream software, and the data were further analysed using the IDEAS v6.2 program.

Techniques: Imaging, Flow Cytometry, Inhibition

Journal: bioRxiv

Article Title: Suppression of Glucosylceramide Synthase Reverses Drug Resistance in Cancer Cells Harbor Homozygous p53 Mutants

doi: 10.1101/2025.11.02.686136

Figure Lengend Snippet: A, Tumor growth of mice treated with oxaliplatin. Mice-bearing tumors generated from WiDr or WiDr/UGCG - cells were treated with vehicle, oxaliplatin (Oxa 2 mg/kg, i.p , once every 6 days) and combination (Oxa 2 mg/kg, i.p, once every 6 days and Genz-161 4 mg/kg, i.p, once every 3 days) for 37 days. *, p <0.01 compared with WiDr tumors treated with vehicle or Oxa. B, Tumor growth of mice treated with irenotecan. Mice-bearing tumors generated from WiDr cells were treated with vehicle, irenotecan (IRI 6 mg/kg, i.p , once every 6 days) and combination (IRI 6 mg/kg i.p , once every 6 days and Genz-161 4 mg/kg, i.p, once every 3 days) for 30 days. *, p <0.01 compared with tumors treated with vehicle; **, p<0.01 compared with tumors treated with IRI. C, H&E and immunofluorescence staining of tumors. Tumor sections were stained with H&E or fluorescent antibodies for CSC markers (CD44v6/CD133). Green, Alexa Fluor 448−CD44v6; red, APC-CD133; blue, DAPI nuclear counterstain. Images were magnified 200x, scale bar represents to 50 μm. D, GCS mRNA levels of tumors. *, p <0.01 compared with WiDr-tumors treated with vehicle or oxaliplatin. E, Tumor stem cell clusters in snRNA. Tumor-bearing mice were treated with Oxa (2 mg/kg, i.p, once every 6 days) for 37 days. *, p <0.001 compared with WiDr tumors treated with Oxa.

Article Snippet: After treatments, suspended cells (10 6 cells/ml) were incubated with human CD44v6 Alexa Fluor ® 488-conjugated antibody (2F10; mouse IgG1; from R&D Systems, Minneapolis, MN, USA) and human CD133 APC-conjugated antibody (170411; mouse IgG2b; from R&D Systems) in 1% BSA-containing PBS at 4°C for 45 min. After washing, cells were resuspended in 1% BSA PBS (5 x 10 5 cells/150 μL) and analysed using an Amnis Imagestream Mark II Imagestream software, and the data were further analysed using the IDEAS v6.2 program.

Techniques: Generated, Immunofluorescence, Staining

Journal: Oncotarget

Article Title: Inhibition of glucosylceramide synthase eliminates the oncogenic function of p53 R273H mutant in the epithelial-mesenchymal transition and induced pluripotency of colon cancer cells

doi: 10.18632/oncotarget.11169

Figure Lengend Snippet: Cells of SW48-Dox and TP53-Dox lines were separately treated with 5 μM PDMP in 5% FBS medium. A. Tumor sphere formation. Scale bar represents 50 μm in photomicrographs (100x magnification). *, p >0.001 compared to SW48-Dox with vehicle; **, p<0.001 compared to TP53-Dox with vehicle. B. CSCs. Cells were incubated with Alexa-Fluor488 CD44v6 and APC-CD133 antibodies and analyzed by using flow cytometry. The detected CD44v6 + /CD133 + cells (CSCs) are identified in the plots by enclosure with an ellipse (upper right), and compared with vehicle controls, as percentages of total cells in the adjacent bar graph.

Article Snippet: Suspended cells (10 6 cells/ml) were incubated with CD44v6 Alexa-Fluor 488-conjugated antibody (2F10; mouse IgG1; purchased from R&D Systems, Minneapolis, MN) and CD133/2 APC-conjugated antibody (293C3, mouse IgG2b; purchased from Miltenyi Biotec, San Diego, CA) in 1% BSA-containing PBS at 4°C for 45 min. After washing, cells were resuspended in 1% BSA PBS (1 mL) and analyzed by flow cytometry, using a FACSCalibur instrument (BD Biosciences, San Jose, CA) operated with CellQuest Pro software (BD Biosciences), and the data were further analyzed by using the FlowJo program (v10; FlowJo, Ashland, OR).

Techniques: Incubation, Flow Cytometry

Journal: Oncotarget

Article Title: Inhibition of glucosylceramide synthase eliminates the oncogenic function of p53 R273H mutant in the epithelial-mesenchymal transition and induced pluripotency of colon cancer cells

doi: 10.18632/oncotarget.11169

Figure Lengend Snippet: A. Flow cytometry analysis of colon CSCs (CD133+/CD44V6) from tumors in mice treated with doxorubicin (Dox, 200 μg/kg, per 6-days, i.p.) alone or combined with PDMP (4.0 mg/kg, per 3-days, i.p.), for 32 days. *, p<0.001 compared to SW48 tumors treated with Dox; **, p<0.001 compared to TP53 tumors treated with Dox. B. Western blotting of pluripotency regulators in tumors. Equal amounts of detergent-soluble proteins (50 μg/lane) extracted were resolved using 4-20% gradient SDS-PAGE and then immunoblotted with corresponding antibodies. Protein levels are represented as mean ± SD of their densities normalized against GAPDH from three blots. *, p <0.01 compared to Dox treatments (200 μg/kg); **, p <0.01 compared to SW48 tumors.

Article Snippet: Suspended cells (10 6 cells/ml) were incubated with CD44v6 Alexa-Fluor 488-conjugated antibody (2F10; mouse IgG1; purchased from R&D Systems, Minneapolis, MN) and CD133/2 APC-conjugated antibody (293C3, mouse IgG2b; purchased from Miltenyi Biotec, San Diego, CA) in 1% BSA-containing PBS at 4°C for 45 min. After washing, cells were resuspended in 1% BSA PBS (1 mL) and analyzed by flow cytometry, using a FACSCalibur instrument (BD Biosciences, San Jose, CA) operated with CellQuest Pro software (BD Biosciences), and the data were further analyzed by using the FlowJo program (v10; FlowJo, Ashland, OR).

Techniques: Flow Cytometry, Western Blot, SDS Page

Journal: Cancer research

Article Title: PPARδ mediates the effect of dietary fat in promoting colorectal cancer metastasis

doi: 10.1158/0008-5472.CAN-19-0384

Figure Lengend Snippet: The NSG mice were treated with a control diet or a high-fat diet after HCT-116/WT or HCT-116/PPARd−/− cells were injected into cecum. (A and C) The representative immunostaining of Nanog (A) and CD44v6 (C) in cecal tumors taken from indicated group of mice (scale bar, 50 μm). (B and D) Cells isolated from cecal tumors taken from indicated group of mice were subjected into flow cytometry analysis. Data represents the percentage of EpCAM+CD45−Nanog+ (B) and EpCAM+CD45−CD44v6+ (D) cells in total cecal tumor epithelial cells. (E) Sphere formation of tumor epithelial cells isolated from cecal tumor cells of indicated group of mice. (F) Average numbers of liver metastatic tumors at different size and total that includes all sizes in indicated group of mice. The error bar indicates ± SEM. *p<0.05.

Article Snippet: Paraffin-embedded tissue sections (5 μm thick; n=5 per animal) were stained with anti-Nanog rabbit antibody (1:100, abcam, Cambridge, MA) and anti-CD44v6 mouse monoclonal antibody (1:50, R&D System) in 4°C overnight.

Techniques: Control, Injection, Immunostaining, Isolation, Flow Cytometry

Journal: Cancer research

Article Title: PPARδ mediates the effect of dietary fat in promoting colorectal cancer metastasis

doi: 10.1158/0008-5472.CAN-19-0384

Figure Lengend Snippet: The NSG mice were treated with vehicle or GW501516 after HCT-116/WT, HCT-116/PPARd−/−, LS-174T/vector, or LS-174T/shPPARδ cells were injected into cecum. (A and C) The representative immunostaining of Nanog (A) and CD44v6 (C) in cecal tumors taken from indicated group of mice (scale bar, 50 μm). (B and D) Cells isolated from cecal tumors of indicated group of mice were subjected into flow cytometry analysis. Data represents the percentage of EpCAM+CD45−Nanog+ (B) and EpCAM+CD45−CD44v6+ (D) cells in total cecal tumor epithelial cells. (E) Sphere formation of tumor epithelial cells isolated from cecal tumor cells of indicated group of mice. The error bar indicates ± SEM. *p<0.05.

Article Snippet: Paraffin-embedded tissue sections (5 μm thick; n=5 per animal) were stained with anti-Nanog rabbit antibody (1:100, abcam, Cambridge, MA) and anti-CD44v6 mouse monoclonal antibody (1:50, R&D System) in 4°C overnight.

Techniques: Plasmid Preparation, Injection, Immunostaining, Isolation, Flow Cytometry

Journal: Cancer research

Article Title: PPARδ mediates the effect of dietary fat in promoting colorectal cancer metastasis

doi: 10.1158/0008-5472.CAN-19-0384

Figure Lengend Snippet: The NSG mice were treated with a control diet or a high-fat diet after LS-174T/vector or LS-174T/shNanog cells were injected into cecum. (A) Cells isolated from cecal tumors taken from indicated group of mice were subjected into flow cytometry analysis. Data represents the percentage of EpCAM+CD45−CD44v6+ cells in total cecal tumor epithelial cells. (B) Sphere formation of tumor epithelial cells isolated from cecal tumor cells of indicated group of mice. (C) Average numbers of liver metastatic tumors at different size and total that includes all sizes in indicated group of mice. The error bar indicates ± SEM. *p<0.05.

Article Snippet: Paraffin-embedded tissue sections (5 μm thick; n=5 per animal) were stained with anti-Nanog rabbit antibody (1:100, abcam, Cambridge, MA) and anti-CD44v6 mouse monoclonal antibody (1:50, R&D System) in 4°C overnight.

Techniques: Control, Plasmid Preparation, Injection, Isolation, Flow Cytometry