Journal: Frontiers in bioengineering and biotechnology

Article Title: Extracellular Vesicles-Mimetic Encapsulation Improves Oncolytic Viro-Immunotherapy in Tumors With Low Coxsackie and Adenovirus Receptor.

doi: 10.3389/fbioe.2020.574007

Figure Lengend Snippet: FIGURE 1 | The relationship between CAR expression level and the Ad5 infection efficiency. (A) A series of cell lines (293T, A549, HCC-LM3, Hepa1-6, B16-F10, CT26.WT, H22, K562, and Jurkat cells) were stained with a monoclonal anti-CAR-PE antibody and subjected to flow cytometry to analyze the CAR expression level. A homologous IgG-PE antibody was used as the isotype control. (B) Genomic diagram of the non-replicative Ad5-GFP adenovirus. (C) 293T, A549, HCC-LM3, Hepa1-6, B16-F10, and CT26.WT cells were infected with Ad5-GFP for 72 h, and then, the cells were monitored under a fluorescence microscope (representative images are shown in the left panel) or subjected to FACS analysis. The infection efficiency in 293T cells was set to 100% to calculate the infection efficiency of Ad5 in each cell line. (D) 293T, H22, K562, and Jurkat cells were infected with Ad5-GFP at the indicated MOI. After 72 h, the cells were harvested and subjected to flow cytometry. The data are shown as the means ± SD. ∗∗∗P < 0.001.

Article Snippet: The mouse hepatocellular carcinoma cell line Hepa1-6, human embryonic kidney cell line 293T, human T-cell leukemia cell line Jurkat, human chronic myeloid leukemia cell line K562, human alveolar adenocarcinoma cell line A549, mouse melanoma cell line B16F10 and colon carcinoma cell line CT26.WT were obtained from the American Type Culture Collection (Manassas, VA, United States).

Techniques: Expressing, Infection, Staining, Cytometry, Control, Microscopy

Journal: Frontiers in bioengineering and biotechnology

Article Title: Extracellular Vesicles-Mimetic Encapsulation Improves Oncolytic Viro-Immunotherapy in Tumors With Low Coxsackie and Adenovirus Receptor.

doi: 10.3389/fbioe.2020.574007

Figure Lengend Snippet: FIGURE 2 | Preparation of extracellular vesicles-mimetic Ad5 (EVM/VSV-G Ad5). (A) Schematic diagram of EVM/VSV-G Ad5 preparation. (B) TEM photographs of Ad5-GFP and EVM/VSV-G Ad5-GFP viruses. The arrow in the lower panel points to the membrane protein spike of the particle. (C) Ad5-GFP and EVM/VSV-G Ad5-GFP viruses were harvested and purified by density gradient centrifugation, and then, CD63 CD9 and VSV-G protein was determined by western blotting. (D) Size distribution analysis of EVM/VSV-G Ad5-GFP by dynamic light scattering (NanoSight NS300). (E) 293T-VSV-G cells were infected with Ad5-GFP for 48 h, and then, the cells were evenly divided and subjected to either freeze-thaw cycles (open bar) or extracellular vesicles-mimetic production (filled bar). The viral titers were measured to calculate the amount of virus recovered. Fold increase compared with the freeze-thaw method is shown. (F) Ad5-GFP or EVM/VSV-G Ad5-GFP were incubated with Ad5 neutralizing antibodies or the VSV-G neutralizing serum then added to 293T cells for another 48 h. Then, green fluorescence-positive cells were either monitored with a fluorescence microscope (left panel) or analyzed by FACS (right panel). (G) 293T, Hepa1-6, B16/F10 and CT26.WT cells were infected with Ad5-GFP or EVM/VSV-G Ad5-GFP viruses at an MOI of 1, and GFP-positive cells were either monitored under a fluorescence microscope (upper panel) or analyzed by flow cytometry (lower panel). (H) H22, Jurkat and K562 cells were infected with Ad5-GFP or EVM/VSV-G Ad5-GFP at an MOI of 1 or 100 for 72 h. Then, GFP-positive cells were analyzed by FACS. Representative images of fluorescent cells from three independent experiments are shown (left panel). The results of the statistical analysis of the flow cytometry data are shown as the means ± SD (right panel). #Not significant and ∗∗∗P < 0.001.

Article Snippet: The mouse hepatocellular carcinoma cell line Hepa1-6, human embryonic kidney cell line 293T, human T-cell leukemia cell line Jurkat, human chronic myeloid leukemia cell line K562, human alveolar adenocarcinoma cell line A549, mouse melanoma cell line B16F10 and colon carcinoma cell line CT26.WT were obtained from the American Type Culture Collection (Manassas, VA, United States).

Techniques: Membrane, Gradient Centrifugation, Western Blot, Infection, Virus, Incubation, Microscopy, Cytometry

Journal: Journal of Biological Chemistry

Article Title: The B Cell Inhibitory Fc Receptor Triggers Apoptosis by a Novel c-Abl Family Kinase-dependent Pathway

doi: 10.1074/jbc.m505308200

Figure Lengend Snippet: FIGURE 3. FcRIIB1 aggregation results in receptor tyrosine phosphorylation, asso- ciation of the FcRIIB1 with c-Abl, and activation of c-Abl in an ITIM- and SHIP- independent fashion. A, DT(FcR), DT(FcR), DT(FcR Y309F), and DT(FcR SHIP) cells (5 107-108 cells) were incubated in medium alone or in medium containing 5 g/ml b-2.4G2 mAb and 3 g/ml avidin for 5 min at 37 °C, washed, lysed, and the lysates subjected to immunoprecipitation (IP) using the phosphotyrosine-specific mouse mAb 4G10 or c-Abl-specific rabbit antibodies. Immunoprecipitates were analyzed by SDS- PAGE and immunoblotting (WB) probing for FcRIIB1 using rabbit antibodies specific for mouse FcR detected using the secondary horseradish peroxidase-conjugated goat antibodies specific for rabbit Ig. The secondary antibodies detected the rabbit c-Abl- specific antibody H chains (Ig) used for immunoprecipitation, which is has a molecular weight similar to that of the FcR(Fc). Alternatively, immunoblots were probed for c-Abl using rabbit antibodies specific for c-Abl detected using horseradish peroxidase-conju- gated to goat antibodies specific for rabbit Ig. Results are representative of at least three independent experiments. B, DT(FcR), DT(FcR Y309F), and DT(FcR SHIP) cells were incubated in medium alone or medium containing 5 g/ml b-2.4G2 mAb and avidin or isotype-matched antibody for 5 min at 37 °C. Cells were washed, fixed, and permeabilized and stained with a phospho-c-Abl (Tyr245)-specific antibody.

Article Snippet: Affinity-purified rabbit antibodies specific for phospho-c-Abl (Tyr245) were obtained from Cell Signaling (Beverly, MA).

Techniques: Phospho-proteomics, Activation Assay, Incubation, Avidin-Biotin Assay, Immunoprecipitation, SDS Page, Western Blot, Molecular Weight, Staining

Journal: The Journal of Cell Biology

Article Title: Adhesion to fibronectin regulates Hippo signaling via the FAK–Src–PI3K pathway

doi: 10.1083/jcb.201501025

Figure Lengend Snippet: PI3K, PDK1, and Src regulation of nuclear YAP via Lats in serum-starved, subconfluent cells. (A) PI3K and PDK1 inhibitors relative to Lats. MCF-10A cells transfected with control, Nf2, or Lats1/2 siRNAs were serum starved and treated with DMSO (solvent control), 10 µM wortmannin (PI3K inhibitor), or 5 µM BX-795 (PDK1 inhibitor) for 30 min. On-target plus nontargeting pool was used as a control siRNA. Localization of endogenous YAP was identified by immunofluorescence staining. (B) SFK inhibitors and YAP localization. Serum-starved, low cell density MCF-10A cells were incubated with SFK inhibitors (10 µM each of PP2, dasatinib, SKI-1, and SU6656) for 30 min. 10 µM each of PP3 and imatinib were used as controls. YAP subcellular localization was determined by immunofluorescence staining. Alexa Fluor 594 secondary antibody was used for SU6656, which has high background green fluorescence. (C) Biochemical effects of Src inhibition. PP3- or PP2-treated MCF-10A cells were analyzed by Western blot using anti-YAP and anti–phospho-YAP (S127) antibodies. Phosphorylated YAP was detected by mobility shift on Phos-tag SDS-PAGE. (D) SFK inhibitors relative to Lats. MCF-10A cells transfected with control, Nf2, or Lats1/2 siRNA were serum starved and treated with 10 µM PP3 or PP2. After 30 min, cells were fixed for immunofluorescence staining with anti-YAP antibody. (E) Depletion of individual SFK. MCF-10A cells were transfected with control, Src, Fyn, or Yes siRNA. After serum starvation, subcellular localization of endogenous YAP was identified by immunofluorescence staining and quantified based on the criteria shown under the graph. More than 120 cells from four random views were quantified. (F) Src knockdown relative to Lats. MCF-10A cells were transfected with control, Src, Lats1/2, or combined siRNA of Src and Lats1/2. Cells were serum starved for 24 h before fixation and stained with anti-YAP antibody. (A, B, and D–F) One of three independent results is presented. Bars, 25 µm.

Article Snippet: The following inhibitors were used: SFK inhibitors SKI-1 and SU6656, Src/Abl inhibitor dasatinib monohydrate, Abl inhibitor imatinib mesylate, PDK1 inhibitor BX-795, FAK inhibitors PF-573228 and PF-562271 (Santa Cruz Biotechnology, Inc.), SFK inhibitor PP2 and its inactive analogue PP3 (EMD Millipore), and PI3K inhibitor wortmannin (Sigma-Aldrich).

Techniques: Transfection, Control, Solvent, Immunofluorescence, Staining, Incubation, Fluorescence, Inhibition, Western Blot, Mobility Shift, SDS Page, Knockdown

Journal: The Journal of Cell Biology

Article Title: Adhesion to fibronectin regulates Hippo signaling via the FAK–Src–PI3K pathway

doi: 10.1083/jcb.201501025

Figure Lengend Snippet: Roles of Src and FAK in the regulation of nuclear YAP by EGF or LPA. (A) Src depletion and FAK inhibition. MCF-10A cells transfected with control or Src siRNA were serum starved for 24 h and treated with EGF or LPA for 30 min. For FAK inhibitor treatment experiment, serum-starved, subconfluent MCF-10A cells were pretreated for 30 min with DMSO or 5 µM PF-573228, followed by a 30-min EGF or LPA treatment. YAP subcellular localization was determined by immunofluorescence staining. One of three independent results is presented. (B) CSK expression to inhibit Src activity. Paired with uninduced cells, doxycycline (Dox)-induced MCF-10A cells expressing myr-CSK-GFP fusion protein were serum starved for 24 h. Cells were treated with EGF or LPA for 30 min before fixation. Subcellular localization of YAP was determined by immunofluorescence staining. One representative out of three independent experiments is shown. Tet, tetracycline (or doxycycline) inducible. (C) Src inhibitors. Serum-starved, subconfluent MCF-10A cells were pretreated with PP3, PP2, imatinib, or dasatinib for 30 min. Cells were then treated with 20 ng/ml EGF for 30 min before fixation. Endogenous YAP was immunofluorescence stained. The result represents three independent experiments. Bars, 25 µm.

Article Snippet: The following inhibitors were used: SFK inhibitors SKI-1 and SU6656, Src/Abl inhibitor dasatinib monohydrate, Abl inhibitor imatinib mesylate, PDK1 inhibitor BX-795, FAK inhibitors PF-573228 and PF-562271 (Santa Cruz Biotechnology, Inc.), SFK inhibitor PP2 and its inactive analogue PP3 (EMD Millipore), and PI3K inhibitor wortmannin (Sigma-Aldrich).

Techniques: Inhibition, Transfection, Control, Immunofluorescence, Staining, Expressing, Activity Assay

Journal: The Journal of Immunology Author Choice

Article Title: c-Abl–Mediated Tyrosine Phosphorylation of PARP1 Is Crucial for Expression of Proinflammatory Genes

doi: 10.4049/jimmunol.1801616

Figure Lengend Snippet: LPS stimulation induces c-Abl’s nuclear transportation. (A) LPS stimulation induces nuclear import of c-Abl. RAW 264.7 cells were challenged with LPS for various lengths of time. Cell lysates from nuclear and cytoplasmic fractions were subjected to Western blotting to determine the subcellular distribution of c-Abl. (B) Immune-fluorescence staining verifies LPS-induced nuclear import of c-Abl. RAW 264.7 cells were mock treated or LPS exposed (±STI571) for 1 h, and then cells were fixed and permeabilized and incubated with anti–c-Abl rabbit polyclonal Ab and TRITC-conjugated secondary Ab. The nuclei of the cells were stained with DAPI. Similar results were obtained from at least three independent experiments. Original magnification ×180.

Article Snippet: Mouse mAb against PARP1 (1:2000, B-10, sc-74470) and rabbit polyclonal Ab against c-Abl (1:3000, K-12, sc-131) were purchased from Santa Cruz Biotechnology (Santa Cruz, CA).

Techniques: Western Blot, Fluorescence, Staining, Incubation

Journal: Molecular Biology of the Cell

Article Title: Nonenzymatic domains of Kalirin7 contribute to spine morphogenesis through interactions with phosphoinositides and Abl

doi: 10.1091/mbc.e13-04-0215

Figure Lengend Snippet: FIGURE 9: Phosphorylation of Kal SR4:6 by Abl1. (A) The purified fragments of Kalirin (0.5 μM) shown in the diagram were each exposed to recombinant Abl1 (10 nM) along with 5 μM ATP and 0.75 μCi [32P-γ]ATP at 32°C for 30 min. (B) The reactions were stopped by boiling each sample into Laemmli sample buffer; after SDS–PAGE and transfer to a polyvinylidene fluoride membrane, phosphorylated proteins were visualized by autoradiography (3-h exposure). In addition to autophosphorylated Abl1 (135 kDa; arrow), KalSR4:6 was phosphorylated (band at ~50 kDa); less extensive phosphorylation of KalSR7:9 and Kal7-CT was also detectable. (C, D) The reaction was scaled up as described in Materials and Methods, and phosphorylated SR4:6 recovered from a silver-stained 4–15% acrylamide gel was subjected to in-gel digestion with trypsin, followed by LC-MS/MS identification of phosphorylated Tyr residues. Sites are identified using the NP_114451.2 numbering scheme for Kalrn. MS/MS fragmentation patterns for the Tyr-591 (C) and Tyr-616 (D) peptides are shown. Manual verification and the site of Tyr modification (red Y) are illustrated in C1 and D1. The b- and y-ion series peak assignments are shown (C2, D2). Extracted ion chromatograms of the parent ion (M+2H)2+ for the IDA and SWATH runs were retention time aligned (C3 and D3). Note that the Cys (blue) in the Tyr-616 peptide was acrylamide gel modified (propionamide). (E) Model illustrates the location of the four Tyr residues in KalSR4:6. For each Tyr, percentage phosphotyrosine (shown in parentheses) was calculated relative to the sum of all of the peptides containing that Tyr residue (phosphorylated or not). The protease-sensitive insert in the B-C loop of SR5 (inset) contains the only predicted calpain-cleavage site (Vishwanatha et al., 2012)

Article Snippet: Purified KalSR4:6 (1.0 μM in a final volume of 25 μl) was incubated with recombinant mouse Abl1 (100 nM) and 2 mM ATP for 1 h at 32°C, as recommended by the supplier (SignalChem, Richmond, Canada).

Techniques: Phospho-proteomics, Purification, Recombinant, SDS Page, Membrane, Autoradiography, Staining, Acrylamide Gel Assay, Liquid Chromatography with Mass Spectroscopy, Tandem Mass Spectroscopy, Modification, Data-independent acquisition, Residue

Journal: Molecular Biology of the Cell

Article Title: Nonenzymatic domains of Kalirin7 contribute to spine morphogenesis through interactions with phosphoinositides and Abl

doi: 10.1091/mbc.e13-04-0215

Figure Lengend Snippet: FIGURE 10: Phosphorylation of KalSR4:6 by Abl1 increases its sensitivity to calpain. (A) KalSR4:6 phosphorylated with [γ-32P]ATP by Abl1 or exposed to the same conditions in the absence of ATP and Abl1 was exposed to the indicated amount of μ-calpain for 60 min at 37°C. Digested samples were fractionated by SDS–PAGE and transferred to polyvinylidene fluoride (PVDF) membranes; 32P-labeled KalSR4:6 was visualized by autoradiography (left), and unlabeled KalSR4:6 was visualized by Western blot (right) using an antibody to SR4:7 (JH2580; Penzes et al., 2000). The amount of intact KalSR4:6 remaining was quantified; error bars show range of duplicate experiments. (B) Purified recombinant KalSR4:6 and KalSR4:6 with a phosphomimetic mutation at position 591 (SR4:6/Y591E) or 616 (SR4:6/Y616E) was fractionated by SDS–PAGE, transferred to a PVDF membrane, and visualized with Coomassie brilliant blue; mutation of either Tyr residue altered the mobility of the protein during electrophoresis. The purified proteins (1 μg each) were exposed to the indicated dose of μ-calpain as described. The reaction was stopped, and the SDS–PAGE fractionated products were visualized with Coomassie; mutation of either site increased the calpain sensitivity of the protein. (C, D) DIV18 cortical neurons from WT (C) and Kal7KO (D) mice were exposed to medium containing 10 μM GNF-5 in DMSO, or an equivalent volume of DMSO, for 6 h and fixed. Images obtained using an Axiovert 200M with Apotome for optical sectioning were coded and scored by a blinded observer. GNF-5 increased the dendritic protrusion density in WT but not Kal7KO neurons (C) while increasing the protrusion length in Kal7KO but not WT neurons (D). N = 9–14 WT neurons and 6–8 Kal7KO neurons; N = 1187–1652 WT protrusions and 306–838 Kal7KO protrusions.

Article Snippet: Purified KalSR4:6 (1.0 μM in a final volume of 25 μl) was incubated with recombinant mouse Abl1 (100 nM) and 2 mM ATP for 1 h at 32°C, as recommended by the supplier (SignalChem, Richmond, Canada).

Techniques: Phospho-proteomics, SDS Page, Labeling, Autoradiography, Western Blot, Purification, Recombinant, Mutagenesis, Membrane, Residue, Electrophoresis