Journal: Oncotarget

Article Title: Ca 2+ /calmodulin-dependent protein kinase IIγ enhances stem-like traits and tumorigenicity of lung cancer cells

doi:

Figure Lengend Snippet: A. Detection of Akt and β-catenin signals in H1299 parental or oncosphere cells by western blots. B. Detection of Akt and β-catenin signals in H1299 cells tranduced with control or CaMKIIγ vector by western blots. C. Detection of Akt and β-catenin signals in ZRLC-1 cells treated with DMSO, KN92, or KN93 by western blots. D. Detection of Oct4 and c-Myc in CaMKIIγ-overexpressed H1299 cells treated with Akt or β-catenin inhibitor by western blots. E. Lysates of CaMKIIγ-overexpressed A549 and H1299 cells were incubated with CaMKIIγ or Akt antibody for immunoprecipitation and the immune complex was then purified, separated by SDS-PAGE, and immunoblotted with Akt or CaMKIIγ antibody. F. Dual luciferase reporter assay of Wnt pathway (TOP, FOP, and the ratio of TOP/FOP) in control or CaMKIIγ-overexpressed H1299 cells. Data are expressed as mean ± SEM of n = 4 independent cell dishes per condition. ** p < 0.01. G. Quantitative western blot analysis of oncosphere formation by control or CaMKIIγ-overexpressed A549 or H1299 cells treated with Akt or β-catenin inhibitor. Data are expressed as mean ± SEM of n = 4 independent cell dishes per condition. * p < 0.05, ** p < 0.01, *** p < 0.001.

Article Snippet: KN93 (Cat. No. 422711), KN92 (Cat. No. 422709), Akt Inhibitor IV (Cat. No. 124011), β-catenin/Tcf Inhibitor V (Cat. No. 219334), and Wnt Pathway inhibitor XI (Cat. No. 681674) were purchased from Merck Millipore.

Techniques: Western Blot, Plasmid Preparation, Incubation, Immunoprecipitation, Purification, SDS Page, Luciferase, Reporter Assay

Journal: Nature Communications

Article Title: Canonical WNT signaling-dependent gating of MYC requires a noncanonical CTCF function at a distal binding site

doi: 10.1038/s41467-021-27868-3

Figure Lengend Snippet: a The position of the CCAT1 -specific CTCFBS within the OSE is indicated (black arrow). The core binding sequence was modified at 8 bases, as marked by gray boxes in the panel, by CRISPR editing. Orange boxes depict enhancer regions. b ChIP analyses of the occupancy of the CTCFBS within the OSE. The MYC promoter and the H19 ICR were used as positive controls. Neg. CTCF negative site (see Methods). c DNA sequences in the edited CTCFBS in comparison to WT HCT-116 cells. d ChIP-seq profiles of CTCF binding patterns to a region encompassing the OSE (upper) and MYC (lower) regions. The ChIP-seq data, which is visualized in relation to a genome browser snapshot, was normalized from three independent experiments for WT HCT-116, D3, and E4 cells. The boxed motif, representing the CCAT1 gene region, is enlarged to identify the edited CTCFBS within its intron. The arrows identify the edited CCAT1 -specific CTCFBS. e Co-cultures of wild type and mutant HCT-116 cells harvested at the indicated time points, followed by qPCR analyses of the proportion of WT and mutant CTCFBSs, respectively. f The effect of BC21 on the relative growth rate of the WT, D3, and E4 cells. All the genomic coordinates use hg19 as a reference genome. The data represent in all instances the average of three independent experiments with indicated standard deviation. The p values were calculated by the two-tailed Student’s t test.

Article Snippet: HCT-116 cells were treated with 10 μM β-Catenin/TCF Inhibitor V, (BC21; Merckmillipore, 219334), or an equivalent amount of the solvent DMSO, for 16 h. Transfection of HCT-116 cells was performed as described .

Techniques: Binding Assay, Sequencing, Modification, CRISPR, Comparison, ChIP-sequencing, Mutagenesis, Standard Deviation, Two Tailed Test

Journal: Nature Communications

Article Title: Canonical WNT signaling-dependent gating of MYC requires a noncanonical CTCF function at a distal binding site

doi: 10.1038/s41467-021-27868-3

Figure Lengend Snippet: a The rate of nuclear export of newly synthesized MYC and FAM49B mRNAs in WT and mutant HCT-116 cells in the absence or presence of BC21 (see Methods). b The transcriptional rate of the MYC and FAM49B genes. The data were generated by qRT-PCR analyses of MYC/FAM49B transcription using newly synthesized (30 min ethynyl-uridine pulse) RNA as template and normalized to ACTB transcription. c The steady state levels of cytoplasmic MYC and FAM49B mRNAs in WT and mutant HCT-116 cells normalized to TBP expression and external “spike in” RNA controls. The levels of TBP and ß-actin mRNA expression, markers used to normalize the mRNA export and transcription rates, were not significantly different between the WT and D3/E4 cells and correctly estimated the number of input cells (Supplementary Fig. ). d Comparison between observed and simulated cytoplasmic MYC RNA levels in WT HCT-116 and E4 cells. The simulation used the parameters of nuclear export of MYC mRNA ( a ), transcriptional rate ( b ), and the kinetics of MYC mRNA decay in the nuclear and cytoplasmic compartments, as described before . The data represent in all instances the average of three independent experiments with indicated standard deviation. The p values were calculated by the two-tailed Student’s t test.

Article Snippet: HCT-116 cells were treated with 10 μM β-Catenin/TCF Inhibitor V, (BC21; Merckmillipore, 219334), or an equivalent amount of the solvent DMSO, for 16 h. Transfection of HCT-116 cells was performed as described .

Techniques: Synthesized, Mutagenesis, Generated, Quantitative RT-PCR, Expressing, Comparison, Standard Deviation, Two Tailed Test

Journal: Nature Communications

Article Title: Canonical WNT signaling-dependent gating of MYC requires a noncanonical CTCF function at a distal binding site

doi: 10.1038/s41467-021-27868-3

Figure Lengend Snippet: a Co-immunoprecipitation analyses of physical interactions between CTCF, ß-catenin, NUP133 and AHCTF1. IgG negative control. b Quantification of the CTCF-bound complexes shown in ( a ). c Co-immunoprecipitation analyses of the physical interactions between AHCTF1 and CTCF in WT HCT-116 cells in the absence or presence of BC21. d ChIP analyses of the binding of AHCTF1 to the oncogenic super-enhancer in DMSO control or BC21-treated WT HCT-116 cells. e ChIP analyses of CTCF and AHCTF1 binding to the CCAT1 -specific CTCFBS in cells transfected with siGFP or siCTCF. The signals were normalized to the siGFP controls. The average siCTCF-mediated reduction in CTCF expression was 85% (Supplementary Fig ). f ChIP analyses of AHCTF1 binding to the CTCFBS and the CCAT1 promoter within the OSE in WT HCT-116 and mutant (D3/E4) cells. g The knock-down of AHCTF1 expression by siRNA using a siGFP as control. h 3D DNA FISH analyses of the proximity between the OSE and the nuclear periphery in HCT-116 cells in the presence or absence of AHCTF1 (reduced to 72% in comparison to controls ). The bars represent the sum of two independent experiments (219 and 201 alleles, respectively) for siGFP and siAHCTF1-treated cells. i Box-and-whisker plots show median values, interquartile ranges and Tukey whiskers of the distribution of the OSE within 0.7 μm from the nuclear periphery. j In situ proximity ligation assay (ISPLA) of the proximity between CTCF and AHCTF1 in the absence or presence of BC21 in WT HCT-116 cells. Overviews of the DMSO, BC21, and no primary antibody control motifs (upper row), with the the lower row shows magnifications of focal planes marked in the upper row. Bar = 5 μm. k The quantification of the ISPLA signals. The data is based on three independent experiments counting a total number of 710 alleles. C CTCF antibody, A AHCTF1 antibody, No ab no primary antibody. All the data (except for h ) represent the average of three independent experiments with indicated standard deviations. The p values for ( b – g , k ) were calculated by the two-tailed Student’s t test whereas the p value for ( i ) was calculated using the two-sided KS test.

Article Snippet: HCT-116 cells were treated with 10 μM β-Catenin/TCF Inhibitor V, (BC21; Merckmillipore, 219334), or an equivalent amount of the solvent DMSO, for 16 h. Transfection of HCT-116 cells was performed as described .

Techniques: Immunoprecipitation, Negative Control, Binding Assay, Control, Transfection, Expressing, Mutagenesis, Knockdown, Comparison, Whisker Assay, In Situ, Proximity Ligation Assay, Two Tailed Test

Journal: Oncotarget

Article Title: CWP232228 targets liver cancer stem cells through Wnt/β-catenin signaling: a novel therapeutic approach for liver cancer treatment

doi: 10.18632/oncotarget.7954

Figure Lengend Snippet: Normal and liver cancer tissues (kindly provided by Dr. Kwan-Kyu Park at the Catholic University, South Korea) were were co-stained with antibodies specific for CD133 and Wnt/β-catenin signaling components WNT1, LEF1, and β-catenin. Wnt/β-catenin signaling components-positive cells largely overlapped with ALDH1- and CD133-positive cells in the cancerous tissues ( A-B ). The chemical structure and molecular mechanism of CWP232228 ( C ). β-catenin responsive TOPFlash luciferase assays revealed that CWP232228 inhibits recombinant Wnt ligand-induced Wnt/β-catenin signaling in Hep3B cells. CWP232228 treatment strongly attenuated ligand-induced TOPFlash activity ( D ). The inhibitory effect of CWP232228 on the expressions of Wnt/β-catenin signaling target gene WNT1 and TCF4 were assessed in Hep3B cells through western blot analysis ( E ). The stimulatory effects of Wnt ligand on the nuclear translocation of β-catenin were successfully attenuated after CWP232228 treatment( F ). Hep3B cells were stained using an antibody specific for β-catenin. These results are suggested that CWP232228 is significantly inhibited the activity of Wnt/β-catenin signaling components in Hep3B cells. Abbreviations: FOPFlash: a reporter plasmid containing mutant Tcf-binding sites, TOPFlash: a reporter plasmid containing multiple copies of wild-type Tcf-binding sites. Topflash and Fopflash (Addgene, Cambridg, MA, USA) reporter constructs is also known as pcDNA3.0 plasmids (Promoter: CMV; Cloning method: restriction enzyme; Size: 5428; Bacterial resistance: Ampicillin; 5′ sequencing 1 primer: CMV-F; 3′ sequencing 1 primer:BGH-rev.) DAPI staining was performed to label the nuclei within each field. β-actin was used as an internal control. The results represent the means ± SD from three independent experiments.

Article Snippet: Wnt signaling inhibitor CWP232228 is designed by JW Pharmaceutical Corporation (Seoul, Korea).

Techniques: Staining, Luciferase, Recombinant, Activity Assay, Western Blot, Translocation Assay, Plasmid Preparation, Mutagenesis, Binding Assay, Construct, Cloning, Sequencing, Control

Journal: Oncotarget

Article Title: CWP232228 targets liver cancer stem cells through Wnt/β-catenin signaling: a novel therapeutic approach for liver cancer treatment

doi: 10.18632/oncotarget.7954

Figure Lengend Snippet: CWP232228 inhibited primary (with CWP232228 treatment) and second sphere formation (without additional CWP232228 treatment) in Hep3B cells. The sphere sizes greater than 100 μm were enumerated, and a representative image of a tumor sphere is shown ( A ). The percentage of ALDH1 and CD133 subpopulations were evaluated by FACS analysis. The treatment of Hep3B cells with CWP232228 for 48 h decreased the percentage of ALDH1 and CD133- double positive cells in the total cell population ( B ). The inhibitory effect of CWP232228 on the expression of stem cell markers OCT4, KLF4, NANOG, and SOX2 was assessed in Hep3B cells through real-time PCR ( C ) and western blot analysis ( D ). These results are indicated that CWP232228 is significantly decreased the mRNA and protein levels of hepatic stem cell markers. Abbreviations: TSFE, Tumor sphere-forming efficiency. β-actin was used as an internal control. The results represent the means ± SD from three independent experiments.

Article Snippet: Wnt signaling inhibitor CWP232228 is designed by JW Pharmaceutical Corporation (Seoul, Korea).

Techniques: Expressing, Real-time Polymerase Chain Reaction, Western Blot, Control

Journal: Oncotarget

Article Title: CWP232228 targets liver cancer stem cells through Wnt/β-catenin signaling: a novel therapeutic approach for liver cancer treatment

doi: 10.18632/oncotarget.7954

Figure Lengend Snippet: Schematic representation of the experimental protocol as described in the materials and methods section ( A ). Anesthetized 7-weeks old male NOD/SCID mice were inoculated with 1:1 mix of matrigel and 5 × 10 5 Hep3B cells into the subcutaneous tissue. Tumor tissue was isolated from mice bearing Hep3B cell tumors that had been treated with CWP232228 (100 mg/kg, intraperitoneally) or vehicle (PBS). Tumor volumes were measured as described in the materials and methods section ( B ). Hep3B cells from monolayer cultures or sphere cultures were dissociated into single cell suspensions and injected into the subcutaneous tissue of mice in limiting dilutions (1,000; 10,000; 100,000). Tumor formation was observed for 4weeks following inoculation. Liver CSC frequency was calculated using extreme limiting dilution assay (ELDA) ( C ). The ALDH1- and CD133-positive subpopulation, as a proportion of the total cell population in the tumor xenografts, was assessed by immunohistochemistry ( D ). The relative expression of Wnt/β-catenin signaling component WNT1, LEF1, and β-catenin in the tumor xenografts was assessed by immunohistochemistry ( E ). CWP232228-mediated apoptotic DNA fragmentation in tumor xenografts was visualized by TUNEL assay ( F ). Inhibitory effects of CWP232228 on the hepatocarcinogenesis were further confirmed by performing proliferating cell nuclear antigen (PCNA) immunohistochemistry to assess tumor xenografts ( G ). In vivo results are suggested that CWP232228 is inhibited tumor progression and decreased the expression levels of hepatic cancer stem markers and Wnt/β-catenin signaling components. DAPI staining was carried out to label the nuclei within each field. The results are presented as the mean ± SD from three independent experiments.

Article Snippet: Wnt signaling inhibitor CWP232228 is designed by JW Pharmaceutical Corporation (Seoul, Korea).

Techniques: Isolation, Injection, Limiting Dilution Assay, Immunohistochemistry, Expressing, TUNEL Assay, In Vivo, Staining

Journal: Oncology Research

Article Title: MINDY1 Induces PD-L1 Deubiquitination to Promote Immune Escape in Hepatocellular Carcinoma by the Wnt/β-Catenin Pathway

doi: 10.32604/or.2025.067638

Figure Lengend Snippet: MINDY1 promotes PD-L1 deubiquitination through Wnt/β-catenin pathway. ( A–C ) MINDY1/Vector and sh-MINDY1/sh-NC were transfected, and Western blot analysis assessed that overexpression of MINDY1 elevated β-catenin and p-GSK3β(Tyr216)/GSK3β levels in HCC cells, and knockdown of MINDY1 did the opposite. ( D – F ) Western blot measured that SKL2001 (Wnt pathway activator) elevated the activating effect of overexpression of MINDY1 on the Wnt/β-catenin pathway, while pathway inhibitor LiCl decreased this phenomenon. (G) Western blot measured that SKL2001 increased PD-L1 protein expression, whereas LiCl led to PD-L1 downregulation. (H) Western blot examined that SKL2001 promoted the deubiquitination of PD-L1 in HCC cells, while LiCl suppressed its deubiquitination. n = 3. * p < 0.05, ** p < 0.01, *** p < 0.001 vs. Vector/sh-NC; & p < 0.05, && p < 0.01, &&& p < 0.001 vs. MINDY1

Article Snippet: Additionally, HCC cells (Hep3B and Huh-7 cells) transfected with MINDY1 were treated with the Wnt signaling pathway activator SKL2001 (20 nM, S793781, Macklin, Shanghai, China) for 24 h, and this group was denoted as the MINDY1+SKL2001 group [ ].

Techniques: Plasmid Preparation, Transfection, Western Blot, Over Expression, Knockdown, Expressing

Journal: Oncology Research

Article Title: MINDY1 Induces PD-L1 Deubiquitination to Promote Immune Escape in Hepatocellular Carcinoma by the Wnt/β-Catenin Pathway

doi: 10.32604/or.2025.067638

Figure Lengend Snippet: MINDY1 hinders CD8 + T cell activation through the Wnt/β-catenin pathway. ( A ) CFSE staining confirmed that co-culture with HCC cells transfected with MINDY1 resulted in diminished proliferation of CD8 + T cells, which was further diminished by SKL2001 treatment, whereas LiCl treatment did the opposite. ( B ) Flow cytometry indicated a rise in CD8 + T cell apoptosis after they were co-cultured with HCC cells overexpressing MINDY1, with an additional rise observed by SKL2001 treatment, while LiCl treatment reduced the apoptosis rate. ( C ) The cytotoxicity of CD8 + T cells was assessed through the LDH kit. ( D , E ) Flow cytometry confirmed that co-culturing with HCC cells overexpressing MINDY1 led to a decrease in Perforin + and TNF-α + T cells. After treatment with SKL2001, this decrease was further exacerbated, while LiCl increased activated CD8 + T cells. ( F , G ) ELISA was used to detect IFN-γ and IL-2 levels. ( H ) CCK-8 assay was employed to examine the survival rates of HCC cells. n = 3. * p < 0.05, *** p < 0.001 vs. Vector; & p < 0.05, && p < 0.01, &&& p < 0.001 vs. MINDY1

Article Snippet: Additionally, HCC cells (Hep3B and Huh-7 cells) transfected with MINDY1 were treated with the Wnt signaling pathway activator SKL2001 (20 nM, S793781, Macklin, Shanghai, China) for 24 h, and this group was denoted as the MINDY1+SKL2001 group [ ].

Techniques: Activation Assay, Staining, Co-Culture Assay, Transfection, Flow Cytometry, Cell Culture, Enzyme-linked Immunosorbent Assay, CCK-8 Assay, Plasmid Preparation