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: iScience

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

doi: 10.1016/j.isci.2021.102664

Figure Lengend Snippet:

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: Control, Recombinant, SYBR Green Assay, Staining, shRNA, Software

Journal: Theranostics

Article Title: Identification of novel CD44v6-binding peptides that block CD44v6 and deliver a pro-apoptotic peptide to tumors to inhibit tumor growth and metastasis in mice

doi: 10.7150/thno.50564

Figure Lengend Snippet: Screening of a phage peptide library and phage cell-binding ELISA to identify CD44v6-binding peptides. (A) Experimental schemes for phage peptide library screening. (B) Immunofluorescence staining of CD44v6 with an anti-CD44v6 antibody (red) in HEK 293T cells transfected or not with a GFP-tagged CD44v6 expression vector (green). DAPI was used for nuclear staining (blue). Scale bars = 40 µm. (C) Western blotting analysis of CD44v6 expression in non-transfected (-) and transfected HEK 293T cells at 24 and 48 h after transfection. (D) Western blotting analysis of CD44v6 expression in tumor cells. (E) Enrichment of phage titers during screening rounds. After each round, the phage titers (plaque-forming units; pfu) were measured by plaque assays. Numbers represent the fold ratios relative to the first round. (F) The phage cell-binding ELISA of individual phage clones was performed using HEK 293T cells transfected or not with a CD44v6 expression vector, MDA-MB231 cells, and MCF7 cells. *, P < 0.05; **, P < 0.01; ***, P < 0.001; n.s, not significant by one-way ANOVA.

Article Snippet: Cells were seeded in each well of an 8-well chamber slide at a density of 1 × 10 5 cells per well, incubated overnight, and then treated with 1% BSA at room temperature for 1 h. Subsequently, the cells were incubated with FITC-conjugated peptides (25 μM) at 4 °C for 1 h, blocked with BSA, fixed, and incubated with an anti-human CD44v6 mouse monoclonal antibody (clone no. VFF-7, 1:100 dilution; Santa Cruz Biotechnology, Dallas, TX) or a rabbit anti-CD44v6 polyclonal antibody (1:100 dilution; Merck).

Techniques: Binding Assay, Enzyme-linked Immunosorbent Assay, Library Screening, Immunofluorescence, Staining, Transfection, Expressing, Plasmid Preparation, Western Blot, Clone Assay

Journal: Theranostics

Article Title: Identification of novel CD44v6-binding peptides that block CD44v6 and deliver a pro-apoptotic peptide to tumors to inhibit tumor growth and metastasis in mice

doi: 10.7150/thno.50564

Figure Lengend Snippet: NLN and NEW bind selectively to CD44v6-overexpressing cells and the CD44v6 protein . (A) Cellular binding of FITC-labeled NLN and NEW (green, 25 µM) and staining of CD44v6 (red) in CD44v6-high MDA-MB231, 4T1, and Panc-1 cells and CD44v6-low MCF7 cells. Nuclei were stained with DAPI (blue). Scale bars = 20 µm. (B) Mean fluorescence intensities (MFIs) of FITC-labeled NLN or NEW (25 µM) bound to MDA-MB231, 4T1, Panc-1 and MCF7 cells. Data represent the mean MFIs ± standard errors (S.E.) of peptide-bound cells from three separate experiments. **, P < 0.01; n.s, not significant compared with the control peptide (Ctrl) by one-way ANOVA. (C) Cellular binding of TAMRA-labeled NLN and NEW (red, 25 µM) and expression of CD44v6 (green) in HEK 293T cells transfected or not with a GFP-tagged CD44v6 expression vector. Nuclei were stained with DAPI (blue). Scale bars = 20 µm. (D-E) SPR analysis of the binding affinity (K D value) of NLN and NEW to recombinant CD44v6-Fc (D) and CD44-Fc (E) proteins. Inset are a binding plot for the peptides. RU, resonance unit.

Article Snippet: Cells were seeded in each well of an 8-well chamber slide at a density of 1 × 10 5 cells per well, incubated overnight, and then treated with 1% BSA at room temperature for 1 h. Subsequently, the cells were incubated with FITC-conjugated peptides (25 μM) at 4 °C for 1 h, blocked with BSA, fixed, and incubated with an anti-human CD44v6 mouse monoclonal antibody (clone no. VFF-7, 1:100 dilution; Santa Cruz Biotechnology, Dallas, TX) or a rabbit anti-CD44v6 polyclonal antibody (1:100 dilution; Merck).

Techniques: Binding Assay, Labeling, Staining, Fluorescence, Control, Expressing, Transfection, Plasmid Preparation, Recombinant

Journal: Theranostics

Article Title: Identification of novel CD44v6-binding peptides that block CD44v6 and deliver a pro-apoptotic peptide to tumors to inhibit tumor growth and metastasis in mice

doi: 10.7150/thno.50564

Figure Lengend Snippet: Cellular binding of NLN and NEW is mediated by CD44v6. (A) Western blotting analysis of CD44v6 and CD44 levels in MDA-MB231 cells after the knockdown (KD) of CD44v6 gene expression for 24, 48, and 72 h in cells treated with CD44v6 siRNA or control siRNA. GAPDH is used as a control protein. (B) The MFI of wild-type (WT) and CD44v6 KD MDA-MB231 cells bound to FITC-labeled NLN and NEW (25 µM). Data are shown as mean MFIs ± S.E. of peptide-bound cells from three separate experiments. *, P < 0.05 by Student's t -test. (C) Confocal microscopic images of WT and KD MDA-MB231 cells bound with FITC-labeled NLN and NEW (green, 25 µM) and stained with an anti-CD44v6 antibody (red) and DAPI (blue). Scale bars = 20 µm. (D) Competitive binding of FITC-labeled NLN and NEW (10 µM) following pre-treatment with anti-CD44v6 and anti-CD44 antibodies and IgG control in MDA-MB231 cells. The mean MFIs ± S.E. of peptide-bound cells from three separate experiments are shown. *, P < 0.05; **, P < 0.01; ***, P < 0.001; n.s, not significant by one-way ANOVA. (E) Competitive binding of an anti-CD44v6 antibody following pre-treatment with NLN and NEW (50 µM) in MDA-MB231 cells. ***, P < 0.001; n.s, not significant compared with untreated control by one-way ANOVA. (F) Pull-down assay of CD44v6 using biotin-labeled NLN and NEW and streptavidin beads, followed by immunoblotting with anti-CD44v6, anti-c-Met, and anti-CD44 antibodies. (G) Pull-down assays of the recombinant human CD44v6-Fc protein using biotin-labeled NLN and NEW and streptavidin followed by immunoblotting (IB) with anti-CD44v6 antibody. Ctrl, control peptide.

Article Snippet: Cells were seeded in each well of an 8-well chamber slide at a density of 1 × 10 5 cells per well, incubated overnight, and then treated with 1% BSA at room temperature for 1 h. Subsequently, the cells were incubated with FITC-conjugated peptides (25 μM) at 4 °C for 1 h, blocked with BSA, fixed, and incubated with an anti-human CD44v6 mouse monoclonal antibody (clone no. VFF-7, 1:100 dilution; Santa Cruz Biotechnology, Dallas, TX) or a rabbit anti-CD44v6 polyclonal antibody (1:100 dilution; Merck).

Techniques: Binding Assay, Western Blot, Knockdown, Gene Expression, Control, Labeling, Staining, Pull Down Assay, Recombinant

Journal: Theranostics

Article Title: Identification of novel CD44v6-binding peptides that block CD44v6 and deliver a pro-apoptotic peptide to tumors to inhibit tumor growth and metastasis in mice

doi: 10.7150/thno.50564

Figure Lengend Snippet: NLN and NEW inhibit HGF-induced c-Met internalization, c-Met phosphorylation, and cell migration and invasion in MDA-MB231 breast tumor cells. (A) A confocal microscopic Z-section analysis of the internalization of NLN and NEW, and CD44v6 into MDA-MB231 cells. Cells were incubated with FITC-labeled peptides (green, 10 µM) at 37 °C for 10, 30, and 60 min and stained with an anti-CD44v6 antibody (red). Nuclei were stained with DAPI (blue), and images were merged. Numbers in the merged images indicate the distances from the cell surfaces in µm. Scale bars = 10 µm. (B) Confocal microscopic analysis of c-Met (red) in MDA-MB231 cells after pre-treatment with NLN and NEW (10 µM) at 37 °C for 10 min and subsequent treatment with 25 ng/mL HGF for 30 or 60 min. Arrows indicate cytoplasmic c-Met. Nuclei were stained with DAPI (blue). Scale bars = 10 µm. (C) Western blotting analysis of c-Met and Erk phosphorylation in MDA-MB231 cells pre-treated or not with NLN and NEW (20 µM) for 10 min and subsequently treated with 25 ng/mL HGF for 10 min. (D) Phosphorylated c-Met and Erk protein levels normalized by c-Met and Erk total protein levels in MDA-MB231 cells. (E-F) Transwell migration (E) and invasion (F) assays of MDA-MB231 cells pre-treated or not with NLN and NEW (20 µM) for 10 min and subsequently treated with 25 ng/mL HGF for 10 min, followed by incubation for 24 h. Scale bars = 20 µm. Graphs (right panels) represent the quantification of the cell numbers in ten randomly selected fields. Data are shown as the means ± S.E. of three independent experiment. ***, P < 0.001; n.s, not significant compared with HGF by one-way ANOVA.

Article Snippet: Cells were seeded in each well of an 8-well chamber slide at a density of 1 × 10 5 cells per well, incubated overnight, and then treated with 1% BSA at room temperature for 1 h. Subsequently, the cells were incubated with FITC-conjugated peptides (25 μM) at 4 °C for 1 h, blocked with BSA, fixed, and incubated with an anti-human CD44v6 mouse monoclonal antibody (clone no. VFF-7, 1:100 dilution; Santa Cruz Biotechnology, Dallas, TX) or a rabbit anti-CD44v6 polyclonal antibody (1:100 dilution; Merck).

Techniques: Phospho-proteomics, Migration, Incubation, Labeling, Staining, Western Blot

Journal: Theranostics

Article Title: Identification of novel CD44v6-binding peptides that block CD44v6 and deliver a pro-apoptotic peptide to tumors to inhibit tumor growth and metastasis in mice

doi: 10.7150/thno.50564

Figure Lengend Snippet: In vivo whole-body fluorescence imaging of NLN and NEW homing to MDA-MB231 breast tumor in mice. (A) In vivo whole-body fluorescence imaging of the homing of Flamma 675 NIR dye-labeled NLN, NEW, or control peptide to tumors at 1-6 h after injection into BALB/c nude mice. Dotted circles represent the tumor region. The scale bar indicates the normalized fluorescent intensity. (B) Ex vivo imaging of the accumulation of Flamma 675 NIR dye-labeled NLN, NEW, or control peptide in the tumors and other organs isolated from mice 6 h after peptide injection. The scale bar indicates the normalized fluorescence intensity. T, tumor; H, heart; L, lung; Li, liver; S, spleen; K, kidney. (C) Quantification of the ex vivo fluorescence intensities in the tumor and organs. Data are shown as the means ± S.E. ( n = 3/group). **, P < 0.01; ***, P < 0.001 compared with the control peptide by one-way ANOVA. (D) Co-localization of CD44v6 (green) with NLN and NEW (red) in tumor tissue sections. Nuclei were counter-stained with DAPI (blue). Scale bars = 20 µm. Ctrl, control peptide.

Article Snippet: Cells were seeded in each well of an 8-well chamber slide at a density of 1 × 10 5 cells per well, incubated overnight, and then treated with 1% BSA at room temperature for 1 h. Subsequently, the cells were incubated with FITC-conjugated peptides (25 μM) at 4 °C for 1 h, blocked with BSA, fixed, and incubated with an anti-human CD44v6 mouse monoclonal antibody (clone no. VFF-7, 1:100 dilution; Santa Cruz Biotechnology, Dallas, TX) or a rabbit anti-CD44v6 polyclonal antibody (1:100 dilution; Merck).

Techniques: In Vivo, Fluorescence, Imaging, Labeling, Control, Injection, Ex Vivo, Isolation, Staining

Journal: Molecular Pharmacology

Article Title: A Novel Gemini Vitamin D Analog Represses the Expression of a Stem Cell Marker CD44 in Breast Cancer

doi: 10.1124/mol.110.068403

Figure Lengend Snippet: Effects of BXL0124 on CD44 protein expression level in MCF10DCIS.com xenograft tumors in vivo. A, MCF10DCIS.com xenografted nu/nu mice were treated with DMSO or BXL0124 (0.1 μg/kg body weight) orally, and mammary tumors were collected at necropsy. Mammary tumors (n = 5) were pooled into either the control group or BXL0124-treated group for Western blot analysis against CD44, CD44v3, CD44v6, PCNA, and β-actin. B, a representative H&E staining in mammary tumors from MCF10DCIS.com xenografted nu/nu mice is shown (original magnification, 400×). C, a representative immunostaining against CD44 and PCNA in mammary tumors from MCF10DCIS.com xenografted nu/nu mice is shown (original magnification, 400×). Three mammary tumors from each group were selected and three representative areas from each tumor were analyzed for the expression of CD44 and PCNA. The mammary tumors immunostained against CD44 and PCNA were scored by four different levels of staining intensity and quantified by using Aperio Scan Scope. The data are presented as the mean ± S.D. (statistical analysis: *, p < 0.05; **, p < 0.01).

Article Snippet: The procedure was described previously ( Lee et al., 2009 ), and the primary antibody, CD44, which recognizes all CD44 splicing variants, was from Santa Cruz Biotechnology (Santa Cruz, CA); CD44 containing variant domain 3 (CD44v3) and CD44 containing variant domain 6 (CD44v6) was from R&D Systems (Minneapolis, MN); vitamin D receptor (VDR) was from Thermo Fisher Scientific (Waltham, MA); β-actin was from Sigma-Aldrich; and secondary antibodies were from Santa Cruz Biotechnology.

Techniques: Expressing, In Vivo, Control, Western Blot, Staining, Immunostaining

Journal: Journal of Experimental & Clinical Cancer Research : CR

Article Title: Musashi2 contributes to the maintenance of CD44v6+ liver cancer stem cells via notch1 signaling pathway

doi: 10.1186/s13046-019-1508-1

Figure Lengend Snippet: MSI2 was closely related to CD44v6 and predicted poor prognosis. a Western blot analysis of CD44v6 and MSI2 protein levels in HCC tissues and adjacent non-tumor tissues selected randomly. β-actin was used as a normalized control. b Analysis of CD44v6 protein levels relative to β-actin in 28 pairs of HCC tissues and adjacent non-tumor tissues ( n = 28, * p = 0.0179, t test). c Analysis of MSI2 protein levels relative to β-actin in 28 pairs of HCC tissues and adjacent non-tumor tissues ( n = 28, ** p = 0.0012, t test). d and e Kaplan–Meier survival analysis of overall survival and disease-free survival were compared according to the expression levels of CD44v6 in HCC tissues. Patients with high CD44v6 expression had shorter overall survival ( d , median survival = 24 months Vs. 36 months, log-rank test, n = 82, * p = 0.0486) and disease-free survival ( e , median survival = 20 months Vs. 36 months, log-rank test, n = 82, ** p = 0.0426). f and g Kaplan–Meier survival analysis of overall survival and disease-free survival were compared according to the expression levels of MSI2 in HCC tissues. Patients with high MSI2 expression had shorter overall survival ( f , median survival = 18 months Vs. 40 months, log-rank test, n = 82, **** p <0.0001) and disease-free survival ( g , median survival = 12 months Vs. 38 months, log-rank test, n = 82, **** p <0.0001). h Representative images of IHC staining of MSI2 in tumor and adjacent non-tumor tissues. And analysis of MSI2 expression in tumor and adjacent non-tumor tissues by paired t test. Scale bars: 200 μm and 50 μm. i The expression of MSI2 and CD44v6 in tumor tissues from the same HCC patient were analyzed by IHC staining and found that MSI2 was positively correlated with CD44v6 ( n = 82, r = 0.6093, **** p <0.0001, Pearson’s correlation). Scale bars: 200 μm and 50 μm

Article Snippet: The cell pellets were suspended in FACS buffer (PBS containing 0.5% fetal bovine serum (FBS)) and then labeled with PE-conjugated human CD44v6 antibody (Clone 2F10, R&D Systems, #FAB3660P) at 4 °C for 30 min.

Techniques: Western Blot, Control, Expressing, Immunohistochemistry

Journal: Journal of Experimental & Clinical Cancer Research : CR

Article Title: Musashi2 contributes to the maintenance of CD44v6+ liver cancer stem cells via notch1 signaling pathway

doi: 10.1186/s13046-019-1508-1

Figure Lengend Snippet: CD44v6+ HCC cells possessed characteristics of cancer stem cells. a Representative images of spheres and histogram analysis in indicated cells. CD44v6+ SNU-398 cells and MHCC-97 h cells processed enhanced self-renewal property than CD44v6- cells. Scale bar, 200 μm. b and c Representative images of transwell migration and invasion in indicated cells. Transwell migration and invasion assays showed that CD44v6+ SNU-398 cells and MHCC-97 h cells displayed higher migratory and invasion capacity than CD44v6- cells. Scale bar, 200 μm. d Representative images of colony formation assays in indicated cells. Colony formation assays demonstrated that CD44v6+ SNU-398 cells and MHCC-97 h cells exhibited higher proliferation and colony formation ability. e CD44v6+ and CD44v6- SNU-398 cells and MHCC-97 h cells were treated with Sorafenib for 24 h and evaluated by CCK8 toxic assay. It showed that CD44v6+ cells were more resistant to Sorafenib than CD44v6- cells. f The expression of cancer stemness-related genes, including Nanog, Oct4 and Sox2 in CD44v6+ and CD44v6- SNU-398 cells. β-actin was used as a normalized control. It showed that elevated stemness-related genes expressed in CD44v6+ SNU-398 cells than CD44v6- cells. g Efficiency of tumor formation of CD44v6+ cells and CD44v6- cells isolated from SNU-398 cell line. Number of injected cells: 1 × 10 5 , 1 × 10 4 , 1 × 10 3 . n = 12. For statistical analysis, * p < 0.05, ** p < 0.01 and *** p < 0.001, t test

Article Snippet: The cell pellets were suspended in FACS buffer (PBS containing 0.5% fetal bovine serum (FBS)) and then labeled with PE-conjugated human CD44v6 antibody (Clone 2F10, R&D Systems, #FAB3660P) at 4 °C for 30 min.

Techniques: Migration, Expressing, Control, Isolation, Injection

Journal: Journal of Experimental & Clinical Cancer Research : CR

Article Title: Musashi2 contributes to the maintenance of CD44v6+ liver cancer stem cells via notch1 signaling pathway

doi: 10.1186/s13046-019-1508-1

Figure Lengend Snippet: Knockdown of MSI2 significantly attenuated the stemness properties of CD44v6+ LCSC. a MSI2 expression levels were tested in CD44v6+ HCC cells and CD44v6- HCC cells by western blot in SNU-398 and MHCC-97 h cell lines. b Immunofluorescence images of CD44v6+ SNU-398 cells and CD44v6- SNU-398 cells for localization of MSI2 (red) and CD44v6 (green). Histogram analysis of the relative fluorescence intensity of CD44v6 and MSI2 in CD44v6+ cells and CD44v6- cells. Scale bar, 50 μm. c Representative images of spheres and histogram analysis in indicated cells. The inhibition of MSI2 decreased self-renewal property in vitro in CD44v6+ LCSCs, Scale bar, 200 μm. d and e Transwell migration and invasion assays showed that knockdown of MSI2 decreased the migration and invasion of CD44v6+ cells. Scale bar, 200 μm. f Colony formation assays showed that the ability of cell proliferation and colony formation of CD44v6+ cells was inhibited when MSI2 was down regulated. g 1 × 10 5 of MSI2 shRNA1 cells and the corresponding controls were injected into the left lobes of liver. Bioluminescence signals from MSI2 shRNA1 groups were weaker than that from corresponding control groups. Red arrows indicated the site of tumor formation. h Efficiency of tumor formation of MSI2 shRNA cells and corresponding control cells. Number of injected cells: 1 × 10 5 . n = 8. Black arrow means Mock group, blue arrow means NC group, red arrow means MSI2 shRNA1 group, and orange arrow means MSI2 shRNA2 group. i The expression of cancer stemness-related genes, including Nanog, Oct4 and Sox2 in MSI2 shRNA cells compared with corresponding control. β-actin was used as a normalized control. It showed the expression of stemness-related genes were decreased when MSI2 was knockdown in CD44v6+ LCSCs. For statistical analysis, * p < 0.05, ** p < 0.01, *** p < 0.001 and **** p < 0.0001, t test

Article Snippet: The cell pellets were suspended in FACS buffer (PBS containing 0.5% fetal bovine serum (FBS)) and then labeled with PE-conjugated human CD44v6 antibody (Clone 2F10, R&D Systems, #FAB3660P) at 4 °C for 30 min.

Techniques: Knockdown, Expressing, Western Blot, Immunofluorescence, Fluorescence, Inhibition, In Vitro, Migration, Injection, Control, shRNA

Journal: Journal of Experimental & Clinical Cancer Research : CR

Article Title: Musashi2 contributes to the maintenance of CD44v6+ liver cancer stem cells via notch1 signaling pathway

doi: 10.1186/s13046-019-1508-1

Figure Lengend Snippet: The inhibition of Notch1 signaling pathway attenuated the stemness properties of CD44v6+ LCSCs. a External dataset from starBase v3.0 project with 374 HCC samples and 50 normal samples was used to analyze the expression of Notch1. The result showed that Notch1 was higher in HCC patients’ samples than that in normal samples (1.77 fold, FDR = 0.0017). b The core components of Notch1 signaling including Notch1 receptor, cleaved Notch1 (NICD), Hey1 and Hes1 were tested in CD44v6+ LCSCs and CD44v6- HCC cells by western blot in SNU-398 cell lines. Western blot showed that CD44v6+ SNU-398 cells expressed more Notch1 signaling pathway key factors. β-actin was used as a normalized control. c Representative images of spheres and histogram analysis in indicated cells. The inhibition of Notch1 decreased self-renewal property in vitro in CD44v6+ LCSCs, Scale bar, 200 μm. d and e Transwell migration and invasion assay showed that knockdown Notch1 decreased the migration and invasion capacity of CD44v6+ cells. Scale bar, 200 μm. f Colony formation assays showed that the ability of cell proliferation and colony formation of CD44v6+ cells was inhibited when Notch1 was down regulated. g and h Efficiency of tumor formation of Notch1 shRNA cells and the corresponding controls. Right flanks of mice were injected with control CD44v6+ cells while left flanks were injected with Notch1 shRNA cells. Number of injected cells: 1 × 10 5 . n = 5. Data are expressed as mean ± SD (error bars). ** p < 0.01, *** p < 0.001 and **** p < 0.0001, t test

Article Snippet: The cell pellets were suspended in FACS buffer (PBS containing 0.5% fetal bovine serum (FBS)) and then labeled with PE-conjugated human CD44v6 antibody (Clone 2F10, R&D Systems, #FAB3660P) at 4 °C for 30 min.

Techniques: Inhibition, Expressing, Western Blot, Control, In Vitro, Migration, Invasion Assay, Knockdown, shRNA, Injection

Journal: Journal of Experimental & Clinical Cancer Research : CR

Article Title: Musashi2 contributes to the maintenance of CD44v6+ liver cancer stem cells via notch1 signaling pathway

doi: 10.1186/s13046-019-1508-1

Figure Lengend Snippet: MSI2 maintained the stemness properties of CD44v6+ LCSCs via activating Notch1 signaling pathway. a External dataset from starBase v3.0 project with 374 HCC samples and 50 normal samples was used to analyze the correlation of MSI2 and Notch1. The results showed that MSI2 was positively related to Notch1 in clinical-pathology ( r = 0.458, p = 8.02e-21). b Immunofluorescence images of MSI2 (red) and Notch1 (green) in HCC tissues. MSI2 was co-localized with Notch1 in HCC tissues. Scale bar, 50 μm. c Immunofluorescence images of CD44v6+ SNU-398 cells and CD44v6- cells for localization of MSI2 (red) and Notch1 (green). Histogram analysis of the relative fluorescence intensity of MSI2 and Notch1 in CD44v6+ cells and CD44v6- cells. Scale bar, 50 μm. d. Silencing MSI2 decreased the expression of Notch1 receptor and Notch1 pathway target genes in CD44v6+ SNU-398 cells. β-actin was used as a normalized control. e Western blot showed that overexpression of MSI2 increased the expression of Notch1 receptor and Notch1 pathway target genes in CD44v6- SNU-398 cells. β-actin was used as a normalized control

Article Snippet: The cell pellets were suspended in FACS buffer (PBS containing 0.5% fetal bovine serum (FBS)) and then labeled with PE-conjugated human CD44v6 antibody (Clone 2F10, R&D Systems, #FAB3660P) at 4 °C for 30 min.

Techniques: Immunofluorescence, Fluorescence, Expressing, Control, Western Blot, Over Expression

Journal: Journal of Experimental & Clinical Cancer Research : CR

Article Title: Musashi2 contributes to the maintenance of CD44v6+ liver cancer stem cells via notch1 signaling pathway

doi: 10.1186/s13046-019-1508-1

Figure Lengend Snippet: MSI2 activated Notch1 signaling through LFNG in CD44v6+ LCSCs. a A Notch RT 2 PCR Array was used to determine mRNA expression profiles between MSI2 shRNA and control CD44v6+ LCSCs. b Hypothesis diagram of MSI2 regulates Notch1 signaling pathway. c Relative mRNA of the most significantly regulated genes were detected by RT-PCR in MSI2 shRNA1 group and the corresponding control group. d Western blot showed that silencing MSI2 decreased the expression of LFNG in CD44v6+ cells while overexpression of MSI2 increased the expression of LFNG in CD44v6- cells. β-actin was used as a normalized control. e Efficiency of tumor formation of LFNG shRNA1 cells and the corresponding controls. Number of injected cells: 1 × 10 5 . n = 4. f Representative images of spheres and histogram analysis in indicated cells. The inhibition of LFNG decreased self-renewal property in vitro in CD44v6+ LCSCs, Scale bar, 200 μm. g The expression of cancer stemness-related genes, including Nanog, Oct4 and Sox2 in LFNG shRNA cells compared with corresponding controls. β-actin was used as a normalized control. The inhibition of LFNG decreased the expression of stemness-related genes in CD44v6+ LCSCs. h Silencing LFNG in CD44v6+ HCC cells decreased the expression of key components of Notch1 pathway (including Notch1, NICD, Hey1 and Hes1) but MSI2 had no significant change. β-actin was used as a normalized control. i Key components of Notch1 signaling reduction caused by MSI2 knockdown could be rescued by LFNG overexpression in CD44v6+ LCSCs. For statistical analysis, * p < 0.05, ** p < 0.01, *** p < 0.001 and **** p < 0.0001, t test

Article Snippet: The cell pellets were suspended in FACS buffer (PBS containing 0.5% fetal bovine serum (FBS)) and then labeled with PE-conjugated human CD44v6 antibody (Clone 2F10, R&D Systems, #FAB3660P) at 4 °C for 30 min.

Techniques: Expressing, shRNA, Control, Reverse Transcription Polymerase Chain Reaction, Western Blot, Over Expression, Injection, Inhibition, In Vitro, Knockdown

Journal: Journal of Experimental & Clinical Cancer Research : CR

Article Title: Musashi2 contributes to the maintenance of CD44v6+ liver cancer stem cells via notch1 signaling pathway

doi: 10.1186/s13046-019-1508-1

Figure Lengend Snippet: MSI2 bound to LFNG mRNA and protein directly. a Schematic representation of MSI2 molecular interaction domains for interaction with RNA and with proteins. RRM, RNA recognition motif; PPD, protein-protein binding domain. b CD44v6+ cells and CD44v6- cells were isolated from SNU-398 cells. CD44v6+ cells were transfected with MSI2 shRNA1 or corresponding control virus, CD44v6- cells were transfected with Lv MSI2 or corresponding control virus. Lysates were precipitated with anti-MSI2 antibody and then immunoblotted (WB) for LFNG. Protein expression of MSI2 and LFNG was also analyzed. c CD44v6+ cells were transfected with MSI2 shRNA1 or corresponding control virus, CD44v6- cells were transfected with Lv MSI2 or corresponding control virus. RIP assays using anti-MSI2 antibody showed that MSI2 interacted with LFNG. The results of agarose electrophoresis of the PCR products were shown. d Schematic illustration the mechanism by which MSI2 activating Notch1 signaling pathway by binding to LFNG mRNA and protein directly

Article Snippet: The cell pellets were suspended in FACS buffer (PBS containing 0.5% fetal bovine serum (FBS)) and then labeled with PE-conjugated human CD44v6 antibody (Clone 2F10, R&D Systems, #FAB3660P) at 4 °C for 30 min.

Techniques: Protein Binding, Isolation, Transfection, Control, Virus, Expressing, Electrophoresis, Binding Assay

Journal: Journal of Experimental & Clinical Cancer Research : CR

Article Title: Musashi2 contributes to the maintenance of CD44v6+ liver cancer stem cells via notch1 signaling pathway

doi: 10.1186/s13046-019-1508-1

Figure Lengend Snippet: Correlation of the expression of CD44v6 and MSI2 with clinical-pathological variables in HCC patients

Article Snippet: The cell pellets were suspended in FACS buffer (PBS containing 0.5% fetal bovine serum (FBS)) and then labeled with PE-conjugated human CD44v6 antibody (Clone 2F10, R&D Systems, #FAB3660P) at 4 °C for 30 min.

Techniques: Expressing

Journal: British Journal of Cancer

Article Title: Butyrate down-regulates CD44 transcription and liver colonisation in a highly metastatic human colon carcinoma cell line

doi: 10.1038/sj.bjc.6600574

Figure Lengend Snippet: Expression of CD44v6 in HM7 cells. Immunocytochemistry: ( A ) (Magnification (M): ×40): HM7 cells strongly expressed CD44v6. ( B ) (M: ×40): In Butyrate-treated HM7 cells a reduced expression of CD44v6 is observed. ( C ) (M: ×40): Addition of the control protein concomitantly with the anti-CD44v6 antibody almost completely abrogated the binding capacity of the antibody to HM7 cellular antigens. Western immunoblotting: ( D ): The CD44v6 antibody distinguished CD44v6 isoforms within the range of 97–220 kDa either in the control protein as well as in HM7 cells incubated under control conditions (CT) or in butyrate-treated HM7 cells (NB). Representative results from four similar experiments.

Article Snippet: The CD44 protein was reacted with mouse monoclonal anti-human CD44v6 antibody (VFF-18 clone, BMS125, Bender MedSystems, Austria) and visualised by enhanced chemiluminescence kit (ECL, Amersham).

Techniques: Expressing, Immunocytochemistry, Control, Binding Assay, Western Blot, Incubation

Journal: British Journal of Cancer

Article Title: Butyrate down-regulates CD44 transcription and liver colonisation in a highly metastatic human colon carcinoma cell line

doi: 10.1038/sj.bjc.6600574

Figure Lengend Snippet: CD44 gene transcription. Nuclear run-off analysis of CD44s and CD44v6 in control HM7 cells (CT) or treated with butyrate (NB) ( A ). HM7 cells transcribed the CD44v6 splice variant. The same results were found for the constant isoform (CD44s). Butyrate inhibited the transcription of both isoforms. RNA loading was verified with β-actin probe. Effect of butyrate treatment on the expression of different splice variants was measured by RT–PCR analysis using primers 10F/17R ( B ), 3F/11R ( C ) and 3F/17R ( D ). Similar results were obtained in 3–5 different experiments.

Article Snippet: The CD44 protein was reacted with mouse monoclonal anti-human CD44v6 antibody (VFF-18 clone, BMS125, Bender MedSystems, Austria) and visualised by enhanced chemiluminescence kit (ECL, Amersham).

Techniques: Control, Variant Assay, Expressing, Reverse Transcription Polymerase Chain Reaction

Journal: British Journal of Cancer

Article Title: Butyrate down-regulates CD44 transcription and liver colonisation in a highly metastatic human colon carcinoma cell line

doi: 10.1038/sj.bjc.6600574

Figure Lengend Snippet: Effect of butyrate and CD44v6-antisense oligonucleotide treatments. CD44v6 isoform including exon 10 was obtained by RT–PCR, using 10F/17R primers in HM7 cells treated with antisense oligonucleotide. Antisense treatment caused a reduction in the mRNA expression of the CD44v6 splice variant. CT: 3, 4 and 5 μg total RNA from control HM7 cells (CT) or CD44v6-antisense treated cells were used in the RT–PCR assays.

Article Snippet: The CD44 protein was reacted with mouse monoclonal anti-human CD44v6 antibody (VFF-18 clone, BMS125, Bender MedSystems, Austria) and visualised by enhanced chemiluminescence kit (ECL, Amersham).

Techniques: Reverse Transcription Polymerase Chain Reaction, Expressing, Variant Assay, Control

Journal: British Journal of Cancer

Article Title: Butyrate down-regulates CD44 transcription and liver colonisation in a highly metastatic human colon carcinoma cell line

doi: 10.1038/sj.bjc.6600574

Figure Lengend Snippet: Liver colonisation model. Livers from athymic nude mice 6 weeks after splenic injection of HM7 control cells ( A ), butyrate-treated cells ( B ) CD44v6-antisense treated cells ( C ) compared to non-injected animals ( D ). Results are representative of three independent identical experiments.

Article Snippet: The CD44 protein was reacted with mouse monoclonal anti-human CD44v6 antibody (VFF-18 clone, BMS125, Bender MedSystems, Austria) and visualised by enhanced chemiluminescence kit (ECL, Amersham).

Techniques: Injection, Control

Journal: International Journal of Clinical and Experimental Pathology

Article Title: Dysregulation of CD44v6 may lead to recurrent spontaneous abortion by inhibiting the proliferation and migration of trophoblast cells

doi:

Figure Lengend Snippet: CD44v6 expression is decreased in villi from RSA patients compared to those from normal pregnant women and inhibition of CD44v6 with CD44v6-specifc siRNA suppresses expression of NF-κB in HTR-8/SVneo cells. A. Representative images of CD44v6 expression in villous tissue. Immunostaining verifies CD44v6 expression in tissue sections, a brownish color represents positive staining, magnification: 200 ×; B and C. Real-time PCR analysis of CD44v6 and NF-κB mRNA in HTR-8/SVneo cells infected with CD44v6 siRNA or control siRNA (**P < 0.01).

Article Snippet: Thereafter, the tissue sections were incubated in mouse anti-human CD44v6 antibody (1:200; VFF-18, Bender MedSystems, Austria), anti-NF-κB p65 (1:1000; NF-12; Sigma, USA), or mouse/rabbit IgG of homologous type overnight at 4°C.

Techniques: Expressing, Inhibition, Immunostaining, Staining, Real-time Polymerase Chain Reaction, Infection, Control

Journal: International Journal of Clinical and Experimental Pathology

Article Title: Dysregulation of CD44v6 may lead to recurrent spontaneous abortion by inhibiting the proliferation and migration of trophoblast cells

doi:

Figure Lengend Snippet: Inhibition of CD44v6 with CD44v6-specifc siRNA suppresses proliferation and migration of HTR-8/SVneo cells. A. Colony-forming ability of HTR-8/SVneo cell was investigated by colony formation assay (**P < 0.01), clone formation rate = clone number/inoculation cell number *100%, 1000 cells were inoculated per hole. B. Representative images of the cells treated with control or CD44v6 siRNA in Transwell migration assay (× 1000). The statistical bar graphs show the summary of three independent experiments (**P < 0.01).

Article Snippet: Thereafter, the tissue sections were incubated in mouse anti-human CD44v6 antibody (1:200; VFF-18, Bender MedSystems, Austria), anti-NF-κB p65 (1:1000; NF-12; Sigma, USA), or mouse/rabbit IgG of homologous type overnight at 4°C.

Techniques: Inhibition, Migration, Colony Assay, Control, Transwell Migration Assay

Journal: International Journal of Clinical and Experimental Pathology

Article Title: Dysregulation of CD44v6 may lead to recurrent spontaneous abortion by inhibiting the proliferation and migration of trophoblast cells

doi:

Figure Lengend Snippet: Knockdown of CD44v6 attenuates nuclear translocation of NF-κB in HTR-8/SVneo cells. Fluorescent signals specific to CD44v6 antibody were visualized as red, NF-κB (green), and the nuclei staining with DAPI (blue).

Article Snippet: Thereafter, the tissue sections were incubated in mouse anti-human CD44v6 antibody (1:200; VFF-18, Bender MedSystems, Austria), anti-NF-κB p65 (1:1000; NF-12; Sigma, USA), or mouse/rabbit IgG of homologous type overnight at 4°C.

Techniques: Knockdown, Translocation Assay, Staining