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: 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: Baseline CD44v6-positive circulating tumor cells to predict first-line treatment failure in patients with metastatic colorectal cancer

doi: 10.18632/oncotarget.27794

Figure Lengend Snippet: Patient characteristics at baseline

Article Snippet: A volume of 7.5 mL of peripheral blood from each participant was collected into CellSave Preservative tubes (Menarini Silicon Biosystems), kept at room temperature and processed through CellSearch ® system within 72 h. To this end, the CellSearch ® CXC kit (Menarini Silicon Biosystems) was employed and anti-human CD44v6 Phycoerythrin (PE)-conjugated antibody (clone FAB3660P; R&D Systems, Minneapolis, USA) was added into user-defined marker channel at a concentration of 0.02 μg/mL.

Techniques: Mutagenesis

Journal: Oncotarget

Article Title: Baseline CD44v6-positive circulating tumor cells to predict first-line treatment failure in patients with metastatic colorectal cancer

doi: 10.18632/oncotarget.27794

Figure Lengend Snippet: Clinical response and CTC number or CD44v6 status at baseline

Article Snippet: A volume of 7.5 mL of peripheral blood from each participant was collected into CellSave Preservative tubes (Menarini Silicon Biosystems), kept at room temperature and processed through CellSearch ® system within 72 h. To this end, the CellSearch ® CXC kit (Menarini Silicon Biosystems) was employed and anti-human CD44v6 Phycoerythrin (PE)-conjugated antibody (clone FAB3660P; R&D Systems, Minneapolis, USA) was added into user-defined marker channel at a concentration of 0.02 μg/mL.

Techniques:

Journal: Cancer Research

Article Title: Osteopontin Promotes Integrin Activation through Outside-In and Inside-Out Mechanisms: OPN-CD44VInteraction Enhances Survival in Gastrointestinal Cancer Cells

doi: 10.1158/0008-5472.can-06-3625

Figure Lengend Snippet: Figure 3. Engagement of CD44 containing exon V6- or V7-encoded sequences by OPN enhances cell survival. A, binding of CD44-expressing cells to OPN. Subconfluent AZ521 cells ectopically expressing designated CD44 isoforms were grown in serum-free medium for 24 h and replated on dishes precoated with OPN (20 Ag/mL) or 1% BSA and allowed to adhere for 30 min. The percentage of cell adhesion was calculated as described in Materials and Methods. B, cell binding to OPN in the presence of RGD peptides and anti-CD44V6 antibody. Subconfluent AZ521/CD44 transfectants were trypsinized, pretreated with RGD peptide (10 Amol/L) or anti-CD44V6 antibody (10 Ag/mL) for 1 h at 37jC, and replated on dishes precoated with OPN for 30 min, and cell adherence was measured. C and D, OPN-mediated survival in CD44-expressing cells. Subconfluent AZ521/CD44 clones (C) and HT29 (D) cells were incubated in serum-free medium with or without added OPN or OPN(RGE) for 4 h and subjected to UV irradiation followed by apoptosis assay as described in the legend of Fig. 2. Columns, mean of three separate experiments; bars, SD. *, P < 0.05, Student’s t test; **, P < 0.01, Student’s t test. E, knockdown of the expression of V6-containing CD44 suppresses OPN-mediated survival. HT29 cells were transfected with CD44 V6-specific shRNA (pSuper-V6) or control shRNA (pSuper), and individual cell clones harboring integrated pSuper-V6 and pSuper control were selected. Top, immunoblot analyses of the selected cell clones using anti-V6 antibody. The subconfluent HT29 cell clones were subjected to UV irradiation at 90 J/m2 in the presence and absence of OPN and subjected to apoptosis assay.

Article Snippet: Mouse monoclonal antibody (mAb) against human CD44V6 (2F10) was from R&D Systems (Minneapolis, MN).

Techniques: Binding Assay, Expressing, Clone Assay, Incubation, Irradiation, Apoptosis Assay, Knockdown, Transfection, shRNA, Control, Western Blot

Journal: Cancer Research

Article Title: Osteopontin Promotes Integrin Activation through Outside-In and Inside-Out Mechanisms: OPN-CD44VInteraction Enhances Survival in Gastrointestinal Cancer Cells

doi: 10.1158/0008-5472.can-06-3625

Figure Lengend Snippet: Figure 4. Synergistic effect of CD44 and integrins on cell adhesion to OPN. A, cell binding to OPN correlates to CD44V expression. AZ521 cells were transiently transfected with increasing amounts of pcDNA-CD44V6 as indicated, and cell attachment to OPN in the presence of RGD peptides (10 Amol/L), anti-V6 antibody (5 Ag/mL), or a control IgG (5 Ag/mL) was determined. B, cell binding to OPN correlates to OPN concentration. AZ521 cells were transiently transfected with 10 Ag of pcDNA-CD44V6 and reseeded to plates precoated with OPN (5, 10, 15, and 20 Ag/mL) in the presence of RGD peptides, anti-V6 antibody, or a control IgG in cell binding assay. C, cell binding to OPN is suppressed by anti-integrin antibodies. Subconfluent AZ521/CD44V6 cells were pretreated with blocking antibodies (5 Ag/mL) against integrins av, h1 (P4C10), avh3, or a5h1 for 1 h at 37jC and replated on OPN (20 Ag/mL) or 1% BSA for 30 min, and cell adhesion was determined. Columns, mean of three independent experiments; bars, SD. **, P < 0.01, Student’s t test.

Article Snippet: Mouse monoclonal antibody (mAb) against human CD44V6 (2F10) was from R&D Systems (Minneapolis, MN).

Techniques: Binding Assay, Expressing, Transfection, Cell Attachment Assay, Control, Concentration Assay, Cell Binding Assay, Blocking Assay

Journal: Cancer Research

Article Title: Osteopontin Promotes Integrin Activation through Outside-In and Inside-Out Mechanisms: OPN-CD44VInteraction Enhances Survival in Gastrointestinal Cancer Cells

doi: 10.1158/0008-5472.can-06-3625

Figure Lengend Snippet: Figure 5. OPN promotes integrin activation through CD44-dependent and CD44-independent pathways. A and B, activation of h1 integrins. Subconfluent AZ521/CD44V6 (A) and HT29 (B) cells were treated with OPN or OPN(RGE) for 1 h at 37jC and incubated with isotype IgG (black shaded area) or antibody (HUTS-21, 5 Ag/mL) specifically recognizing the active conformation of integrin h1 followed by labeling with Alexa Fluor 488–conjugated secondary antibody. Fluorescence intensity was determined by flow cytometry. Cells preincubated with 2 mmol/L MnCl2 for 30 min at 37jC were included to serve as a positive control. C, effects of RGD peptide and anti-CD44V6 antibody on OPN-induced integrin activation. AZ521/CD44V6 cells were pretreated with RGD peptide (10 Amol/L) and/or anti-CD44V6 antibody for 1 h at 37jC before the incubation with OPN. After incubation with antibody specifically recognizing the active conformation of integrin h1, fluorescence intensity was determined. D, OPN/CD44-induced integrin activation is mediated through Src activation. AZ521/CD44V6 cells were preincubated with RGD peptide (10 Amol/L) and then treated with PP2 (30 Amol/L) for 30 min at 37jC. Alternatively, cells were transfected with a control plasmid or plasmid encoding dominant-negative Src (K297D) and incubated with RGD peptide (10 Amol/L). Cells were then incubated with OPN (10 Ag/mL) for 1 h at 37jC and incubated with antibody specifically recognizing the active conformation of integrin h1 as described above. Y axis, cell numbers that were stained with antibodies in each logarithmic scale of fluorescence amplifier. Similar results were obtained from three independent experiments and a representative histogram is shown. Specific fluorescence intensity (SFI) was calculated as the ratio of the mean fluorescence values obtained with the specific antibody recognizing the active h1 integrin (Act-b1) and the isotype control antibody. Columns, mean of three separate experiments; bars, SD. *, P < 0.05, Student’s t test; **, P < 0.01, Student’s t test.

Article Snippet: Mouse monoclonal antibody (mAb) against human CD44V6 (2F10) was from R&D Systems (Minneapolis, MN).

Techniques: Activation Assay, Incubation, Labeling, Fluorescence, Flow Cytometry, Positive Control, Transfection, Control, Plasmid Preparation, Dominant Negative Mutation, Staining

Journal: Cancer Research

Article Title: Osteopontin Promotes Integrin Activation through Outside-In and Inside-Out Mechanisms: OPN-CD44VInteraction Enhances Survival in Gastrointestinal Cancer Cells

doi: 10.1158/0008-5472.can-06-3625

Figure Lengend Snippet: Figure 6. OPN/CD44V-elicited cell survival is derived from ECM. A, OPN promotes cell adhesion to fibronectin. Subconfluent HT29, AZ521/Mock, and AZ521/CD44V6 cells were incubated with or without OPN (10 Ag/mL) for 1 h at 37jC, replated on fibronectin (FN), poly-D-lysine (PDL), or BSA, and allowed to adhere for 30 min. B, OPN-elicited cell adhesion is suppressed by antibodies against CD44V6 and integrins (5 Ag/mL). AZ521/CD44V6 cells were trypsinized and pretreated with blocking antibodies as indicated for 1 h at 37jC before the incubation with OPN. Cell adhesion to fibronectin was determined as described in Materials and Methods. C, anti-integrin h1 blocking antibody blocks OPN/CD44V- mediated cell survival. Subconfluent AZ521/Mock and AZ521/CD44 clones were pretreated with or without blocking antibody against integrin h1 (P4C10) for 1 h at 37jC and further incubated in the same medium supplemented with or without OPN. Cells were UV irradiated and subjected to apoptosis assay as described above. D, fibronectin supports OPN-mediated antiapoptosis, which is blocked by the expression of the dominant-negative FAK. Subconfluent AZ521/CD44V6 cells were transfected with a control plasmid or plasmid encoding FRNK, cultured in serum-free medium for 24 h, and replated on fibronectin or poly-D-lysine in the presence of medium with or without OPN (10 Ag/mL) for 4 h. The cells were then subjected to UV irradiation followed by apoptosis assay as described above. Columns, mean of three separate experiments; bars, SD. **, P < 0.01, Student’s t test.

Article Snippet: Mouse monoclonal antibody (mAb) against human CD44V6 (2F10) was from R&D Systems (Minneapolis, MN).

Techniques: Derivative Assay, Incubation, Blocking Assay, Clone Assay, Irradiation, Apoptosis Assay, Expressing, Dominant Negative Mutation, Transfection, Control, Plasmid Preparation, Cell Culture

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