Journal: Cell reports

Article Title: Activation of GPR44 decreases severity of myeloid leukemia via specific targeting of leukemia initiating stem cells

doi: 10.1016/j.celrep.2023.112794

Figure Lengend Snippet:

Article Snippet: Mouse anti-mouse CD45.1 (FITC) , Miltenyi Biotec , Cat# 130–124-211; RRID: AB_2857674.

Techniques: Virus, Recombinant, Enzyme-linked Immunosorbent Assay, Binding Assay, Staining, Modification, Saline, Concentration Assay, Over Expression, Protein Extraction, Membrane, SYBR Green Assay, Bicinchoninic Acid Protein Assay, Protease Inhibitor, CCK-8 Assay, Reverse Transcription, Selection, Plasmid Preparation, Knock-Out, Software, Real-time Polymerase Chain Reaction, Flow Cytometry

Journal: The Journal of investigative dermatology

Article Title: α(1,3) Fucosyltransferases IV and VII are essential for the initial recruitment of basophils in chronic allergic inflammation.

doi: 10.1038/jid.2013.160

Figure Lengend Snippet: Figure 4. Selectin-dependent cooperative recruitment of basophils and effector cells. (a) Irradiated a(1, 3) fucosyltransferase-IV (FT-IV)( / )/FT-VII( / ) mice received wild-type (WT) basophils in combination with CD49b( ) bone marrow cells (effector cells) from either WT or FT-IV( / )/FT-VII( / ) mice. They were then immunized with trinitrophenyl-IgE (TNP-IgE) and challenged with TNP-OVA. The untreated group comprised FT-IV( / )/FT-VII( / ) mice without cell transfer. (b) Cell populations in inflammatory skin. *Po0.05 compared with WT effector cells. BM, basement membrane.

Article Snippet: In vitro selectin binding assay of basophils CD49b (þ ) basophil–enriched bone marrow cells were suspended in phosphate-buffered saline containing 5% fetal calf serum, 0.1% NaN3, 1 mmol l 1 Ca2þ , and 1 mmol l 1 Mg2þ , followed by incubation with 10mg ml 1 of murine P-, E-, or L-selectin-human IgG Fc chimera or control human IgG1 Fc (R&D Systems) for 40 minutes at 4 1C.

Techniques: Irradiation, Membrane

Journal: The Journal of investigative dermatology

Article Title: α(1,3) Fucosyltransferases IV and VII are essential for the initial recruitment of basophils in chronic allergic inflammation.

doi: 10.1038/jid.2013.160

Figure Lengend Snippet: Figure 5. Expression of a(1, 3) fucosyltransferase (FT) messenger RNA (mRNA) and FT-dependent selectin binding in basophils. (a) Primary basophils were subjected to further purification by positive selection with CD123 (purity 499%). Transcripts for FT-IV and FT-VII mRNA were quantified by real-time PCR. (b) Binding of soluble P- and E-selectins to primary basophils assessed by flow cytometry. BMBa, bone marrow–derived basophil; BMMCs, bone marrow–derived mast cells.

Article Snippet: In vitro selectin binding assay of basophils CD49b (þ ) basophil–enriched bone marrow cells were suspended in phosphate-buffered saline containing 5% fetal calf serum, 0.1% NaN3, 1 mmol l 1 Ca2þ , and 1 mmol l 1 Mg2þ , followed by incubation with 10mg ml 1 of murine P-, E-, or L-selectin-human IgG Fc chimera or control human IgG1 Fc (R&D Systems) for 40 minutes at 4 1C.

Techniques: Expressing, Binding Assay, Selection, Real-time Polymerase Chain Reaction, Cytometry, Derivative Assay

Journal: The Journal of investigative dermatology

Article Title: α(1,3) Fucosyltransferases IV and VII are essential for the initial recruitment of basophils in chronic allergic inflammation.

doi: 10.1038/jid.2013.160

Figure Lengend Snippet: Figure 6. Basophil P-selectin glycoprotein-1 (PSGL-1)–L-selectin interaction involvement in IgE-mediated chronic allergic inflammation (IgE-CAI). (a) PSGL-1 and L-selectin expressions on basophils. (b) PSGL-1 Ab effect on selectin binding to primary basophils. (c) L-selectin binding to basophils from wild-type (WT) and a(1, 3) fucosyltransferase-IV (FT-IV)( / )/FT-VII( / ) mice. (d) Effects of L-selectin and/or PSGL-1 Abs on IgE-CAI in WT mice. *Po0.05 compared with control IgG. **Po0.05 compared with L-selectin or PSGL-1 Ab alone.

Article Snippet: In vitro selectin binding assay of basophils CD49b (þ ) basophil–enriched bone marrow cells were suspended in phosphate-buffered saline containing 5% fetal calf serum, 0.1% NaN3, 1 mmol l 1 Ca2þ , and 1 mmol l 1 Mg2þ , followed by incubation with 10mg ml 1 of murine P-, E-, or L-selectin-human IgG Fc chimera or control human IgG1 Fc (R&D Systems) for 40 minutes at 4 1C.

Techniques: Binding Assay, Control

Journal: Nature medicine

Article Title: An AKT3-FOXG1-Reelin Network Underlies Defective Migration in Human Focal Malformations of Cortical Development

doi: 10.1038/nm.3982

Figure Lengend Snippet: ( a ) Quantitative real-time PCR was performed to measure RELN expression in hNPCs expressing AKT3 E17K and compared to vector-expressing hNPCs. Three independent experiments were quantified in triplicate. ( b ) Mouse embryos at E14.5 were electroporated with indicated DNA constructs. Brains isolated at E18.5 used for reelin expression. Arrowheads: reelin immunostaining in pericellular area of ectopic neurons (intensity-per-pixel: 622.55 ± 45.80 compared to 4092.77 ± 9.46, marginal zone). ( c ) E14.5 embryos were co-electroporated with indicated constructs or RNAs. ( d ) Localization of GFP + neurons at E18.5 was quantified in each cortical region ( n = 3 for each). ( e, f ) Developing cortices were in utero electroporated (IUE) at E14.5 with RFP vector. After 15 min, embryos were sequentially electroporated with either GFP vector ( n = 3) or GFP also expressing AKT3 E17K (A3) with or without Reln siRNA ( n = 6 and 4, respectively). At E18.5, brains were sectioned for imaging. Localization of RFP + GFP − cells was quantified in each cortical region. Values: mean ± s.d. n.s., not significant; **, P < 0.01; ***, P < 0.001, Student’s t -test ( a ); G-test of goodness-of-fit ( d, f ). Scale bars: 25 μm ( b ); 100 μm ( c, e ). ( g ) Enrichment of FOX-binding site. See Online Methods for details. ( h ) Model of effect of AKT3 mutation on surrounding cells. Mutant cell, pink; surrounding cells, gray. AKT3* activating somatic mutation leads to mTOR activation and phosphorylation and cytoplasmic sequestration of FOXG1, relieving repression of RELN , which is then secreted by mutant cells to act in an autocrine (cell autonomous) and paracrine (non-cell autonomous) fashion.

Article Snippet: Paraffin embedded tissues were sectioned, rehydrated and retrieved for antigen (citrated buffer, pH 6.0) before immunostaining for GFP (1:200; Rockland; cat. no. 600-106-215) followed by detection by DAB (Vector Labs).

Techniques: Real-time Polymerase Chain Reaction, Expressing, Plasmid Preparation, Construct, Isolation, Immunostaining, In Utero, Imaging, Binding Assay, Mutagenesis, Activation Assay, Phospho-proteomics

Journal: Oncotarget

Article Title: c-Cbl mediates the degradation of tumorigenic nuclear β-catenin contributing to the heterogeneity in Wnt activity in colorectal tumors

doi: 10.18632/oncotarget.12107

Figure Lengend Snippet: A. Expression and localization of β-catenin and c-Cbl in two stage IV CRC patients showing a reverse expression pattern in β-catenin and c-Cbl. A set of consecutive slides stained for c-Cbl and β-catenin were examined at different magnifications (200X and 400X). Patient # 88290 showed a strong nuclear β-catenin (a black arrow), while c-Cbl expression was low (a yellow arrow). In contrast, patient # 3862 showed predominantly membrane and cytoplasmic β-catenin (a white arrow), and a high c-Cbl expression (a yellow arrow). B. Overview of the image-processing pipeline to estimate the amounts of nuclear β-catenin and c-Cbl. Two stages including RGB color image conversion to L*a*b space followed by segmentation of the color image into three sub-regions using a clustering algorithm are shown. Three basic sub-regions within each image: nuclei and its neighborhood, luminal area, and the interstitial space. (i) For the case of c-Cbl, the k -means algorithm ( k =3) was used on each transformed color image in a*b* space and segmented into three clusters. From the three segmented clusters, the encapsulated cytoplasmic area within the entire image was identified. A size-based filtering operation was then performed on the identified cluster to eliminate all connected components that have fewer than 5000 pixels were removed from the identified cytoplasmic cluster. (ii) For nuclear β-catenin, the same clustering algorithm was used to first divide the transformed color image in a*b* space into three clusters. Each of the three clusters derived from the color image in a*b* space was divided into two sub-groups using the same k -means ( k =2) clustering approach. The final outcome of this 2-tier clustering approach was 6 non-overlapping images. The surgical pathologists then identified the images with nuclear β-catenin. C. An inverse correlation between c-Cbl and nuclear β-catenin in CRC. A correlation between average normalized nuclear β-catenin and normalized c-Cbl was performed in 83 stage IV CRC patients. The dotted lines correspond to the averages of each parameter. Error bars = SEM. The highlighted areas represent groups with an inverse relationship between two parameters, and the divided cohort is depicted in 2×2 contingency table .

Article Snippet: Primary antibodies were β-catenin (H-102; 1:250; Santa Cruz Biotechnology) and PCNA (D3H8P; 1:2500; Cell Signaling Technology). c-Cbl antibody was validated using a blocking peptide.

Techniques: Expressing, Staining, Membrane, Transformation Assay, Derivative Assay

Journal: Oncotarget

Article Title: c-Cbl mediates the degradation of tumorigenic nuclear β-catenin contributing to the heterogeneity in Wnt activity in colorectal tumors

doi: 10.18632/oncotarget.12107

Figure Lengend Snippet: A. c-Cbl silenced (c -Cbl - sil ) HT29 cells that were used for the xenograft model. HT 29 cells stably expressing control (pSuper) and c-Cbl shRNA( c-Cbl -sil) were lyzed and probed. The β-catenin band was normalized with actin for this figure and in all of the figures in this manuscript. A representative of the image from two repeat experiments is shown. B. c-Cbl silencing significantly increased the CRC tumor growth. HT29 cells (1×10 7 /animal) stably expressing control and silenced c-Cbl were mixed with Matrigel and injected subcutaneously into nude mice ( N = 5). The growth of tumor cells was measured every seven days for three weeks. An average of tumor volume is shown. Error bars = SEM. C. Xenografts were removed on the day of harvest and dissected. The explants are shown. D. The tumor areas in c-Cbl silenced and control xenografts. Paraffin-embedded xenograft tumors were processed and stained for H & E. Ten slides per each xenograft were prepared ( N = 5 xenografts from each group), and five random images were captured from each slide at 10X magnification. The area of the tumor, which includes adenocarcinoma without the interstitium or luminal regions, was measured using image quantification technique shown in Figure . In the first tier, this algorithm segments the area into three fundamental compartments including adenocarcinoma, interstitium, and lumen. The control tumors showed adenocarcinoma (the dotted line) interspersed with a small interstitial area, while in c-Cbl silenced xenografts showed predominant tumor area without interstitium. A representative from each group is shown, where I = interstitium and T = tumor, marked with the broken white line. Scale bar = 100 μM. E. The tumor area was significantly higher in c-Cbl silenced xenograft. A color-based segmentation pipeline was used to quantitate area of tumor in each slide as described above. Ten slides were prepared randomly from each xenograft ( N = 5 for each group) and five random images per slides were analyzed. An average of tumor area is shown with error bars = SD. p values were calculated using Mann-Whitney U test. F. An increase in the proliferating cells in c-Cbl silenced CRC xenografts. Ten slides per each xenograft were processed and stained for PCNA, and counter-stained with H & E. Five random images were captured from each slide at 40X magnification and a representative from each group is shown. The black arrow indicates PCNA positive nuclei. Scale bar = 100 μM. G. PCNA positive nuclei were significantly higher in c-Cbl silenced xenografts. A color-based segmentation pipeline to quantitate PCNA positive nuclei was developed, as described above and expressed as percent of PCNA positive nuclei to the total nuclei. Ten slides were prepared randomly from each xenograft and five random images per slides were analyzed, and an average of number of PCNA positive nuclei is shown with error bars = SD. p values calculated using Mann-Whitney U test. H. An increase in the nuclear β-catenin in c-Cbl silenced xenograft. Xenograft slides processed and stained for β-catenin were analyzed as above. The yellow/white arrow indicates β-catenin positive nuclei. Scale bar = 100 μM. I. The cells with nuclear β-catenin were significantly higher in c-Cbl silenced xenografts. A color-based segmentation pipeline was used to quantitate β-catenin positive nuclei and normalized to a total number of nuclei. The percentage of β-catenin positive nuclei to the total number of nuclei is shown with the error bars = SD. p values calculated using Mann-Whitney U test.

Article Snippet: Primary antibodies were β-catenin (H-102; 1:250; Santa Cruz Biotechnology) and PCNA (D3H8P; 1:2500; Cell Signaling Technology). c-Cbl antibody was validated using a blocking peptide.

Techniques: Stable Transfection, Expressing, Control, shRNA, Injection, Staining, MANN-WHITNEY

Journal: Oncotarget

Article Title: c-Cbl mediates the degradation of tumorigenic nuclear β-catenin contributing to the heterogeneity in Wnt activity in colorectal tumors

doi: 10.18632/oncotarget.12107

Figure Lengend Snippet: A. The binding of recombinant c-Cbl with different species of β-catenin. Recombinant GST or GST-tagged c-Cbl (1-358 and 359-909) were purified from E. coli and tethered to GST™ beads. The lysates from different CRC cell lines including RKO (wild-type β-catenin), HCT116 (phospho-resistant S33A mutant β-catenin) and HCT15 (active β-catenin in the milieu of loss of APC ) were incubated for 4 hours with the beads, which then were washed with lysis buffer containing 0.2M NaCl. The eluents were probed using β-catenin antibody. Five percent of cell lysates and GST-tagged c-Cbl truncations stained using Coomassie dye are shown as inputs. Representative immunoblot from three experiments is shown. B. A co-localization of c-Cbl with wild-type, S33A mutant and active β-catenin in different CRC cell lines. CRC cells after 24 hours of seeding were fixed and stained for endogenous β-catenin and c-Cbl. DAPI was used for nuclear staining. Images obtained from the confocal microscope, were analyzed using ImageJ to generate the profile plots to demonstrate the distribution of the signal. C. c-Cbl silencing increased β-catenin in CRC cells. CRC cells were stably transduced with control (pSuper) or c-Cbl-silenced ( c-Cbl-sil ) shRNA and lyzed and probed using c-Cbl and β-catenin antibodies. Actin served as a loading control. A representative of three different experiments is shown. D. c-Cbl downregulated different species of β-catenin, while c-Cbl-70Z, an E3 UB ligase-deficient form of c-Cbl, served as a dominant negative. CRC cells stably expressing Flag-tagged c-Cbl (cbl) or c-Cbl-70Z or control (Ctr) were fractionated. Tubulin and fibrillarin served both as loading controls and the markers for the respective fractions. A representative of three different experiments is shown. E. c-Cbl destabilized active β-catenin in the milieu of APC loss. HCT15 cells stably transduced with the control and c-Cbl pretreated with 20μM emetine were harvested at the indicated time. A representative of three different experiments is shown. F. The stability of β-catenin was determined by the half-life, which is the time point corresponding to 50% of original amount of β-catenin after blocking the protein translation with emetine. β-catenin bands were normalized to tubulin and then to the amount of β-catenin at time zero. An average of three experiments is shown. Error bars = SD.

Article Snippet: Primary antibodies were β-catenin (H-102; 1:250; Santa Cruz Biotechnology) and PCNA (D3H8P; 1:2500; Cell Signaling Technology). c-Cbl antibody was validated using a blocking peptide.

Techniques: Binding Assay, Recombinant, Purification, Mutagenesis, Incubation, Lysis, Staining, Western Blot, Microscopy, Stable Transfection, Transduction, Control, shRNA, Dominant Negative Mutation, Expressing, Blocking Assay

Journal: Oncotarget

Article Title: c-Cbl mediates the degradation of tumorigenic nuclear β-catenin contributing to the heterogeneity in Wnt activity in colorectal tumors

doi: 10.18632/oncotarget.12107

Figure Lengend Snippet: A. The c-Cbl silencing increased the Wnt activity in CRC cells with the S33A mutant β-catenin and the active β-catenin due to APC loss. The CRC cell lines were transfected with TCF-responsive promoter-reporter pBARLS and nonresponsive control reporter pfuBARLS tethered to a luciferase reporter (17). The Wnt activity was quantified by measuring relative firefly luciferase units normalized to protein concentration. An average of three independent experiments was shown. A Student's t-test was performed to determine the statistical significance. Error bars = SEM. B. The loss of c-Cbl activity upregulated Wnt target genes in CRC cells harboring the S33A mutant β-catenin. HCT116 cells stably expressing control or c-Cbl shRNA (c -Cbl -sil) were harvested within 24 hours of seeding. qRT-PCR reactions were run in duplicate for each sample and quantified using real-time PCR in triplicates for detecting AXIN-2, C-MYC and CYCLIND1 mRNAs and the Ct values were generated. The values were normalized to the control and actin. An average of three independent experiments performed in duplicates is shown. Error bars = SEM. C. Doubling of c-Myc with c-Cbl silencing in CRC cell lines. The CRC silenced for c-Cbl were probed for c-Myc. Actin served as a loading control. A representative of two independent experiments is shown.

Article Snippet: Primary antibodies were β-catenin (H-102; 1:250; Santa Cruz Biotechnology) and PCNA (D3H8P; 1:2500; Cell Signaling Technology). c-Cbl antibody was validated using a blocking peptide.

Techniques: Activity Assay, Mutagenesis, Transfection, Control, Luciferase, Protein Concentration, Stable Transfection, Expressing, shRNA, Quantitative RT-PCR, Real-time Polymerase Chain Reaction, Generated

Journal: Oncotarget

Article Title: c-Cbl mediates the degradation of tumorigenic nuclear β-catenin contributing to the heterogeneity in Wnt activity in colorectal tumors

doi: 10.18632/oncotarget.12107

Figure Lengend Snippet: A. c-Cbl silencing increased the proliferation of CRC cells harboring active β-catenin due to APC loss. HCT15 cells stably expressing control (pSuper) or c-Cbl shRNA (c -Cbl -sil) were serum starved for 24 hours and stimulated with 5% FBS. 3 [H] incorporation assay was performed after 24 hours using The LabLogic 300SL Liquid Scintillation Counter. An average of six samples done in duplicates is shown. A Student's t-test was performed. Error bars = SEM. B. c-Cbl-70Z, an E3 UB ligase-deficient and a dominant negative form of c-Cbl increased the proliferation of CRC cells with S33A mutant β-catenin. HCT116 cells stably expressing control or c-Cbl-70Z construct were serum starved for 24 hours and stimulated with 5% FBS. 3 [H] incorporation assay was performed after 24 hours. An average of six samples done in duplicates is shown. A Student's t-test was performed. Error bars = SEM. C. c-Cbl regulated the CRC proliferation in a TCF-4 dependent manner. Ls174T cells with inducible dominant negative (dnTCF4) were stably transduced with control (pSuper) or c-Cbl shRNA (c -Cbl -sil). The dnTCF4 was induced with doxycycline treatment for 5 days and the cells were processed as above. An average of six samples done in duplicates is shown. A Student's t-test was performed. The p values are shown for different comparisons. Compared to control, c-Cbl silenced cells showed a significant increase in the CRC proliferation. Induction of dnTC4 significantly reduced the Wnt activity in both the groups. Even in the dnTCF4 group, c-Cbl silenced cells showed a higher Wnt activity compared to the control cells ( p =0.003). Error bars = SEM. D. The expression of dnTCF-4 and β-catenin are shown. The lysates from the above experiments were probed and the actin served as a loading control. E. c-Cbl continued β-catenin regulation even in the milieu of RTK suppression. LS174T cells stably expressing control (pSuper) or c-Cbl silenced shRNA ( c-Cbl- sil) were treated with Sorafenib 25 μM, Foretinib 20 μM and same concentrations of Sorafenib + Foretinib for 24 hours based on IC50 . β-catenin bands were normalized to actin. A representative of three independent experiments is shown. A Student's t-test was performed to determine the statistical significance. Compared to pSuper, c-Cbl silenced cells had a 2-fold increase in β-catenin, p = 0.004. The group averages of treated cells were compared with untreated cells in pSuper and Silenced group using Wilcoxon rank sum test. A significant lower reduction was noted in treated c -Cbl silenced cells compared to the treated control cells ( p = 0.0003). F. c-Cbl continued to regulate the Wnt activity in the milieu of RTK suppression. HT29 cells co-expressing TCF-responsive promoter-reporter pBARLS or nonresponsive control reporter pfuBARLS tethered to a luciferase reporter along with control (pSuper) or c-Cbl shRNA silenced cells were seeded in 96-well plates and serum starved overnight. The cells were stimulated with 5% FBS with Sorafenib or Foretinib or the same concentrations of Sorafenib + Foretinib, as described above. The cell lysates were subjected to luciferase assay using Promega Dual Luciferase Assay Kit ® according to the manufacturer's instructions and normalized to the protein content. An average of six independent samples is shown. A Student's t-test was performed to determine the statistical significance. Compared to the control cells (pSuper), the c-Cbl silenced (c -Cbl -sil) cells had 77% increased in the Wnt activity, (#) p = 0.00001. In the pSuper group, compared to the untreated cells, the cells treated with Sorafenib had (*) p = 0.02, Foretinib = 0.14 and Sorafenib + Foretinib treated cells had p = 0.0001. In the c-Cbl silenced group, compared to the untreated cells, the cells treated with Sorafenib (*) had p = 0.02, Foretinib p = 0.23 and Sorafenib + Foretinib p = 0.049. The group averages of treated cells were compared in pSuper and Silenced cells using Wilcoxon rank sum test (the dotted line, p = 0.046). Error bars = SEM. G. c-Cbl regulated the CRC proliferation even in the presence of RTK suppression. HT29 cells stably transduced with the control (pSuper) or c-Cbl shRNA (c -Cbl -sil) were seeded in a 96-well plate and serum starved. 3 [H] thymidine incorporation assay (cpm/well) was performed after treating the cells with Sorafenib or Foretinib or Sorafenib + Foretinib as described above. An average of six samples is shown. A Student t-test was performed. Compared to the pSuper, the c-Cbl silenced cells showed a 44.69% increase in the cell proliferation, (#) p = 0.009. In the pSuper group, compared to the control, the cells treated with Sorafenib had (*) p = 0.02, Foretinib p = 0.109 and Sorafenib + Foretinib p = 0.011. In the Silenced group, compared to untreated cells, Sorafenib-treated cells had (*) p = 0.002, Foretinib p = 0.03 and Sorafenib + Foretinib p = 0.007. The group averages of treated cells were compared in pSuper and Silenced cells using Wilcoxon rank sum test (the dotted line, p <0.0001). Error bars = SEM.

Article Snippet: Primary antibodies were β-catenin (H-102; 1:250; Santa Cruz Biotechnology) and PCNA (D3H8P; 1:2500; Cell Signaling Technology). c-Cbl antibody was validated using a blocking peptide.

Techniques: Stable Transfection, Expressing, Control, shRNA, Dominant Negative Mutation, Mutagenesis, Construct, Transduction, Activity Assay, Luciferase, Thymidine Incorporation Assay