Journal: Journal of Biological Chemistry

Article Title: The tetraspanin Tspan15 is an essential subunit of an ADAM10 scissor complex

doi: 10.1074/jbc.ra120.012601

Figure Lengend Snippet: Figure 1. Generation of human Tspan15-expressing MEFs as an immunogen and validation of resulting mouse anti-human Tspan15 mAbs. (A) ADAM10-knockout MEFs (–) and ADAM10- knockout MEFs stably overexpressing FLAG-tagged Tspan15 (+) were lysed in 1% Triton X-100 lysis buffer and subjected to anti-FLAG (top panel) and anti-α-tubulin (bottom panel) western blotting. (B) Wild-type (WT) and Tspan15-knockout (KO) Jurkat human T cells were analysed by flow cytometry with tissue culture supernatant for each of the four mouse anti-human Tspan15 hybridomas (1C12, 4A4, 5D4 or 5F4; solid line), or with mouse IgG1 as a negative control (dotted line). Histograms are representative of two independent experiments. (C) HEK-293T cells were transfected with FLAG-tagged human TspanC8 expression constructs (except for Tspan10, which was of mouse origin) or an empty vector control (–),

Article Snippet: Mouse ADAM10 tagged at the C-terminus with the C-terminal half of superfolder GFP was generated using a twostep PCR approach in which the GFP tag was at U C L L ibrary Services on M arch 1, 2020 http://w w w .jbc.org/ D ow nloaded from subcloned into pcDNA3.1 mouse ADAM10 (42). pRK5M human ADAM10 was a gift from Rik Derynck (Addgene plasmid # 31717) (43).

Techniques: Expressing, Biomarker Discovery, Knock-Out, Stable Transfection, Lysis, Western Blot, Flow Cytometry, Negative Control, Transfection, Construct, Plasmid Preparation, Control

Journal: Journal of Biological Chemistry

Article Title: The tetraspanin Tspan15 is an essential subunit of an ADAM10 scissor complex

doi: 10.1074/jbc.ra120.012601

Figure Lengend Snippet: Figure 3. Tspan15 mAbs 1C12 and 4A4 partially inhibit ADAM10/Tspan15 activity. (Ai) Wild-type (WT), ADAM10-knockout (A10 KO) and Tspan15-knockout (T15 KO) HEK-293T cells were transfected with a VE-cadherin expression construct. Cells were treated with 10 μM DAPT to prevent post-ADAM10 proteolysis by γ-secretase, followed by 2 mM NEM for 30 minutes to activate ADAM10. Cells were lysed in 1% Triton X-100 lysis buffer and subjected to western blotting with an antibody against the cytoplasmic tail of VE-cadherin. No C-terminal fragment was detected in the absence of NEM (data not shown). (Aii) VE-cadherin cleavage data were quantitated to calculate the percentage cleaved. Data were arcsine- transformed and statistically analysed by a one-way ANOVA with a Dunnett’s multiple comparisons test (***p<0.001 compared to WT). Error bars represent standard error of the mean from three independent experiments. (B) Wild-type HEK-293T cells were transfected with VE-cadherin, treated with Tspan15 mAbs or MOPC-21 negative control mAb for 30 minutes, and stimulated with NEM as described for panel

Article Snippet: Mouse ADAM10 tagged at the C-terminus with the C-terminal half of superfolder GFP was generated using a twostep PCR approach in which the GFP tag was at U C L L ibrary Services on M arch 1, 2020 http://w w w .jbc.org/ D ow nloaded from subcloned into pcDNA3.1 mouse ADAM10 (42). pRK5M human ADAM10 was a gift from Rik Derynck (Addgene plasmid # 31717) (43).

Techniques: Activity Assay, Knock-Out, Transfection, Expressing, Construct, Lysis, Western Blot, Transformation Assay, Negative Control

Journal: Journal of Biological Chemistry

Article Title: The tetraspanin Tspan15 is an essential subunit of an ADAM10 scissor complex

doi: 10.1074/jbc.ra120.012601

Figure Lengend Snippet: Figure 4. Tspan15 and ADAM10 co-localise on the cell surface. (Ai) A549 cells were fixed and stained with anti-ADAM10 mAb (red) and either anti-Tspan15 mAb 5D4 (green) or anti-CD9 mAb 1AA2 (green). ADAM10, Tspan15 and CD9 on the basal membrane were imaged using TIRF microscopy. Images shown are representative of 48 fields of view from four independent experiments (scale bar 10 µm). (Aii) The degree of co-localisation between ADAM10 and Tspan15 or CD9 was determined using Manders’ coefficients to measure the proportion of overlapping pixels contained within total ADAM10 signal in the red channel (M1) and total Tspan15 or CD9 signal in the green channel (M2). Data were arcsine- transformed and statistically analysed by a one-way ANOVA with a Tukey’s multiple comparisons test to compare M1 and M2, within and between Tspan15 and CD9 (***p<0.001 for all pairwise comparisons). Error bars represent standard error of the mean.

Article Snippet: Mouse ADAM10 tagged at the C-terminus with the C-terminal half of superfolder GFP was generated using a twostep PCR approach in which the GFP tag was at U C L L ibrary Services on M arch 1, 2020 http://w w w .jbc.org/ D ow nloaded from subcloned into pcDNA3.1 mouse ADAM10 (42). pRK5M human ADAM10 was a gift from Rik Derynck (Addgene plasmid # 31717) (43).

Techniques: Staining, Membrane, Microscopy, Transformation Assay

Journal: Journal of Biological Chemistry

Article Title: The tetraspanin Tspan15 is an essential subunit of an ADAM10 scissor complex

doi: 10.1074/jbc.ra120.012601

Figure Lengend Snippet: Figure 5. ADAM10 is the principal Tspan15-interacting protein in HEK-293T cells. Wildtype (WT) and Tspan15-knockout (KO) HEK-293T cells were lysed in 1% digitonin lysis buffer and immunoprecipitated with Tspan15 mAb 1C12 cross-linked to protein G sepharose beads. Proteins were identified by liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS). Proteomic profiles of WT and Tspan15 KO HEK-293T immunoprecipitates are presented in a volcano plot to identify differentially expressed proteins. The minus log10 transformed p-value of each protein was plotted against the log2 transformed protein label free quantification ratio between the Tspan15 co-immunoprecipitation of WT samples and the control co-immunoprecipitation of Tspan15 KO samples. Proteins with significant fold change (p<0.05) are depicted in red; blue dots represent proteins with no significant changes in expression. A permutation-based false discovery rate estimation was applied and visualised as hyperbolic curves in grey.

Article Snippet: Mouse ADAM10 tagged at the C-terminus with the C-terminal half of superfolder GFP was generated using a twostep PCR approach in which the GFP tag was at U C L L ibrary Services on M arch 1, 2020 http://w w w .jbc.org/ D ow nloaded from subcloned into pcDNA3.1 mouse ADAM10 (42). pRK5M human ADAM10 was a gift from Rik Derynck (Addgene plasmid # 31717) (43).

Techniques: Knock-Out, Lysis, Immunoprecipitation, Liquid Chromatography, Mass Spectrometry, Liquid Chromatography with Mass Spectroscopy, Transformation Assay, Quantitative Proteomics, Control, Expressing

Journal: Journal of Biological Chemistry

Article Title: The tetraspanin Tspan15 is an essential subunit of an ADAM10 scissor complex

doi: 10.1074/jbc.ra120.012601

Figure Lengend Snippet: Figure 6. Tspan15 protein expression requires ADAM10. (A) Tspan15 surface expression in wildtype (WT), Tspan15-knockout (KO) and ADAM10 KO Jurkat, HEK-293T and A549 cell lines were analysed

Article Snippet: Mouse ADAM10 tagged at the C-terminus with the C-terminal half of superfolder GFP was generated using a twostep PCR approach in which the GFP tag was at U C L L ibrary Services on M arch 1, 2020 http://w w w .jbc.org/ D ow nloaded from subcloned into pcDNA3.1 mouse ADAM10 (42). pRK5M human ADAM10 was a gift from Rik Derynck (Addgene plasmid # 31717) (43).

Techniques: Expressing, Knock-Out

Journal: Journal of Biological Chemistry

Article Title: The tetraspanin Tspan15 is an essential subunit of an ADAM10 scissor complex

doi: 10.1074/jbc.ra120.012601

Figure Lengend Snippet: Figure 7. The requirement of Tspan15 for ADAM10 surface expression is cell type dependent. (A) ADAM10 surface expression in WT, ADAM10 KO and Tspan15 KO Jurkat, HEK-293T and A549 cells was measured by flow cytometry and quantitated as described in Figure 4A. (B) HUVECs were transfected with two different Tspan15 siRNAs or negative control siRNA and surface expression of ADAM10 was measured by flow cytometry and analysed as described in Figure 6A.

Article Snippet: Mouse ADAM10 tagged at the C-terminus with the C-terminal half of superfolder GFP was generated using a twostep PCR approach in which the GFP tag was at U C L L ibrary Services on M arch 1, 2020 http://w w w .jbc.org/ D ow nloaded from subcloned into pcDNA3.1 mouse ADAM10 (42). pRK5M human ADAM10 was a gift from Rik Derynck (Addgene plasmid # 31717) (43).

Techniques: Expressing, Flow Cytometry, Transfection, Negative Control

Journal: Journal of Biological Chemistry

Article Title: The tetraspanin Tspan15 is an essential subunit of an ADAM10 scissor complex

doi: 10.1074/jbc.ra120.012601

Figure Lengend Snippet: Figure 8. ADAM10 and Tspan15 form dynamic bimolecular fluorescence complementation (BiFC) complexes. (A) Schematic representation of ADAM10 tagged with the C-terminal half of superfolder GFP (sfGFP-C), Tspan15 tagged with the N-terminal half of superfolder GFP (sfGFP-N) and the predicted ADAM10/Tspan15 BiFC dimer. Solid ovals represent N-glycosylation. (B) HEK-293T cells were transfected with the ADAM10 and Tspan15 BiFC expression constructs, fixed and stained with Alexa Fluor® 647-conjugated Tspan15 mAb 5D4, and analysed by confocal microscopy. The image shown is representative of middle plane sections taken from two independent experiments (scale bar 10 µm). (C-D) Fluorescence correlation spectroscopy (FCS) measurements from the upper membrane of HEK-293T expressing the ADAM10/Tspan15 BiFC complexes were used to determine the average particle concentration (C) and diffusion co-efficient (D) of the complexes. (E) Fluorescence fluctuations from the FCS reads were also subjected to photon counting histogram (PCH) analysis to obtain the average molecular brightness (ε) of particles within the confocal volume. The FCS data were separated into groups that preferentially fit to a one-component or a two-component PCH model with dimmer and brighter subcomponents. Data were obtained from 43 individual measurements from three independent experiments. Error bars represent standard errors of the mean, N is the number of particles, and cpm is the counts per molecule. Data were log-transformed and statistically analysed by a one-way ANOVA followed by Tukey’s multiple comparisons test (***p<0.001).

Article Snippet: Mouse ADAM10 tagged at the C-terminus with the C-terminal half of superfolder GFP was generated using a twostep PCR approach in which the GFP tag was at U C L L ibrary Services on M arch 1, 2020 http://w w w .jbc.org/ D ow nloaded from subcloned into pcDNA3.1 mouse ADAM10 (42). pRK5M human ADAM10 was a gift from Rik Derynck (Addgene plasmid # 31717) (43).

Techniques: Fluorescence, Glycoproteomics, Transfection, Expressing, Construct, Staining, Confocal Microscopy, Spectroscopy, Membrane, Concentration Assay, Diffusion-based Assay, Transformation Assay

Journal: Journal of Biological Chemistry

Article Title: The tetraspanin Tspan15 is an essential subunit of an ADAM10 scissor complex

doi: 10.1074/jbc.ra120.012601

Figure Lengend Snippet: Figure 9. A synthetic ADAM10/Tspan15 fusion protein is a functional scissor. (A) Schematic representation of the synthetic ADAM10/Tspan15 fusion protein that has the C-terminus of ADAM10

Article Snippet: Mouse ADAM10 tagged at the C-terminus with the C-terminal half of superfolder GFP was generated using a twostep PCR approach in which the GFP tag was at U C L L ibrary Services on M arch 1, 2020 http://w w w .jbc.org/ D ow nloaded from subcloned into pcDNA3.1 mouse ADAM10 (42). pRK5M human ADAM10 was a gift from Rik Derynck (Addgene plasmid # 31717) (43).

Techniques: Functional Assay

Journal: PLoS ONE

Article Title: Microvesicles from Mesenchymal Stromal Cells Are Involved in HPC-Microenvironment Crosstalk in Myelodysplastic Patients

doi: 10.1371/journal.pone.0146722

Figure Lengend Snippet: (A) Percentage of CD34 + cells isolated by immunomagnetic beads and the purity determined by flow cytometry. (B) Flow cytometry characterization of MVs released from MSCs of MDS and HD. The upper images are dot-plots of forward and side scatter of MVs. The gate was defined as elements of smaller size than the 1μm beads. The histograms represent the MVs stained with negative (CD34 and CD45) and positive markers for MVs from HD and MDS-MSC (CD90, CD44, CD73) and for MVs markers (CD81 and CD63). Controls (unstained MVs) are shown in gray; the MVs stained with the different antibodies are shown in black. Images on the left are those of the MVs from MSC-HD, while those on the right images are of the MVs from MSC-MDS. (C) Representative images of transmission electronic microscopy of MVs released by MSC from HD(left) and MDS (right) as revealed by TEM. Scale bar, 200nm. Original magnification: x 8000. (D) MVs characterization by Western Blot assay for the expression of CD63. HD-MVs: microvesicles from healthy donors. MDS-MVs: microvesicles from patients with myelodysplastic syndrome.

Article Snippet: After isolation, the purity and viability of CD34 + cells was evaluated by flow cytometry using fluorescein isothiocyanate (FITC)-conjugated CD34 (11-0349-42, eBioscience Inc. San Diego, CA, USA) and APC H7 Annexin V DY634.

Techniques: Isolation, Flow Cytometry, Staining, Transmission Assay, Microscopy, Western Blot, Expressing

Journal: PLoS ONE

Article Title: Microvesicles from Mesenchymal Stromal Cells Are Involved in HPC-Microenvironment Crosstalk in Myelodysplastic Patients

doi: 10.1371/journal.pone.0146722

Figure Lengend Snippet: (A) Representative images of MVs incorporation by CD34 + cells stained with anti-CD90 Ab (red) and anti-CD45 Ab (green). (B) Representative images of MVs previously labeled with Vybrant-Dil cell-labeling solution (red) that were incorporated into CD34 + cells and stained with anti-CD45 Ab (green). (A-B) Images in the top row are from CD34 + cells that incorporated the MVs released from MSC-HD. Images on the middle row show the incorporation of MVs released from MSC-MDS. In the lower row, images of the CD34 + cells (without incorporation) are shown. Nuclei were counterstained with DAPI (blue). Scale bar, 7.5μm. Revealed by confocal microscopy and acquired in layers (z-Stacks) of 1μm.

Article Snippet: After isolation, the purity and viability of CD34 + cells was evaluated by flow cytometry using fluorescein isothiocyanate (FITC)-conjugated CD34 (11-0349-42, eBioscience Inc. San Diego, CA, USA) and APC H7 Annexin V DY634.

Techniques: Staining, Labeling, Confocal Microscopy

Journal: PLoS ONE

Article Title: Microvesicles from Mesenchymal Stromal Cells Are Involved in HPC-Microenvironment Crosstalk in Myelodysplastic Patients

doi: 10.1371/journal.pone.0146722

Figure Lengend Snippet: (A) Variations in microRNAs expression when CD34 + cells were co-cultured with MDS-MVs or HD-MVs. Ratio was calculated dividing the expression of each microRNA from CD34 + + HD-MVs or CD34 + + MDS-MVs by that of CD34 + cells without MVs. Results were summarized as the median. (B) Expression by RT-PCR of TP53 and MDM2 in CD34 + cells cultured with MDS-MVs from patients (grey) and expression of CD34 + cells without MVs (black). Results were summarized as the mean and standard deviation. (C) Capillary Electrophoresis Immunoassay of MDM2 vs Actin as control. CD34 + cells (without MVs), CD34 + cells with MDS-MVs and with HD-MVs. Each bar of the lower graph represents the value of quantified MDM2 protein expression normalized to actin protein abundance. Each bar represents the quantification of both bands of MDM2 from the pseudo-blots, control CD34 + cells vs CD34 + cells + MDS-MVs or HD-MVs.

Article Snippet: After isolation, the purity and viability of CD34 + cells was evaluated by flow cytometry using fluorescein isothiocyanate (FITC)-conjugated CD34 (11-0349-42, eBioscience Inc. San Diego, CA, USA) and APC H7 Annexin V DY634.

Techniques: Expressing, Cell Culture, Reverse Transcription Polymerase Chain Reaction, Standard Deviation, Electrophoresis, Control, Quantitative Proteomics

Journal: PLoS ONE

Article Title: Microvesicles from Mesenchymal Stromal Cells Are Involved in HPC-Microenvironment Crosstalk in Myelodysplastic Patients

doi: 10.1371/journal.pone.0146722

Figure Lengend Snippet: (A): Representative FACS plots of annexinV/7AAD staining on CD34 + cells with and without MVs. Percentage of each subset (dead, live, early and late apoptosis) within the total number of CD34 + cells. (B) Percentage of live CD34 + cells. An increase on the percentage of CD34 + viable cells (annexinV - /7AAD - ) was observed when cells were cultured with MDS-MVs compared with the other groups is shown. (C) Clonogenic assays. Results are expressed as the ratio between CFU-GM obtained with CD34 + cells that had been cultured with MVs and CD34 + cells without MVs.

Article Snippet: After isolation, the purity and viability of CD34 + cells was evaluated by flow cytometry using fluorescein isothiocyanate (FITC)-conjugated CD34 (11-0349-42, eBioscience Inc. San Diego, CA, USA) and APC H7 Annexin V DY634.

Techniques: Staining, Cell Culture

Journal: Blood

Article Title: RUNX1 is required for oncogenic Myb and Myc enhancer activity in T-cell acute lymphoblastic leukemia

doi: 10.1182/blood-2017-03-775536

Figure Lengend Snippet: RUNX1 is ubiquitously expressed in human T-ALL cells, and RUNX1 or CBFβ knockdown results in apoptosis. (A) Protein was isolated from human T-ALL cell lines and RUNX1, RUNX3, CBFβ, TAL1, MYB, NOTCH1, and MYC protein levels were determined by immunoblotting. Extracellular signal–regulated kinase 1/2 (ERK1/2) was used as a loading control. (B) The human T-ALL cell line Jurkat was infected with lentiviruses expressing a control shRNA or 2 shRNAs specific for RUNX1. RUNX1 mRNA and protein levels were examined by qRT-PCR and immunoblotting. (C) RUNX1 knockdown results in leukemic cell apoptosis. Control (GFP) and RUNX1 shRNA-transduced Jurkat cells were stained with Annexin V-FITC and 7AAD and analyzed by flow cytometry 6 days after infection. A representative flow profile is shown (left). The percentage of apoptotic cells was determined by Annexin V/7AAD staining and analyzed by flow cytometry. Four independent experiments were performed, and data are shown as means ± SD (right). (D) CBFβ knockdown also induces apoptosis. Control (GFP) or CBFβ shRNA-transduced Jurkat cells were stained with Annexin V-FITC and 7AAD and analyzed by flow cytometry. Four independent experiments were performed, and data are shown as means ± SD (right). (E) CBFβ protein levels in control and knockdown cells were analyzed by immunoblotting. **P < .005; ***P < .0005; ****P < .0001, one-way ANOVA multiple comparisons test.

Article Snippet: 2 Immunoblotting and coimmunoprecipitation To examine protein expression in human T-ALL cells, cells were lysed in modified radioimmunoprecipitation assay buffer, transferred to a membrane, and probed with antibodies to RUNX1 (ab23980, Abcam), RUNX3 (MAB3765, R&D Systems), TAL1 (sc-12984, Santa Cruz Biotechnology), MYB (05-175, EMD Millipore), NOTCH1 (Val1744, Cell Signaling Technology), MYC (N262, Santa Cruz Biotechnology), or extracellular signal-regulated kinase 1/2 (9102, Cell Signaling Technology).

Techniques: Knockdown, Isolation, Western Blot, Control, Infection, Expressing, shRNA, Quantitative RT-PCR, Staining, Flow Cytometry

Journal: Blood

Article Title: RUNX1 is required for oncogenic Myb and Myc enhancer activity in T-cell acute lymphoblastic leukemia

doi: 10.1182/blood-2017-03-775536

Figure Lengend Snippet: RUNX1 is required for intracellular NOTCH1 binding and for chromatin accessibility at the N-Me. (A) H3K27ac, NOTCH1, and RUNX1 enrichment at the human MYC super-enhancer is shown in genome browser tracks (genome.ucsc.edu, human hg19). (B) The mouse genomic region (mm10) encompassing Myc and its enhancer loci located 1.27 Mb from the TSS are shown. The RBPJ- and RUNX-binding sites are depicted. (C) Recruitment of RUNX1, intracellular NOTCH1, H3K27ac, and H3 to the mouse Myc enhancer was determined by ChIP-qPCR in control or Runx1-deleted mouse T-ALLs. (D) The degree of open chromatin at the N-Me enhancer region in control or Runx1-deleted mouse T-ALLs was determined by ATAC-quantitative PCR. Data are the mean of 3 or 4 independent experiments and error bars represent ± SEM. *P < .05; **P < .005; ***P < .0005, two-way ANOVA multiple comparisons test.

Article Snippet: 2 Immunoblotting and coimmunoprecipitation To examine protein expression in human T-ALL cells, cells were lysed in modified radioimmunoprecipitation assay buffer, transferred to a membrane, and probed with antibodies to RUNX1 (ab23980, Abcam), RUNX3 (MAB3765, R&D Systems), TAL1 (sc-12984, Santa Cruz Biotechnology), MYB (05-175, EMD Millipore), NOTCH1 (Val1744, Cell Signaling Technology), MYC (N262, Santa Cruz Biotechnology), or extracellular signal-regulated kinase 1/2 (9102, Cell Signaling Technology).

Techniques: Binding Assay, ChIP-qPCR, Control, Real-time Polymerase Chain Reaction

Journal: Blood

Article Title: RUNX1 is required for oncogenic Myb and Myc enhancer activity in T-cell acute lymphoblastic leukemia

doi: 10.1182/blood-2017-03-775536

Figure Lengend Snippet: RUNX1 mediates survival in a TAL1-negative, TLX3-transformed human T-ALL cell line, and RUNX3 also contributes to human T-ALL survival. (A) The human T-ALL cell line HPB-ALL was transduced with lentiviruses expressing shRNAs against GFP or RUNX1. Apoptotic cells were quantified by Annexin V/7AAD staining followed by flow cytometry. Data are shown as the mean of 3 independent experiments with error bars representing ± SEM. (B) Gene expression in control or RUNX1 knockdown cells was determined by qRT-PCR. Three independent experiments were performed, and data are shown as means with error bars representing ± SEM. (C) RUNX1 or RUNX3 knockdown in KOPTK1 cells induces cell death. The human T-ALL KOPTK1 cell line was transduced with lentiviruses expressing shRNAs against GFP, RUNX1, or RUNX3. Apoptotic cells were quantified by Annexin V/7AAD staining followed by flow cytometry. (D) RUNX1 and RUNX3 binding to N-Me, MYB +14-kb, and MYB –93-kb enhancer loci was determined by ChIP-qPCR. Data are shown as the mean of 4 independent experiments with error bars representing ± SEM (*P < .05, multiple Student t tests). (E-F) The expression of MYB and MYC in RUNX1- or RUNX3-silenced KOPTK1 cells was determined by qRT-PCR. Data are shown as the mean of 3 or 4 independent experiments with error bars representing ± SEM. *P <.05; **P < .005; ***P < .0005, one-way ANOVA multiple comparisons test.

Article Snippet: 2 Immunoblotting and coimmunoprecipitation To examine protein expression in human T-ALL cells, cells were lysed in modified radioimmunoprecipitation assay buffer, transferred to a membrane, and probed with antibodies to RUNX1 (ab23980, Abcam), RUNX3 (MAB3765, R&D Systems), TAL1 (sc-12984, Santa Cruz Biotechnology), MYB (05-175, EMD Millipore), NOTCH1 (Val1744, Cell Signaling Technology), MYC (N262, Santa Cruz Biotechnology), or extracellular signal-regulated kinase 1/2 (9102, Cell Signaling Technology).

Techniques: Transformation Assay, Transduction, Expressing, Staining, Flow Cytometry, Gene Expression, Control, Knockdown, Quantitative RT-PCR, Binding Assay, ChIP-qPCR

Journal: PLOS ONE

Article Title: Critical domains for NACC2-NTRK2 fusion protein activation

doi: 10.1371/journal.pone.0301730

Figure Lengend Snippet: (A) Schematic presents NACC2, NTRK2, and NACC2-NTRK2 fusion with K572R kinase dead mutation (KD). “L” refers to Leucine-rich domain, “IG1” and “IG2” refer to Immunoglobulin-like domains 1 and 2, “BTB” refers to Broad-Complex, Tramtrack and Bric a brac domain, “BEN” refers to an adapter domain found in BANP, E5R, and NACC1 proteins, “TM” refers to the Transmembrane helix, and “Kinase” refers to the tyrosine kinase domain of NTRK3. The kinase dead (KD) mutation K572R is also shown in NTRK2 and NACC2-NTRK2. Placement of domains is only approximate. (B) Representative plates of NIH3T3 cell transformation assays are shown for NTRK2, NACC2-NTRK2, and NACC2-NTRK2(KD). BCR-FGFR1 is included as positive control. In this experiment, all constructs were assayed in three independent replicates, except for NACC2-NTRK2 which was assayed six times, and BCR-FGFR1 which was assayed five times. (C) Results of NIH3T3 focus assays are presented. Each construct was assayed a minimum of three times, and the ratio of foci/G418-resistant colonies was calculated as a percentage of transformation +/- SEM relative to BCR-FGFR1. The P value of a two-tailed paired t test comparing NACC2-NTRK2 with BCR-FGFR1 was 0.011, and is shown as * = P ≤0.05. (D) The activation of downstream signaling pathways is presented. HEK293T cell lysates expressing NTRK2, NTRK2(KD), NACC2-NTRK2, and NACC2-NTRK2(KD) were analyzed by SDS-PAGE and immunoblotted for P-NTRK (top panel), total NTRK2 (2 nd panel), P-MAPK (3 nd panel), total MAPK (4 th panel), P-PLCγ1 (5th panel, upper band), total PLCγ1(6 th panel), P-STAT3(7 th panel) and total STAT3(8 th panel). Note that in this figure, and other immunoblots of P-PLCγ1, the band of interest migrates above the 130 kD marker; the prominent band near the bottom of this blot represents antiserum cross-reactivity with the activated NTRK2 kinase domain.

Article Snippet: Antibodies were purchased as following: NACC2 (Bethyl A304-991A), P-NTRK (Cell Signaling 4621S), NTRK2 (Invitrogen PA5-86241), P-MAPK (Cell Signaling 4370), MAPK (Cell Signaling 9102), P-PLCγ1 (Cell Signaling 2821), PLCγ1 (Santa Cruz sc-81), P-STAT3 (Cell Signaling 9145), STAT3 (Cell Signaling 9139).

Techniques: Mutagenesis, Transformation Assay, Positive Control, Construct, Two Tailed Test, Activation Assay, Protein-Protein interactions, Expressing, SDS Page, Western Blot, Marker

Journal: PLOS ONE

Article Title: Critical domains for NACC2-NTRK2 fusion protein activation

doi: 10.1371/journal.pone.0301730

Figure Lengend Snippet: (A) The interaction of NACC2 and NACC2-NTRK2 is examined. Lysates of HEK293T cells expressing NACC2-NTRK2 (designated as WT), or with the mutations D31N/R45Q or Y86A, were coexpressed with NACC2 as indicated (Lanes 4–6). Samples were prepared in E1A buffer to preserve protein-protein interactions. The 3 rd panel presents the results of immunoprecipitation using antibodies against NTRK2, to recover the NACC2-NTRK fusion proteins, followed by immunoblotting to detect NACC2 present in the immune complexes. Lane 4 clearly shows binding of NACC2 to NACC2-NTRK2, which is reduced in the presence of the mutations D31N/R45Q (lane 5), or Y86A (lane 6). Control immunoblots are shown for NTRK2 (top panel) and NACC2 (middle panel). (B) The ratio of the intensity of NACC2 relative to the NACC2-NTRK2 fusion with which it was co-immunoprecipitated is shown, using intensities of multiple replicates quantitated by ImageJ. The P values of two-tailed paired t tests are shown: For NACC2 bound to NACC2-NTRK2 versus NACC2(D31N/R45Q)-NTRK2, P = 0.034, indicated as * = P ≤0.05; for NACC2 bound to NACC2-NTRK2 versus NACC2(Y86A)-NTRK2, P = 0.003, indicated as ** = P ≤0.01. (C) HEK293T cells expressing NACC2-NTRK2, NACC2-NTRK2(KD), NACC2(D31N/R45Q)-NTRK2, NACC2(Y86A)-NTRK2 or NACC2(ΔBTB)-NTRK2 were examined for the activation of downstream signaling pathways. Cell lysates were analyzed by SDS-PAGE and immunoblotted for P-NTRK (top panel), total NTRK2 (2 nd panel), P-MAPK (3 nd panel), total MAPK (4 th panel), P-PLCγ1 (5th panel, upper band), total PLCγ1 (6 th panel), P-STAT3(7 th panel) and total STAT3(8 th panel). (D) The immunoblots presented in (C), together with additional independent replicates, were quantitated by ImageJ and used to calculate the changes in P-NTRK2, P-MAPK, P-PLCγ1, and P-STAT3, after normalization of each sample in comparison to total NTRK2, MAPK, PLCγ1, and STAT3. The P values of two-tailed paired t tests are shown for wild-type NACC2-NTRK2 versus each of the mutants KD, D31N/R45Q, Y86A, and ΔBTB. Statistical significance is indicated as follows: ns = not significant; * = P ≤0.05; ** = P ≤0.01; *** = P ≤0.001; **** = P ≤0.0001. Four independent replicates were used to calculate the changes in P-NTRK2 relative to NTRK2, and three independent replicates were used for changes in P-MAPK, P-PLCγ1, and P-STAT3. (E) The BTB domain mutants of NACC2-NTRK2 were examined for NIH3T3 cell transformation activity. In this experiment, all constructs were assayed in three independent replicates, except for NACC2-NTRK2 which was assayed six times. The ratio of foci/G418-resistant colonies was calculated as a percentage of transformation +/- SEM relative to NACC2-NTRK2, which was set to 100%.

Article Snippet: Antibodies were purchased as following: NACC2 (Bethyl A304-991A), P-NTRK (Cell Signaling 4621S), NTRK2 (Invitrogen PA5-86241), P-MAPK (Cell Signaling 4370), MAPK (Cell Signaling 9102), P-PLCγ1 (Cell Signaling 2821), PLCγ1 (Santa Cruz sc-81), P-STAT3 (Cell Signaling 9145), STAT3 (Cell Signaling 9139).

Techniques: Expressing, Protein-Protein interactions, Immunoprecipitation, Western Blot, Binding Assay, Control, Two Tailed Test, Activation Assay, SDS Page, Comparison, Transformation Assay, Activity Assay, Construct

Journal: PLOS ONE

Article Title: Critical domains for NACC2-NTRK2 fusion protein activation

doi: 10.1371/journal.pone.0301730

Figure Lengend Snippet: (A) NACC2-NTRK2 is shown schematically with the BTB domain from residues 20–120, a partial BEN domain at residues 351–418, and the disorder region (DR) in between. Also shown in comparison is the deletion of residues 120–418 of NACC2, which retains only the N-terminal BTB domain, designated BTB-NTRK2. (B) Downstream activation by BTB-NTRK2. HEK293T cells expressing NACC2-NTRK2 or BTB-NTRK2 were examined for activation of downstream signaling pathways, as described previously for Figs and . (C) The immunoblots presented in (B), together with additional independent replicates, were quantitated by ImageJ and used to calculate the changes in P-NTRK2, P-MAPK, P-PLCγ1, and P-STAT3, after normalization of each sample in comparison to total NTRK2, MAPK, PLCγ1, and STAT3. The P values of two-tailed paired t tests are shown for wild-type NACC2-NTRK2 versus BTB-NTRK2. Statistical significance is indicated as follows: ns = not significant; * = P ≤0.05; ** = P ≤0.01. Three independent replicates were used for each condition, except for P-STAT3 for which five independent replicates were available. (D) Lysates of HEK293T cells expressing NACC2-NTRK2 or BTB-NTRK2 were examined for homo-multimerization. Samples were loaded using non-reducing sample buffer (left panel), or reducing sample buffer containing β-mercaptoethanol (right panel), and then analyzed by SDS-PAGE and immunoblotted for NTRK2. (E) Hetero-multimerization assay of NACC2-NTRK2 and BTB-NTRK2. Fusion clones were cotransfected with NACC2 (lanes 4 and 5) and examined for their ability to bind to NACC2 in an immune complex prepared using antibodies against NTRK2. The 3 rd panel shows clearly the binding of NACC2 to NACC2-NTRK2 (lane 4), indicated by a red arrowhead. NACC2 also clearly binds to BTB-NTRK2 (lane 5), again indicated by a red arrowhead. Control lysate blots are shown for NTRK2 (1 st panel) and NACC2 (2 nd panel). Control IP immunoblot is shown in 4 th panel for NTRK2. (F) The BTB-NTRK2 fusion was compared with NACC2-NTRK2 in NIH3T3 cells transformation assays. In this experiment, NACC2-NTRK2 was assayed in six independent replicates, and BTB-NTRK2 was assayed three times. The ratio of foci/G418-resistant colonies was calculated as a percentage of transformation +/- SEM relative to NACC2-NTRK2, which was set to 100%. The P value of a two-tailed paired t test comparing NACC2-NTRK2 with BTB-NTRK2 was 0.00091, and is shown as *** = P ≤0.001.

Article Snippet: Antibodies were purchased as following: NACC2 (Bethyl A304-991A), P-NTRK (Cell Signaling 4621S), NTRK2 (Invitrogen PA5-86241), P-MAPK (Cell Signaling 4370), MAPK (Cell Signaling 9102), P-PLCγ1 (Cell Signaling 2821), PLCγ1 (Santa Cruz sc-81), P-STAT3 (Cell Signaling 9145), STAT3 (Cell Signaling 9139).

Techniques: Comparison, Activation Assay, Expressing, Protein-Protein interactions, Western Blot, Two Tailed Test, SDS Page, Clone Assay, Binding Assay, Control, Transformation Assay

Journal: PLOS ONE

Article Title: Critical domains for NACC2-NTRK2 fusion protein activation

doi: 10.1371/journal.pone.0301730

Figure Lengend Snippet: (A) NACC2-NTRK2 examined thus far in this work is shown as NACC2-NTRK2(ex4:ex13), in comparison with two derivatives: NACC2-NTRK2-ΔTM, deleting just the transmembrane helix, and NACC2-NTRK2(ex4:ex15) which deletes NTRK2 exons 13 and 14. (B) HEK293T cells expressing NACC2-NTRK2, NACC2-NTRK2-ΔTM and NACC2-NTRK2(ex4:ex15) were examined for activation of downstream signaling pathways, as described previously for Figs and . (C) The immunoblots presented in (B), together with additional independent replicates, were quantitated by ImageJ and used to calculate the changes in P-NTRK2, P-MAPK, P-PLCγ1, and P-STAT3, after normalization of each sample in comparison to total NTRK2, MAPK, PLCγ1, and STAT3. The P values of two-tailed paired t tests are shown for wild-type NACC2-NTRK2 versus each of the mutants KD and ΔTM. Statistical significance is indicated as follows: ns = not significant; * = P ≤0.05; ** = P ≤0.01; *** = P ≤0.001; **** = P ≤0.0001. Three independent replicates were used for each condition. (D) The biological activity of NACC2-NTRK2(ex4:ex13) was compared to NACC2-NTRK2-ΔTM using NIH3T3 cell transformation assays. In this experiment, both NACC2-NTRK2(ex4:ex13) and NACC2-NTRK2-ΔTM were assayed in six independent replicates. The ratio of foci/G418-resistant colonies was calculated as a percentage of transformation +/- SEM relative to NACC2-NTRK2(ex4:ex13), which was set to 100%. The P value of a two-tailed paired t test comparing NACC2-NTRK2(ex4:ex13) with NACC2-NTRK2-ΔTM was 0.0096, and is shown as ** = P ≤0.01. (E) Lysates prepared from cells expressing either NACC2-NTRK2 (left lanes) or NACC2-NTRK2-ΔTM (right lanes) and treated with cycloheximide for the indicated time points were examined by SDS-PAGE and immunoblotted for NTRK2 The left lanes show decreasing amounts of NACC2-NTRK2 in the presence of cycloheximide, while the right lanes reveal little or no change in NACC2-NTRK2-ΔTM under identical conditions. (F) The half-life of NACC2-NTRK2(ex4:13) was determined to be approximately 18 hours, that of NACC2-NTRK2(ex4:15) approximately 42 hours, while the half-life of NACC2-NTRK2-ΔTM could not be determined due to its stability over the time course of this experiment. Quantitation was accomplished using ImageJ with a minimum of three replicates for each sample, and half-lives were determined by least squares analysis.

Article Snippet: Antibodies were purchased as following: NACC2 (Bethyl A304-991A), P-NTRK (Cell Signaling 4621S), NTRK2 (Invitrogen PA5-86241), P-MAPK (Cell Signaling 4370), MAPK (Cell Signaling 9102), P-PLCγ1 (Cell Signaling 2821), PLCγ1 (Santa Cruz sc-81), P-STAT3 (Cell Signaling 9145), STAT3 (Cell Signaling 9139).

Techniques: Comparison, Expressing, Activation Assay, Protein-Protein interactions, Western Blot, Two Tailed Test, Activity Assay, Transformation Assay, SDS Page, Quantitation Assay

Journal: mBio

Article Title: Unraveling the cryptic functions of mitogen-activated protein kinases Cpk2 and Mpk2 in Cryptococcus neoformans

doi: 10.1128/mbio.01156-24

Figure Lengend Snippet: Mpk2 functions as an Mpk1 paralog for maintaining the CWI. ( A ) The wild-type strain (H99) was grown to an OD 600 of 0.8 before transitioning to a fresh yeast extract-peptone-dextrose (YPD) medium containing 1% CFW. Cells were harvested at intervals of 0, 30, 60, 90, and 120 min and subjected to RNA extraction and cDNA synthesis. Expression levels of MPK1 and MPK2 were determined via quantitative reverse transcription-PCR (qRT-PCR) in three biological replicates. ( B ) MPK2 expression levels were quantified by qRT-PCR with three biological replicates in the wild-type (H99) and mpk1 Δ P H3 :MPK2 (YSB9585 and YSB9586) strains. ( C ) Following overnight growth, cells from wild-type (H99), mpk1 Δ (KK3), mpk2 Δ (YSB3236), mpk1 Δ mpk2 Δ (YSB8245), and mpk1 Δ P H3 :MPK2 (YSB9575) strains were serially diluted to 10 −4 and spotted on solid media containing various stressors: 0.03% Congo Red (CR), 2.5 mg/mL CFW, 32 µg/mL anidulafungin, or 32 µg/mL micafungin. After 2 days, the plates were imaged. ( D ) Wild-type, mpk1 Δ, mpk2 Δ, and mpk1 Δ mpk2 Δ strains were cultured overnight, synchronized to OD 600 0.2, and further grown to 0.8 with fresh YPD. The strains were then treated with 1% CFW for 0, 30, and 60 min. After specified times, 40 mL of cells was collected and frozen with liquid nitrogen. Whole proteins were extracted and immunoblotted with phospho-p44/42 antibody. Phosphorylated Mpk1 and Mpk2 levels were measured using the chemiluminescence method, after which the membranes were stripped and reprobed with anti-actin antibody to quantify total proteins. Protein quantification was conducted using the ImageLab program. ( E ) Wild-type, mpk1 Δ, mpk2 Δ, mpk1 Δ mpk2 Δ, and mpk1 Δ P H3 :MPK2 strains were cultured overnight and adjusted to an OD 600 of 0.2 and then grown for 24 h in YPD at 30°C and fixed with formaldehyde solution. Cells were stained with CFW in the dark, washed, and imaged using differential interference contrast (DIC) and DAPI (4′,6-diamidino-2-phenylindole) filters. Chitin content in the membrane of randomly chosen 50 cells was assessed using ImageJ. ( F ) The aforementioned strains in panels D and E were grown in capsule-inducing Littman medium for 2 days. Cells were then stained with India ink, and the capsular thickness of 50 randomly selected cells was measured using NIS-elements software. The indicated white scale bar is 10 µm. Statistical differences were determined using one-way ANOVA and Tukey’s test: *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001; NS = not significant.

Article Snippet: First antibodies were used as follows: phospho-p44/42 MAPK antibody (Cell signaling, #4370, 2,000:1), anti β-actin antibody (sc-47778, 1,000:1), and anti α-tubulin antibody (sc-8035, 1,000:1).

Techniques: RNA Extraction, cDNA Synthesis, Expressing, Reverse Transcription, Quantitative RT-PCR, Cell Culture, Staining, Membrane, Software

Journal: mBio

Article Title: Unraveling the cryptic functions of mitogen-activated protein kinases Cpk2 and Mpk2 in Cryptococcus neoformans

doi: 10.1128/mbio.01156-24

Figure Lengend Snippet: Mpk2 is regulated by MAP2K Mkk2 and MAP3Ks Ssk2 and Ste11. ( A and B ) Wild-type, mpk1 Δ, mpk2 Δ, bck1 Δ (YSB273), mkk2 Δ (YSB330), ssk2 Δ (YSB264), and ste11 Δ (YSB313) strains were grown overnight, resuspended with fresh yeast extract-peptone-dextrose (YPD) medium at an OD 600 of 0.2, and further incubated to an OD of 0.8. Subsequently, these strains underwent a treatment with 1% CFW for intervals of 0, 30, and 60 min. Following each designated time point, 40 mL of the cell cultures was spun down and flash-frozen in liquid nitrogen. Total proteins were then extracted for immunoblot analysis using an anti phospho-p44/42 antibody. The levels of phosphorylated Mpk1 and Mpk2 were detected by chemiluminescence. After detection, the blots were stripped and re-probed for actin or tubulin to assess the total protein levels. ( C ) A schematic of the proposed Mpk1/Mpk2 regulatory mechanisms for maintaining CWI in C. neoformans .

Article Snippet: First antibodies were used as follows: phospho-p44/42 MAPK antibody (Cell signaling, #4370, 2,000:1), anti β-actin antibody (sc-47778, 1,000:1), and anti α-tubulin antibody (sc-8035, 1,000:1).

Techniques: Incubation, Western Blot

Journal: Oncotarget

Article Title: P73 tumor suppressor and its targets, p21 and PUMA, are required for madin-darby canine kidney cell morphogenesis by maintaining an appropriate level of epithelial to mesenchymal transition

doi:

Figure Lengend Snippet: A. Knockdown of TAp73 decreases, whereas knockdown of ΔNp73 increases, the expression of p21 and PUMA. The levels of p21, PUMA, and actin were measured in parental MDCK, MDCK-TAp73-KD, and MDCK-ΔNp73-KD cells by Western blotting. B. Generation of MDCK cell lines in which p21 was stably knocked down. Parental MDCK and MDCK-p21-KD cells were mock-treated or treated with camptothecin for 18h and the level of p21 and actin protein was measured by Western blotting. C. Representative microscopic images of parental MDCK and MDCK-p21-KD cells in 2-D cultures. D. Top panel: colony formation assay was performed with parental MDCK or MDCK-p21-KD cells. Bottom panel: the number of colonies was counted and presented as Mean ± S.D. from three separate experiments. E. Wound healing assay was performed with parental MDCK and MDCK-p21-KD cells. Top panel: cell migration was determined by visual assessment of cells migrating into the wound for 24 h using a phase-contrast microscopy. The dashed lines indicate the wound edge. Bottom panel: the time required for would closure was measured and presented as Mean ± s.d. from three separate experiments. F. Representative images of parental MDCK and MDCK-p21-KD cells in 3-D culture for 6 d or 12 d. Scale bar: 50 μM.

Article Snippet: Antibodies used were purchased from Bethyl (anti-p73), ProSci (anti-PUMA), Santa Cruz Biotechnology (anti-p21 (H164), anti-β-catenin (E-5), anti-Snail-1, anti-Twist), BD Transduction Laboratories (anti-E-cadherin, San Jose, CA), Sigma (anti-actin, St. Louis, MO), and BioRad (secondary antibodies against rabbit or mouse IgG conjugated with HRP, Life Science Research, Hercules, CA).

Techniques: Expressing, Western Blot, Stable Transfection, Colony Assay, Wound Healing Assay, Migration, Microscopy

Journal: Oncotarget

Article Title: P73 tumor suppressor and its targets, p21 and PUMA, are required for madin-darby canine kidney cell morphogenesis by maintaining an appropriate level of epithelial to mesenchymal transition

doi:

Figure Lengend Snippet: A. - D. The levels of β-catenin, E-cadherin, Snail, Twist, and actin were determined by Western blotting with extracts from parental MDCK (A-D), MDCK-TAp73-KD A. , MDCK-p21-KD B. , MDCK-PUMA-KD C. , and MDCK-ΔNp73-KD D. cells. E. The levels of E-cadherin, Snail and Twist transcripts were measured by qRT-PCR in parental MDCK cells and MDCK cells with knockdown of TAp73, ΔNp73, p21 or PUMA. The level of Actin was measured as an internal control. F. A model for the role of p73, p21 and PUMA in MDCK cell morphogenesis.

Article Snippet: Antibodies used were purchased from Bethyl (anti-p73), ProSci (anti-PUMA), Santa Cruz Biotechnology (anti-p21 (H164), anti-β-catenin (E-5), anti-Snail-1, anti-Twist), BD Transduction Laboratories (anti-E-cadherin, San Jose, CA), Sigma (anti-actin, St. Louis, MO), and BioRad (secondary antibodies against rabbit or mouse IgG conjugated with HRP, Life Science Research, Hercules, CA).

Techniques: Western Blot, Quantitative RT-PCR

Journal: Frontiers in Pharmacology

Article Title: Pterostilbene attenuates lung ischemia-reperfusion injury: integrative insights from network pharmacology, molecular dynamics, and experimental validation

doi: 10.3389/fphar.2026.1747977

Figure Lengend Snippet: Chemical structure of PTE and Venn analysis. (A) The 2D chemical structure of Pterostilbene (C 16 H 16 O 3 ), CAS number: 537-42-8. (B) Venn diagram representing the overlapping of PTE targets (blue) and LIRI targets (yellow). PTE: pterostilbene, 2D: two-dimensional, CAS: chemical abstracts service.

Article Snippet: Pterostilbene (PTE, HY-N0828), LY294002 (LY, HY-10108), and Anisomycin (Ani, HY-18982) were all purchased from MedChemExpress (MCE, United States), and dissolved in dimethyl sulfoxide (DMSO, D8371, Solarbio, China) before dilution to the desired concentrations for subsequent experiments.

Techniques:

Journal: Frontiers in Pharmacology

Article Title: Pterostilbene attenuates lung ischemia-reperfusion injury: integrative insights from network pharmacology, molecular dynamics, and experimental validation

doi: 10.3389/fphar.2026.1747977

Figure Lengend Snippet: PTE mitigates IR-induced lung injury in rats. (A) Representative images showing gross morphology, HE staining, MPO immunohistochemistry (IHC), and TUNEL staining of rat lungs following 1 h of left hilar clamping and 2 h of reperfusion. PTE was administered as a single intraperitoneal injection at the onset of reperfusion (30 mg/kg for the low-dose group, PTE-L; 60 mg/kg for the high-dose group, PTE-H). The Sham group underwent no hilar occlusion, whereas the IR group received an equivalent volume of saline. (B) Pulmonary edema after reperfusion was assessed by the lung W/D ratio. (C–E) Histopathological damage was quantitatively evaluated using lung injury score, the number of MPO-positive cells, and the number of TUNEL-positive cells. (F–I) Concentrations of TNF-α, IL-1β, IL-6, and IL-10 in lung tissue homogenates were measured by ELISA. (J–M) Relative mRNA expression levels of EGFR, MAPK8, PIK3CB and SRC were determined by qRT-PCR. HE staining: scale bar = 100 μm; MPO staining: scale bar = 50 μm; TUNEL staining: scale bar = 100 μm. Data are presented as the mean ± SD (n = 6). * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001, vs. Sham group; # P < 0.05, ## P < 0.01, ### P < 0.001, #### P < 0.0001, vs. IR group; n.s = non-significant. PTE: pterostilbene, IR: ischemia-reperfusion, W/D: wet to dry, HE: hematoxylin and eosin, MPO: myeloperoxidase, TUNEL: terminal deoxynucleotidyl transferase dUTP nick end labeling, ELISA: enzyme-linked immunosorbent assay, qRT-PCR: quantitative real-time polymerase chain reaction.

Article Snippet: Pterostilbene (PTE, HY-N0828), LY294002 (LY, HY-10108), and Anisomycin (Ani, HY-18982) were all purchased from MedChemExpress (MCE, United States), and dissolved in dimethyl sulfoxide (DMSO, D8371, Solarbio, China) before dilution to the desired concentrations for subsequent experiments.

Techniques: Staining, Immunohistochemistry, TUNEL Assay, Injection, Saline, Enzyme-linked Immunosorbent Assay, Expressing, Quantitative RT-PCR, Real-time Polymerase Chain Reaction

Journal: Frontiers in Cellular and Infection Microbiology

Article Title: Host MicroRNA hsa-miR-494-3p Promotes EV71 Replication by Directly Targeting PTEN

doi: 10.3389/fcimb.2018.00278

Figure Lengend Snippet: Effects of over-expression of hsa-miR-494-3p on EV71 infection. RD and HEK 293 cells were transfected with hsa-miR-494-3p mimics or the negative control (NC) for 48 h, and then followed by EV71 infection for 2, 4, 6, 12 h at an MOI of 2. Virus titers in culture supernatants from RD (A) and HEK 293 (B) were measured at the indicated time post-infection. The cells were collected at different time points as indicated. RD (C) and HEK 293 (D) cell lysates were blotted with anti-EV71 VP1 and anti-β actin antibodies. β actin was used as an internal control. Next, qRT-PCR was employed to determine the EV71 VP1 mRNA levels in RD (E) and HEK 293 (F) cells. The data shown are the mean ± standard deviation. The experiments were repeated three times († P < 0.05, * p < 0.01 by ANOVA).

Article Snippet: The mouse polyclonal antibody against EV71 VP1 (~36 kDa, Abcam), the mouse monoclonal antibodies against PTEN (~54 kDa, Cell Signaling) and β-actin (~42 kDa, Abcam), the rabbit monoclonal antibodies against Akt (~60 kDa, Cell Signaling) and phosphor-Akt (~60 kDa, Cell Signaling), and the goat anti-mouse and anti-rabbit IgGs conjugated to horseradish peroxidase (HRP).

Techniques: Over Expression, Infection, Transfection, Negative Control, Virus, Control, Quantitative RT-PCR, Standard Deviation

Journal: Frontiers in Cellular and Infection Microbiology

Article Title: Host MicroRNA hsa-miR-494-3p Promotes EV71 Replication by Directly Targeting PTEN

doi: 10.3389/fcimb.2018.00278

Figure Lengend Snippet: Effects of hsa-miR-494-3p inhibitors on EV71 infection. RD and HEK 293 cells were transfected with hsa-miR-494-3p inhibitors or the negative control (NC) for 48 h, and then followed by EV71 infection for 2, 4, 6, 12 h at an MOI of 2. Virus titers in culture supernatants from RD (A) and HEK 293 (B) were measured at the indicated time post-infection. The cells were collected at different time points as indicated. RD (C) and HEK 293 (D) cell lysates were blotted with anti-EV71 VP1 and anti-β actin antibodies. β actin was used as an internal control. Next, qRT-PCR was employed to determine the EV71 VP1 mRNA levels in RD (E) and HEK 293 (F) cells. The data shown are the mean ± standard deviation. The experiments were repeated three times ( * p < 0.01 by ANOVA).

Article Snippet: The mouse polyclonal antibody against EV71 VP1 (~36 kDa, Abcam), the mouse monoclonal antibodies against PTEN (~54 kDa, Cell Signaling) and β-actin (~42 kDa, Abcam), the rabbit monoclonal antibodies against Akt (~60 kDa, Cell Signaling) and phosphor-Akt (~60 kDa, Cell Signaling), and the goat anti-mouse and anti-rabbit IgGs conjugated to horseradish peroxidase (HRP).

Techniques: Infection, Transfection, Negative Control, Virus, Control, Quantitative RT-PCR, Standard Deviation

Journal: Frontiers in Cellular and Infection Microbiology

Article Title: Host MicroRNA hsa-miR-494-3p Promotes EV71 Replication by Directly Targeting PTEN

doi: 10.3389/fcimb.2018.00278

Figure Lengend Snippet: PTEN is a target of EV71-induced hsa-miR-494-3p. (A) The putative hsa-miR-494-3p binding site within the PTEN 3′ UTR is predicted by TargetScan, and the mutation form is indicated. RD and HEK 293 cells were transiently co-transfected with luciferase reporter plasmids containing the hsa-miR-494-3p target sites (wt PTEN 3′UTR) or corresponding mutants (mt PTEN 3′UTR) and hsa-miR-494-3p mimics, or negative control, and cultured in a standard growth medium. After 24 h, luciferase reporter assays were performed in RD (B) and HEK 293 cells (C) . The data shown are the mean ± standard deviation. The cells were transiently transfected with hsa-miR-494-3p mimics, inhibitors, or negative control. After 24 h, the RD (D) and HEK 293 (E) cells were collected, and then the lysates were blotted with anti-phospho Akt, anti-PTEN, anti-Akt, and anti-β actin antibodies. The cells were exposed to EV71 infection for 0.5, 3, 6, 12 h at an MOI of 2. The RD (F) and HEK 293 (G) cell lysates were blotted with anti-PTEN and anti-β actin antibodies. β actin was used as an internal control. The experiments were repeated three times ( p < 0.01 by ANOVA).

Article Snippet: The mouse polyclonal antibody against EV71 VP1 (~36 kDa, Abcam), the mouse monoclonal antibodies against PTEN (~54 kDa, Cell Signaling) and β-actin (~42 kDa, Abcam), the rabbit monoclonal antibodies against Akt (~60 kDa, Cell Signaling) and phosphor-Akt (~60 kDa, Cell Signaling), and the goat anti-mouse and anti-rabbit IgGs conjugated to horseradish peroxidase (HRP).

Techniques: Binding Assay, Mutagenesis, Transfection, Luciferase, Negative Control, Cell Culture, Standard Deviation, Infection, Control

Journal: Frontiers in Cellular and Infection Microbiology

Article Title: Host MicroRNA hsa-miR-494-3p Promotes EV71 Replication by Directly Targeting PTEN

doi: 10.3389/fcimb.2018.00278

Figure Lengend Snippet: Infection with EV71 resulted in the activation of PI3K/Akt signaling pathway in RD and HEK 293 cells. (A,B) RD and HEK293 cells were infected with EV71 at an MOI of 2. At the indicated time points post-infection, the cells were collected and lysed for blotting analysis to detect phospho Akt (p-Akt) and β actin. (C,D) RD and HEK293 cells were pre-incubated without or with Wortmannin (100 nM) for 1 h, and then followed by infection with EV71 at an MOI of 2. Wortmannin was present throughout the experiments. The cells and supernatants were collected separately at different time points as indicated. Cell lysates were blotted with anti-phospho Akt and anti-β actin antibodies. β actin was used as an internal control. (E,F) Virus titers in the supernatants collected in panels (C,D) were measured by TCID 50 . (G,H) qRT-PCR was employed to determine the EV71 VP1 mRNA levels in RD and HEK 293 cells. The data shown are the mean ± standard deviation. The experiments were repeated three times ( p < 0.01 by ANOVA).

Article Snippet: The mouse polyclonal antibody against EV71 VP1 (~36 kDa, Abcam), the mouse monoclonal antibodies against PTEN (~54 kDa, Cell Signaling) and β-actin (~42 kDa, Abcam), the rabbit monoclonal antibodies against Akt (~60 kDa, Cell Signaling) and phosphor-Akt (~60 kDa, Cell Signaling), and the goat anti-mouse and anti-rabbit IgGs conjugated to horseradish peroxidase (HRP).

Techniques: Infection, Activation Assay, Incubation, Control, Virus, Quantitative RT-PCR, Standard Deviation

Journal: Scientific Reports

Article Title: Riluzole regulates pancreatic cancer cell metabolism by suppressing the Wnt-β-catenin pathway

doi: 10.1038/s41598-022-13472-y

Figure Lengend Snippet: Inhibition of cell viability and colony formation, and induction of apoptosis by riluzole. ( A , B ), Pancreatic cancer cell lines (AsPC-1, PANC-1, and MIA PaCa-2), pancreatic cancer stem cells (Pan CSCs), and human normal pancreatic ductal epithelial (HPNE) cells were seeded in 96-well plates, treated with or without riluzole (0–40 μM) for 72 h and cell viability was measured by CellTiter-Glo Luminescent Cell Viability Assay (Promega). ( C ), Pancreatic cancer cell lines (AsPC-1, PANC-1, MIA PaCa-2 and Hs 766 T) and Pan CSCs were seeded in dishes and treated with riluzole (0–40 μM). Number of colonies formed at 21 days were counted as described in Material and Methods. ( D ), Pancreatic cancer cell lines (AsPC-1, PANC-1, MIA PaCa-2 and Hs 766 T) and CSCs were seeded and treated with riluzole (0–40 µM) for 72 h. Apoptosis was measured by TUNEL assay. Data represent mean ± SD (n = 4). *, #, and % = significantly different from control and each other; p < 0.05.

Article Snippet: Human pancreatic cancer stem cells (CSCs, CD24 + /CD44 + /CD133 +) were isolated from primary tumors and grown in stem cell culture medium as per the supplier’s instructions (obtained from Celprogen, Torrance, CA).

Techniques: Inhibition, Cell Viability Assay, TUNEL Assay, Control

Journal: Scientific Reports

Article Title: Riluzole regulates pancreatic cancer cell metabolism by suppressing the Wnt-β-catenin pathway

doi: 10.1038/s41598-022-13472-y

Figure Lengend Snippet: Riluzole regulates Wnt-β-catenin and mitochondrial pathways by RNA-Seq. Pancreatic cancer cells (MIA PaCa-2) were treated with Riluzole for 36 h. RNA was isolated. RNA-Seq was performed as described in Materials and methods. ( A ), Heat Map. Differential expression of genes in MIA PaCa-2 cells treated with or without riluzole. ( B ), Data analysis identified modulation of Wnt-β-catenin and mitochondrial pathways by riluzole in pancreatic cancer cells.

Article Snippet: Human pancreatic cancer stem cells (CSCs, CD24 + /CD44 + /CD133 +) were isolated from primary tumors and grown in stem cell culture medium as per the supplier’s instructions (obtained from Celprogen, Torrance, CA).

Techniques: RNA Sequencing, Isolation, Quantitative Proteomics

Journal: Scientific Reports

Article Title: Riluzole regulates pancreatic cancer cell metabolism by suppressing the Wnt-β-catenin pathway

doi: 10.1038/s41598-022-13472-y

Figure Lengend Snippet: Inhibition of β-catenin/TCF-LEF1 pathway by riluzole. ( A – F ), Pancreatic cancer MIA PaCa-2 cells were treated with riluzole (0–40 µM) for 36 h. RNA was extracted and the expression of β-catenin, Wnt3a, Wnt5a, WSP, TCF and LEF was measured by q-RT-PCR. Data represent mean ± SD (n = 4). *, and # = significantly different from control and each other; p < 0.05. ( G ), MIA PaCa-2 cells were stably transduced with TCF/LEF1-responsive GFP/firefly luciferase viral particles (pGreen Fire1-TCF/LEF1 with EF1, System Biosciences). After transduction, cells were treated with riluzole (0–40 µM) for 36 h. TCF/LEF1 reporter activity was measured by luciferase assay as we described . Data represent mean ± SD (n = 4). *, # and % = significantly different from control and each other; p < 0.05. ( H ), MIA PaCa-2 cells were treated with riluzole (0–40 µM) for 36 h. Cells were harvested and cytoplasmic and nuclear fractions were prepared. The expression of β-catenin in the cytoplasmic and nuclear fractions was measured by the Western blot analysis. β-Actin and Lamin B1 were used as loading controls in cytoplasmic and nuclear fractions, respectively. ( I–N ), Pancreatic cancer AsPC-1 cells were treated with riluzole (0–40 µM) for 36 h. RNA was extracted and the expression of β-catenin, Wnt3a, Wnt5a, WSP, TCF and LEF was measured by q-RT-PCR. Data represent mean ± SD (n = 4). *, # and % = significantly different from control and each other; p < 0.05. ( O ), AsPC-1 cells were stably transduced with TCF/LEF1-responsive GFP/firefly luciferase viral particles (pGreen Fire1-TCF/LEF1 with EF1, System Biosciences). After transduction, cells were treated with riluzole (0–40 µM) for 36 h. TCF/LEF1 reporter activity was measured by luciferase assay as we described . Data represent mean ± SD (n = 4). *, # and % = significantly different from control and each other; p < 0.05. ( P ), AsPC-1 cells were treated with riluzole (0–40 µM) for 36 h. Cells were harvested and cytoplasmic and nuclear fractions were prepared. The expression of β-catenin in the cytoplasmic and nuclear fractions was measured by the Western blot analysis. β-Actin and Lamin B1 were used as loading controls in cytoplasmic and nuclear fractions, respectively.

Article Snippet: Human pancreatic cancer stem cells (CSCs, CD24 + /CD44 + /CD133 +) were isolated from primary tumors and grown in stem cell culture medium as per the supplier’s instructions (obtained from Celprogen, Torrance, CA).

Techniques: Inhibition, Expressing, Reverse Transcription Polymerase Chain Reaction, Control, Stable Transfection, Transduction, Luciferase, Activity Assay, Western Blot

Journal: Scientific Reports

Article Title: Riluzole regulates pancreatic cancer cell metabolism by suppressing the Wnt-β-catenin pathway

doi: 10.1038/s41598-022-13472-y

Figure Lengend Snippet: Inhibition of TCF-LEF1 target genes and induction of growth arrest at G2/M stage by riluzole. ( A ), Pancreatic cancer MIA PaCa-2 and AsPC-1 cells were treated with riluzole (0–40 µM) for 36 h. RNA was isolated and the expression of cyclin D1 was measured by q-RT-PCR. Data represent mean ± SD (n = 4). *, # and % = significantly different from control and each other; p < 0.05. ( B ), Cell cycle analyses. MIA PaCa-2 and AsPC-1 cells were treated with riluzole (0–40 µM) for 48 h. Cells were harvested, fixed, stained with propidium iodide (PI) and analysed with flow cytometer. *, # and % = significantly different from control and each other; p < 0.05. ( C ), MIA PaCa-2 cells were treated with riluzole (0–40 µM) for 36 h. RNA was isolated and the expression of cMyc, Axin1, GSK3β, PDK1, and MCT1 was measured by q-RT-PCR. Data represent mean ± SD (n = 4). *, # and % = significantly different from control and each other; p < 0.05. ( D ), AsPC-1 cells were treated with riluzole (0–40 µM) for 36 h. RNA was isolated and the expression of cMyc, Axin1, GSK3β, PDK1, and MCT1 was measured by q-RT-PCR. Data represent mean ± SD (n = 4). *, # and % = significantly different from control and each other; p < 0.05.

Article Snippet: Human pancreatic cancer stem cells (CSCs, CD24 + /CD44 + /CD133 +) were isolated from primary tumors and grown in stem cell culture medium as per the supplier’s instructions (obtained from Celprogen, Torrance, CA).

Techniques: Inhibition, Isolation, Expressing, Reverse Transcription Polymerase Chain Reaction, Control, Staining, Flow Cytometry

Journal: Scientific Reports

Article Title: Riluzole regulates pancreatic cancer cell metabolism by suppressing the Wnt-β-catenin pathway

doi: 10.1038/s41598-022-13472-y

Figure Lengend Snippet: Riluzole inhibits Glut2 expression, glucose uptake, LDHA-A expression, and NAD + level. ( A ), Pancreatic cancer (PANC-1, Hs 766 T, MIA PaCa-2 and AsPC-1) cells and Pan CSCs were treated with riluzole (0–10 µM) for 36 h. RNA was extracted and the expression of Glut-2 was measured by q-RT-PCR. Data represent mean ± SD (n = 4). * = significantly different from control; p < 0.05. ( B ), Glucose uptake. Pancreatic cancer cells and CSCs were labelled with 2.5 µg/ml of 2-deoxy-2-[(7-nitro-2,1,3-benzoxadiazol-4-yl)amino]-D-glucose (2-NBD Glucose) for 30 min and treated with riluzole for 24 h. Cells were washed and resuspended in PBS. Fluorescence was measured (excitation 544 nm and emission at 590 nm). Data represent mean ± SD (n = 4). * = significantly different from control; P < 0.05. ( C ), Pancreatic cancer ( PANC-1, Hs 766 T, MIA PaCa-2 and AsPC-1) cells, and Pan CSCs were treated with riluzole (0–10 µM) for 36 h. RNA was isolated and the expression of LDH-A was measured by q-RT-PCR. Data represent mean ± SD (n = 4). * = significantly different from control; p < 0.05. ( D ), Pancreatic cancer ( PANC-1, Hs 766 T, MIA PaCa-2 and AsPC-1) cells were treated with riluzole (0–10 µM) for 1 h and total cellular NAD + concentration was measured at 450 nm by a NAD/NADH assay kit (Cayman). Data represent mean ± SD (n = 4). * = significantly different from control; p < 0.05.

Article Snippet: Human pancreatic cancer stem cells (CSCs, CD24 + /CD44 + /CD133 +) were isolated from primary tumors and grown in stem cell culture medium as per the supplier’s instructions (obtained from Celprogen, Torrance, CA).

Techniques: Expressing, Reverse Transcription Polymerase Chain Reaction, Control, Fluorescence, Isolation, Concentration Assay, Nad NADH Assay

Journal: Scientific Reports

Article Title: Riluzole regulates pancreatic cancer cell metabolism by suppressing the Wnt-β-catenin pathway

doi: 10.1038/s41598-022-13472-y

Figure Lengend Snippet: Requirement of glutamine, inhibition of glutamate efflux and GSH level, and upregulation of reactive oxygen species (ROS) in PDAC. ( A ), PANC1 cells were grown in the absence or presence of glutaminase (GLS) inhibitor (BPTES or DON, 5 mM) with glutamine (2 mM) in the medium. After 48 h, apoptosis was measured by TUNEL assay. Data represent mean ± SD (n = 4). * = significantly different from control, p < 0.05. ( B ), PANC-1 cells were grown in the presence (2 mM) or absence of glutamine for 48 h. Apoptosis was measured by TUNEL assay. Data represent mean ± SD (n = 4). * = significantly different from control, p < 0.05. ( C ), PANC-1, Hs 766 T, MIA PaCa-2, AsPC-1, and Pan CSCs were treated with riluzole (0–10 µM) for 24 h. Glutamate release was measured by the Amplex Red Glutamic Acid/Glutamate Oxidase Assay Kit with a fluorometer using excitation at 540 nm and emission at 590 nm (Invitrogen). Data represent mean ± SD (n = 4). * = significantly different from control, p < 0.05. ( D ), Pancreatic cancer PANC-1, Hs 7ssT, MIA PaCa-2, AsPC-1 cells and CSCs were treated with riluzole (0–10 µM) for 24 h. Intracellular total GSH was detected by measuring the product of glutathionylated DTNB at 405 nm (GSH assay kit, Cayman Chemical). Data represent mean ± SD (n = 4). * = significantly different from control, P < 0.05. ( E and F ), AsPC-1 and PANC-1 cells were pre-treated with NAC (3 mM) for 2 h, followed by treatment with riluzole (0–10 µM) for 24 h. Cells were labelled with 2’,7’-dichlorofluorescein diacetate (DCFDA / H2DCFDA) and ROS production was measured for various time points (0- 360 min) with a fluorometer using excitation at 495 nm and emission at 529 nm (Cellular Reactive Oxygen Species Detection Assay Kit, Abcam). Data represent mean ± SD (n = 4). *, @, #, $. %, &, and ** = significantly different from control; p < 0.05. ( G ), AsPC-1 and Pan CSCs were pre-treated with NAC (3 mM) for 2 h, followed by treatment with riluzole (0–10 µM) for 24 h. Cells were labelled with 2’,7’-dichlorofluorescein diacetate (DCFDA / H2DCFDA) and ROS production was measured at 120 min with a fluorometer using excitation at 495 nm and emission at 529 nm. Data represent mean ± SD (n = 4). * = significantly different from control and each other; p < 0.05.

Article Snippet: Human pancreatic cancer stem cells (CSCs, CD24 + /CD44 + /CD133 +) were isolated from primary tumors and grown in stem cell culture medium as per the supplier’s instructions (obtained from Celprogen, Torrance, CA).

Techniques: Inhibition, TUNEL Assay, Control, GSH Assay, Detection Assay

Journal: Scientific Reports

Article Title: Riluzole regulates pancreatic cancer cell metabolism by suppressing the Wnt-β-catenin pathway

doi: 10.1038/s41598-022-13472-y

Figure Lengend Snippet: Riluzole inhibits pancreatic cancer growth in KPC (Pdx1-Cre, LSL-Trp53 R172H , and LSL-Kras G12D ) transgenic mice. KPC mice (about 6 weeks old) were injected ip with riluzole (20 mg/kg, Monday through Friday) for 12 weeks. At the end of the treatment, mice were sacrificed. ( A ) Pancreas Weight. Mice were euthanized and pancreas weight was taken from Cre mice (normal, untreated), KPC mice (control, untreated), and KPC mice (treated with riluzole 20 mg/kg). Data represent mean ± SD (n = 7). * and # = significantly different from control group; p < 0.05. ( B ), Histological examination of the pancreas was performed by H&E staining. Numbers of PanINs and PDAC were quantified. Data represent mean ± SD (n = 7). *, #, %, and & = significantly different from control group; p < 0.05. X = not detected.

Article Snippet: Human pancreatic cancer stem cells (CSCs, CD24 + /CD44 + /CD133 +) were isolated from primary tumors and grown in stem cell culture medium as per the supplier’s instructions (obtained from Celprogen, Torrance, CA).

Techniques: Transgenic Assay, Injection, Control, Staining