Journal: bioRxiv

Article Title: A Novel miR-4745 -KLC2 Axis Regulates Cancer Stem Cell Traits in Colorectal Cancer

doi: 10.64898/2026.01.07.697660

Figure Lengend Snippet: (a) Quantification of the CD44v6-APC-positive population in CPP1 (n=7) and CTC44 (n=5) cells transfected with miR-4745-3p ( miR4745 ) or miRVana microRNA Mimic Negative Control #1 (miCTRL) mimics (right panel). Percentage of CD44v6-APC positive CPP1 cells is indicated in inset boxes for a representative experiment (left panel). (b) Representative images of tumorspheres (upper panel) and the percentage (lower panel) of sphere-forming cells in patient-derived CPP1 and circulating tumor (CTC44) colon cancer cells transfected with miR-4745-3p ( miR4745 ) or miCTRL mimics. Data from individual replicates (n=15 per experiment) are presented, along with the mean ± SEM from six independent experiments. (c) Percentage of sphere-forming cells in patient-derived CPP1 colon cancer cells transfected with miR4745 or negative control (miCTRL) mimics and, maintained as first-generation colonospheres (S1) or after several passages (S2, S3). Data from individual replicates (n=12 per experiment) are presented, along with the mean ± SEM from three independent experiments. (d) Percentage of surviving cancer stem cells (Aldefluor-positive) measured 48 hours following treatment with specified concentrations of FIRI (1X = 50 µM 5-FU + 500 nM SN38) in vitro . Sorted CPP1 Aldefluor-positive cells were first transfected with 50 nM miR-4745-3p ( miR-4745 ) or miRVana microRNA Mimic Negative Control #1 (miCTRL) mimics, 24 hours prior to FIRI treatment. Data are expressed as mean ± SEM (n = 3). (e) Percentage of Aldefluor-positive cells (% ALDH + cells) in CPP1 cells transfected with miR-4745 or miCTRL mimics and then exposed for 72 hours to chemotherapy (Firi=5µM 5-FU + 50nM SN38). Data are expressed as mean ± SEM (n=6). *, p<0.05; **, p<0.005, ***, p < 0.001, Mann Whitney test.

Article Snippet: CD44v6-APC (clone REA706, 130-111-238; Macs Miltenyi) antibody was incubated with 100 000 cells in PBS containing 5% FBS for 20 min at 4°C.

Techniques: Transfection, Negative Control, Derivative Assay, In Vitro, MANN-WHITNEY

Journal: iScience

Article Title: CHK1 inhibitor sensitizes resistant colorectal cancer stem cells to nortopsentin

doi: 10.1016/j.isci.2021.102664

Figure Lengend Snippet: CR-CSphCs are endowed with innate chemoresistance (A) Cell growth kinetics of CR-CSphCs treated with a vehicle or 5-FU in combination with oxaliplatin, up to 4 days. Data represent the mean ± SD (n = 3) using 2 different CR-CSphC lines for each subgroup (wt, #7 and #21; Braf , #3 and #5; Kras #8 and #9; Res #R3 and #R4). Statistical significance between 2 groups was determined by unpaired Student's t-test (2-tailed). ns, nonsignificant. (B) Cell cycle analysis in CR-CSphCs treated as in (A) for 24 h. Data show percentage of cell number in G0–G1, S, and G2–M phases. Data are expressed as mean ± SD of three independent experiments performed in CR-CSphCs isolated from patients with wt (#7 and #21), Braf (#3 and #5), Kras (#8 and #9) and chemoresistant (#R3 and #R4) CRC. (Right panels) Representative cell cycle analysis of CR-CSphCs treated with a vehicle or 5-FU in combination with oxaliplatin, for 24 h (blue color = G0-G1; yellow color = S; green color = G2-M). (C) Percentage of viability in flow-cytometry-sorted TOP-GFP CR-CSphCs, treated as in (A) up to 120 h. Data are expressed as mean ± SD of three independent experiments performed using two different CR-CSphC lines (#8, #9). (Right panel) Representative flow cytometry analysis of TOP-GFP expression in spared CR-CSphCs, at 5 days. Statistical significance between 2 groups was determined by unpaired Student's t-test (2-tailed). ∗∗p ≤ 0.01. (D) Cell viability percentage of CR-CSphCs enriched for CD44v6 expression and treated with vehicle or 5-FU in combination with oxaliplatin up to 120 h. Data are expressed as mean ± SD of three independent experiments performed using four different CR-CSphCs lines (#3, #9, #21, #R4). (Right panel) Representative flow cytometry analysis of the percentage of CD44v6 positivity in spared CR-CSphCs, at 5 days. Statistical significance between 2 groups was determined by unpaired Student's t-test (2-tailed). ∗p ≤ 0.05. (E) Tumor size of CR-CSphCs subcutaneously injected into immunocompromized mice, treated for 4 weeks (from sixth to ninth week) with vehicle or 5-FU in combination with oxaliplatin. Data represent the mean ± SD of tumor size measured in six mice per group, using 2 different CR-CSphC lines (#8, #21). Black arrowheads indicate weeks of treatment. Statistical significance between 2 groups was determined by unpaired Student's t-test (2-tailed). ns, nonsignificant. (F) Cell viability analysis of healthy cells (IMEC and AD-MSCs) treated as in (D), for 3 days. Data are expressed as mean ± SD of three independent experiments. Statistical significance between 2 groups was determined by unpaired Student's t-test (2-tailed).∗∗p ≤ 0.01; ∗∗∗∗p ≤ 0.0001. See also Figure S1 .

Article Snippet: Double staining was performed using antibodies against CD44v6 (2F10 APC, mouse IgG1, R&D systems) and p-CHK1 (Ser345, 133D3, Rabbit IgG, Cell Signaling Technology), revealed by the MACH 2 double stain 2 kit conjugated goat antimouse polymer horseradish peroxidase (HRP) and the conjugated goat antirabbit polymer alkaline phosphatase (Biocare Medical), and detected by DAB and Vulcan Fast Red chromogen.

Techniques: Cell Cycle Assay, Isolation, Flow Cytometry, Expressing, Injection

Journal: iScience

Article Title: CHK1 inhibitor sensitizes resistant colorectal cancer stem cells to nortopsentin

doi: 10.1016/j.isci.2021.102664

Figure Lengend Snippet: CD44v6 + CR-CSCs acquire resistance to NORA234 treatment (A) Kinetics of cell growth of CR-CSphCs treated with vehicle or NORA234, up to 4 days. Data represent the mean ± SD (n = 3) using CR-CSphCs isolated from wt (#7 and #21), Braf (#3 and #5), Kras (#8 and #9) and chemoresistant(#R3 and #R4) CRC patients. Statistical significance between 2 groups was determined by unpaired Student's t-test (2-tailed). ∗p ≤ 0.05; ∗∗p ≤ 0.01. (B) Viability percentage of low and high TOP-GFP cell fraction treated as in (A) up to 120 h. Data are expressed as mean ± SD of three independent experiments performed using two different CR-CSphC lines (#8, #9). (C) Viability percentage of CR-CSphCs enriched for CD44v6 expression and treated as in (A) up to 120 h. Data are expressed as mean ± SD of three independent experiments performed using four different CR-CSphC lines (#3, #9, #21, #R4). (D) Colony-forming assay of CR-CSphCs treated with a vehicle or NORA234, at 21 days. The number of colonies represents mean ± SD of 3 independent experiments performed with cells isolated from 4 different patients with CRC (CR-CSphCs #3, #9, #21, #R4). Statistical significance between 2 groups was determined by unpaired Student's t-test (2-tailed). ns, nonsignificant; ∗∗p ≤ 0.01. See also Figure S1 .

Article Snippet: Double staining was performed using antibodies against CD44v6 (2F10 APC, mouse IgG1, R&D systems) and p-CHK1 (Ser345, 133D3, Rabbit IgG, Cell Signaling Technology), revealed by the MACH 2 double stain 2 kit conjugated goat antimouse polymer horseradish peroxidase (HRP) and the conjugated goat antirabbit polymer alkaline phosphatase (Biocare Medical), and detected by DAB and Vulcan Fast Red chromogen.

Techniques: Isolation, Expressing

Journal: iScience

Article Title: CHK1 inhibitor sensitizes resistant colorectal cancer stem cells to nortopsentin

doi: 10.1016/j.isci.2021.102664

Figure Lengend Snippet: Inhibition of CHK1 activity sensitized CR-CSphCs to NORA234 by synthetic lethality (A) Fold variation of cell number in CR-CSphCs transduced with shCHK1 or ns shRNA and treated with vehicle, 5-FU in combination with oxaliplatin, or NORA234, up to 72 h. Data are mean ± S.D. of 3 independent experiments performed with cells isolated from patients with wt (#21), Braf (#3), Kras (#9), or chemoresistant (#R4) CRC. (B) Cell viability analysis of cells treated as in (A) for 72 h. Data are mean ± S.D. of 3 independent experiments performed with cells isolated from 4 different CRC patients (CR-CSphCs #3, #9, #21, #R4). Statistical significance between 2 groups was determined by unpaired Student's t-test (2-tailed). ns, nonsignificant; ∗p ≤ 0.05; ∗∗p ≤ 0.01; ∗∗∗p ≤ 0.001. (C) Representative colony forming assay of CR-CSphCs (R#4) transduced with shCHK1 or ns shRNA and treated with vehicle or NORA234, at 21 days. n represents the number of colonies. Scale bars, 1000 μm. (D) Percentage of Annexin V positivity in CR-CSphCs treated with vehicle or rabusertib (LY2603618), for 24 h. Data represent mean ± S.D. of 3 independent experiments performed with cells isolated from patients with wt (#21), Braf (#3), Kras (#9) or chemoresistant (#R4) CRC. Statistical significance between 2 groups was determined by unpaired Student's t-test (2-tailed). ∗∗p ≤ 0.01; ∗∗∗p ≤ 0.001; ∗∗∗∗p ≤ 0.0001. (E) Cell cycle analysis in CR-CSphCs treated with vehicle or NORA234 in combination with Rabusertib (LY2603618), for 48 h. Data show percentage of cell number in subG0 (red color), G0–G1 (blue color), S (brow color), and G2–M (green color) cell cycle phase. Data are expressed as mean ± SD of three independent experiments using cells isolated from 4 different patients with CRC (CR-CSphCs #3, #9, #21, #R4). (F) Flow cytometry analysis of GFP and CD44v6 positivity percentage in CR-CSphCs transduced with TOP-GFP and treated with a vehicle, NORA234, alone or in combination with rabusertib (LY2603618), for 48 hr. Data are expressed as mean ± SD of three independent experiments using cells isolated from 2 different patients with CRC (CR-CSphCs #8, #9). (G) 3D synergy map of viability in CR-CSphCs treated alone or in combination with NORA234 and rabusertib (LY2603618) at the indicated doses, for 48 h. Data are mean of 3 independent experiments using cells isolated from patients with wt (#21), Braf (#3), Kras (#9) or chemoresistant (#R4) CRC. See also and .

Article Snippet: Double staining was performed using antibodies against CD44v6 (2F10 APC, mouse IgG1, R&D systems) and p-CHK1 (Ser345, 133D3, Rabbit IgG, Cell Signaling Technology), revealed by the MACH 2 double stain 2 kit conjugated goat antimouse polymer horseradish peroxidase (HRP) and the conjugated goat antirabbit polymer alkaline phosphatase (Biocare Medical), and detected by DAB and Vulcan Fast Red chromogen.

Techniques: Inhibition, Activity Assay, Transduction, shRNA, Isolation, Cell Cycle Assay, Flow Cytometry

Journal: Journal of Investigative Dermatology

Article Title: Fibroblast Invasive Migration into Fibronectin/Fibrin Gels Requires a Previously Uncharacterized Dermatan Sulfate-CD44 Proteoglycan

doi: 10.1046/j.0022-202x.2004.22205.x

Figure Lengend Snippet: Figure 5 Adult human dermal fibroblasts only express CD44 isoform CD44H. Fluor- escence-activated cell sorter analysis showed that fibroblasts expressed CD44H (A) whereas CD44v3 (B), CD44v4/5 (C), and CD44v6 (D) expres- sion was not detectable as the pattern was the same as second antibody alone (arrows represent the maximum level of cell fluorescence in the absence of primary antibody). Human keratinocytes were negative for CD44H (E) but ex- pressed CD44v3 (F) and CD44v6 (H).

Article Snippet: Monoclonal antibodies against human CD44H, CD44v3, CD44v4/ 5, and CD44v6 were obtained from R&D Systems (Minneapolis, MN).

Techniques:

Journal: Stem Cells International

Article Title: CD44v6+ Hepatocellular Carcinoma Cells Maintain Stemness Properties through Met/cJun/Nanog Signaling

doi: 10.1155/2022/5853707

Figure Lengend Snippet: CD44v6+ HCC cells possess stemness properties. (a) CD44v6 negative/positive SNU398 cells were subcutaneously injected into the BALB/c mice. Red arrow means CD44v6 positive group, black arrow means negative group. The tumor volume from each group were tested. For statistical analysis, ∗ p < 0.05, ∗∗ p < 0.01, t -test. The representative images of IHC staining of CD44v6 and Ki67 in CD44v6 positive and negative group tumor tissues. Bars: 200 μ m. (b) The liver and the lung of the SNU398 cell subcutaneously treated mice were dissected. Red circle indicates the site of metastatic tumor. Hematoxylin-eosin (HE) staining showed the metastatic tumor in the liver. (c) Representative images of transwell migration and invasion in indicated cells. Scale bar, 200 μ m. ∗∗ p < 0.01, t -test. (d) The colony formation assay showed that the colony formation ability of CD44v6+ cells was stronger than CD44v6- cells. ∗∗ p < 0.01, t -test. (e) CCK8 assay showed the cell proliferation in CD44v6+ cells was stronger than CD44v6- cells. ∗ p < 0.05, ∗∗∗ p < 0.001, t -test. (f) The representative images of spheres and histogram analysis in indicated cells. Scale bar, 200 μ m. ∗∗ p < 0.01, t -test. (g) Cell viability assay was performed using CCK8 assay in SNU398 cells, which were treated with indicated concentrations of sorafenib for 24 hours. And the number of viable cells represented the susceptibility of the CD44v6+ cells to antisorafenib. ∗ p < 0.05, t -test.

Article Snippet: Cultured cells (5 × 10 5 ) were centrifuged, and the cell pellets suspended in FACS buffer (PBS containing 0.5% FBS), then labeled with PE-conjugated human CD44v6 antibody (R&D Systems, #FAB3660P) (4°C, 30 min).

Techniques: Injection, Immunohistochemistry, Staining, Migration, Colony Assay, CCK-8 Assay, Viability Assay

Journal: Stem Cells International

Article Title: CD44v6+ Hepatocellular Carcinoma Cells Maintain Stemness Properties through Met/cJun/Nanog Signaling

doi: 10.1155/2022/5853707

Figure Lengend Snippet: The HGF/MET signaling pathway contributes to maintenance of stemness in CD44v6+ HCC Cells. (a) Western blot analysis of the Met protein levels in 18 paired HCC tissues and adjacent nontumor tissues selected randomly. β -Actin was used as a normalized control. (b) Kaplan–Meier survival analysis of overall survival were compared according to the expression levels of the Met in HCC tissues. Patients with Met expression median survival = 32 months vs. 48 months, log-rank test, n = 53, ∗ p = 0.0415. The immunohistochemical staining shows the expressions of Met in surgical specimen from patients with HCC. The expression of Met in tumor tissue was significantly higher than in adjacent nontumor tissue. Scale bar, 20 μ m. (c) The expression of EMT-related genes, including Snail1, Slug, and Twist in CD44v6+ and CD44v6- cells. β -Actin was used as a normalized control. (d) The expression of Met and the stemness relative genes, including Nanog, Sox2, and Oct4 in CD44v6+ and CD44v6- cells. β -Actin was used as a normalized control. (e) shMet CD44v6+ cells and scrambled cells were subcutaneously injected into the BALB/c mice ( n = 5 in each group). Black arrow means NC group, red arrow means Met shRNA group. The tumor volume from each group was tested. For statistical analysis, ∗ p < 0.05, ∗∗ p < 0.01, t -test. The representative images of IHC staining of Met in NC and shMet group tumor tissues. Bars: 200 μ m. (f) The representative images of spheres and histogram analysis in indicated cells. Scale bar, 200 μ m. ∗ p < 0.05, t -test. (g) The representative images of the wound-healing experiment and histogram analysis in indicated cells. Scale bar, 100 μ m. ∗∗∗ p < 0.001, t -test. (h) Representative images of transwell migration and invasion in indicated cells. Scale bar, 200 μ m. ∗ p < 0.05, ∗∗ p < 0.01, t -test.

Article Snippet: Cultured cells (5 × 10 5 ) were centrifuged, and the cell pellets suspended in FACS buffer (PBS containing 0.5% FBS), then labeled with PE-conjugated human CD44v6 antibody (R&D Systems, #FAB3660P) (4°C, 30 min).

Techniques: Western Blot, Control, Expressing, Immunohistochemical staining, Staining, Injection, shRNA, Immunohistochemistry, Migration

Journal: Stem Cells International

Article Title: CD44v6+ Hepatocellular Carcinoma Cells Maintain Stemness Properties through Met/cJun/Nanog Signaling

doi: 10.1155/2022/5853707

Figure Lengend Snippet: The HGF/MET signaling pathway activates Nanog expression in CD44v6+ HCC cells. (a) Silencing Met decreased the expression of stemness relative genes, including Oct4, Sox2, and Nanog in CD44v6+ SNU-398 cells. β -Actin was used as a normalized control. (b) The expression of EMT-related genes, including Snail1, Slug, and Twist in CD44v6+ SNU-398 cells. β -Actin was used as a normalized control. (c) Western blot showed that silencing Met decreased the expression of Nanog in CD44v6+ cells. But LvNanog did not affect the Met expression. β -Actin was used as a normalized control. (d) 1 × 10 5 of cells were injected into the left lobes of the liver. Bioluminescence signals from Met shRNA groups were weaker than that from corresponding control groups. But overexpressed Nanog could rescue the bioluminescence signals. The central luminescence intensity: 8.8e + 7 vs. 1.6e + 6; ∗ p < 0.05, ∗∗ p < 0.01; t -test. The representative images of Ki67 immunohistochemistry. Scale bar, 50 μ m. (e) The representative images of spheres and histogram analysis in indicated cells. Scale bar, 200 μ m. (f) and (g) transwell migration and invasion assays showed the knockdown of Met decreased the migration and invasion of CD44v6+ cells, while overexpressed Nanog could rescue the migration and invasion ability. Scale bar, 200 μ m. ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001; t -test.

Article Snippet: Cultured cells (5 × 10 5 ) were centrifuged, and the cell pellets suspended in FACS buffer (PBS containing 0.5% FBS), then labeled with PE-conjugated human CD44v6 antibody (R&D Systems, #FAB3660P) (4°C, 30 min).

Techniques: Expressing, Control, Western Blot, Injection, shRNA, Immunohistochemistry, Migration, Knockdown

Journal: Stem Cells International

Article Title: CD44v6+ Hepatocellular Carcinoma Cells Maintain Stemness Properties through Met/cJun/Nanog Signaling

doi: 10.1155/2022/5853707

Figure Lengend Snippet: Schematic illustration of the mechanism by which the transcription factor cJun is binding in the transcription start site of Nanog and then promotes the Nanog expression, contributing to the maintenance of stemness of CD44v6+ HCC cells.

Article Snippet: Cultured cells (5 × 10 5 ) were centrifuged, and the cell pellets suspended in FACS buffer (PBS containing 0.5% FBS), then labeled with PE-conjugated human CD44v6 antibody (R&D Systems, #FAB3660P) (4°C, 30 min).

Techniques: Binding Assay, Expressing

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