Journal: Bioengineering & Translational Medicine

Article Title: Utilizing induced neural stem cell‐based delivery of a cytokine cocktail to enhance chimeric antigen receptor‐modified T‐cell therapy for brain cancer

doi: 10.1002/btm2.10538

Figure Lengend Snippet: RANTES/IL‐15‐secreting GBM tumor cells enhance T‐cell trafficking and expansion. (a) RANTES and IL‐15 were detected in culture supernatant collected from transduced U87 tumor cells. (b) U87 or U87 RANTES‐IL‐15 were seeded and cultured for 24 or 48 h. Transwell inserts were added and CSPG4‐CAR‐T or control T cells were seeded therein. T cells were allowed to migrate through inserts for 5 h. Afterward, inserts were removed and media was collected for flow cytometry. (c) Migration of CSPG4‐CAR‐T or control T cells toward U87 or U87 RANTES‐IL‐15 cultured for 24 or 48 h was evaluated by flow cytometry ( n = 3). (d) NSG mice were implanted i.c. with U87, U87 RANTES‐IL‐15 , or PBS in the right hemisphere of the brain. Five days later, mice were infused i.v. with CSPG4‐CAR‐T. Mice were sacrificed 1, 4, or 7 days following CSPG4‐CAR‐T infusion. Brains were harvested, bisected along the interhemispheric fissure, and processed for flow cytometry ( n = 3). (e) CSPG4‐CAR‐T concentrations in single cell suspensions generated from bisected mouse brains (contralateral hemisphere vs tumor hemisphere) were analyzed by flow cytometry. Schematic(s) created with BioRender.com . * p < 0.05, ** p < 0.01, *** p < 0.001. CAR‐T, chimeric antigen receptor‐modified T cell; CSPG4‐CAR‐T, CAR‐T cells targeting chondroitin sulfate proteoglycan 4 antigen; GBM, glioblastoma; IL‐15, interuekin 15; NSG, NOD.Cg‐ Prkdc scid Il2rg tm1Wjl /SzJ.

Article Snippet: The U87 glioma cell line and human telomerase reverse transcriptase (hTERT)‐immortalized normal human fibroblasts (NHF1) were obtained from the American Type Culture Collection and as previously described.

Techniques: Cell Culture, Control, Flow Cytometry, Migration, Generated, Modification

Journal: Bioengineering & Translational Medicine

Article Title: Utilizing induced neural stem cell‐based delivery of a cytokine cocktail to enhance chimeric antigen receptor‐modified T‐cell therapy for brain cancer

doi: 10.1002/btm2.10538

Figure Lengend Snippet: Development and characterization of hiNeuroS RANTES‐IL‐15 . (a) Schematic representation of lentivirus encoding human RANTES and IL‐15. (b) Human fibroblasts were transduced with LV‐RANTES‐IL‐15‐GFP, LV‐Sox2, and LV‐rtTA in succession. Reprogramming and primary hiNeuroS formation were initiated by switching to NSC growth media supplemented with growth factors and tetracycline. Three rounds of cell cluster selection were performed by serial seeding on laminin‐coated plates. After each round of selection, nonadhering cells were collected and expanded to yield secondary hiNeuroS. (c) Fluorescence imaging of hiNeuroS RANTES‐IL‐15 confirms successful LV‐RANTES‐IL‐15‐GFP transduction. Scale bar represents 100 μm. (d) RANTES and IL‐15 were detected in culture supernatant collected from transduced hiNeuroS. (e) Migration of CSPG4‐CAR‐T toward U87 ± NS‐CM or control media using a transwell migration assay was evaluated by flow cytometry ( n = 3). (f) CSPG4‐CAR‐T counts after 3 days in co‐culture with U87 ± NS‐CM ( n = 3). (g) Anti‐tumor activity of CSPG4‐CAR‐T after 3 days in co‐culture with U87 ± NS‐CM was assessed by bioluminescence ( n = 8–10). Schematic(s) created with BioRender.com . * p < 0.05, ** p < 0.01, *** p < 0.001. CAR‐T, chimeric antigen receptor‐modified T cell; CSPG4‐CAR‐T, CAR‐T cells targeting chondroitin sulfate proteoglycan 4 antigen; NSC, neural stem cell.

Article Snippet: The U87 glioma cell line and human telomerase reverse transcriptase (hTERT)‐immortalized normal human fibroblasts (NHF1) were obtained from the American Type Culture Collection and as previously described.

Techniques: Transduction, Selection, Fluorescence, Imaging, Migration, Control, Transwell Migration Assay, Flow Cytometry, Co-Culture Assay, Activity Assay, Modification

Journal: Bioengineering & Translational Medicine

Article Title: Utilizing induced neural stem cell‐based delivery of a cytokine cocktail to enhance chimeric antigen receptor‐modified T‐cell therapy for brain cancer

doi: 10.1002/btm2.10538

Figure Lengend Snippet: Combination hiNeuroS RANTES‐IL‐15 + CAR‐T therapy increases CAR‐T infiltration and eradicates non‐invasive GBM. (a) Schematic representation of study design. NSG mice were implanted i.c. with FLuc + U87 ± hiNeuroS RANTES‐IL‐15 in the right hemisphere of the brain. Four days later, mice were infused i.v. with CSPG4‐CAR‐T or PBS ( n = 5–7). (b) Representative bioluminescent images depict U87 tumor growth. (c) Luminescence was quantified by calculating total flux within a region of interest centered on the head, then normalizing to Day 0 values. Dotted line signifies CAR‐T infusion. (d) Kaplan–Meier survival curves. (e) AUC was calculated for each normalized tumor growth curve. (f) CAR‐T tumor infiltration (brown) was assessed by immunohistochemistry. Schematic(s) created with BioRender.com . *** p < 0.001. CAR‐T, chimeric antigen receptor‐modified T cell; CSPG4‐CAR‐T, CAR‐T cells targeting chondroitin sulfate proteoglycan 4 antigen; GBM, glioblastoma; NSG, NOD.Cg‐ Prkdc scid Il2rg tm1Wjl /SzJ.

Article Snippet: The U87 glioma cell line and human telomerase reverse transcriptase (hTERT)‐immortalized normal human fibroblasts (NHF1) were obtained from the American Type Culture Collection and as previously described.

Techniques: Immunohistochemistry, Modification

Journal: Carcinogenesis

Article Title: Aberrant activation of hedgehog signaling pathway in ovarian cancers: effect on prognosis, cell invasion and differentiation

doi: 10.1093/carcin/bgn230

Figure Lengend Snippet: ( A ) Kaplan–Meier plot on cumulative survival of patients with ovarian cancer with high and low Gli1 expression showed that high Gli1 expression was associated with poor prognosis. ( B ) Representative mRNA expression of Shh, Patched, Smo and Gli1 in various ovarian tissues detected by RT–PCR. (N1 and N2, normal tissues; B1, benign tumor; B2, borderline tumor; C1–C6, cancers; Ptch, Patched.) Elevated expression of Shh mRNA was observed in cancer samples, whereas sustained expression of Patched mRNA and heterogeneous expression of Gli1 mRNA was present in ovarian cancers. ( C ) Results of RT–PCR for Shh, Patched, Smo and Gli1 and western blotting for Patched and Gli1 in immortalized normal ovarian epithelial cell lines (HOSE 6-3, HOSE 11-12) and ovarian cancer cell lines (OVCA420, OVCA429, OVCA433, OVCAR3 and SKOV3). Sustained expression of Patched and variable expression of Gli1 mRNA and protein was observed in all seven cell lines (Ptch, Patched). ( D ) Mesothelial marker calretinin (Cal) and mesenchymal marker vimentin (Vim) were expressed in normal cell lines, whereas epithelial marker E-cadherin (E-Cad) and cytokeratin 7 (CK 7) were present in cancer cell lines. Snail expression was observed in all the normal and cancer cell lines.

Article Snippet: Two human ovarian cancer cell lines SKOV3 and OVCAR3 were purchased from the American Type Culture Collection (Manassas, VA).

Techniques: Expressing, Reverse Transcription Polymerase Chain Reaction, Western Blot, Marker

Journal: Carcinogenesis

Article Title: Aberrant activation of hedgehog signaling pathway in ovarian cancers: effect on prognosis, cell invasion and differentiation

doi: 10.1093/carcin/bgn230

Figure Lengend Snippet: Ectopic overexpression of Gli1 promoted proliferation, migration and invasion of ovarian cancer cells. ( A ) Transient transfection of pcDNA3.1-HisB-hGli1 promotes cancer cell proliferation as assessed by 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide. Error bars, SD. ( B ) Increased cell number was found to migrate through Matrigel-coated chamber after transfection of pcDNA3.1-HisB-hGli1 (OVCAR3 cells were shown). Bar chart below showed an increase in percentage of cells migrating through Matrigel-coated chamber in Gli1-transfected cells when compared with empty vector-transfected cells and untransfected (untreated) cells (* P < 0.01; ** P < 0.05). (U, untransfected cells; V, vector; G, pcDNA3.1-HisB-hGil) Error bars, SD. ( C ) Wound-healing assay shows increased cell migration ability of SKOV3 with ectopic overexpression of Gli1. ( D ) Increased expression of Bcl-2, total form of caspases-3, 7, 9 and PARP as well as β1-integrin was present in Gli1-transfected cancer cells. (Casp-3, 7, 9, caspases-3, 7, 9, respectively; U, untransfected; V, empty vector; G, pcDNA3. 1-HisB-hGli1.)

Article Snippet: Two human ovarian cancer cell lines SKOV3 and OVCAR3 were purchased from the American Type Culture Collection (Manassas, VA).

Techniques: Over Expression, Migration, Transfection, Plasmid Preparation, Wound Healing Assay, Expressing

Journal: Carcinogenesis

Article Title: Aberrant activation of hedgehog signaling pathway in ovarian cancers: effect on prognosis, cell invasion and differentiation

doi: 10.1093/carcin/bgn230

Figure Lengend Snippet: Ectopic Gli1 overexpression induced increased expression of MT1-MMP and VEGF mRNA as well as epithelial differentiation markers in ovarian cancer cells. (V, empty vector; His-Gli1 and G, pcDNA3.1-HisB-hGli1.) ( A ) Real-time PCR showed increased expression of MT1-MMP in OVCAR3 and VEGF in all cancer cells. Error bars, SD. ( B ) Increased expression of E-cadherin (E-Cad) and vimentin (Vim) was present in cancer cells after transfection of pcDNA3.1-HisB-hGli1 (V, empty vector, G, pcDNA3.1-HisB-hGli1). ( C ) E-cadherin mRNA expression was increased with ectopic overexpression of Gli1 in SKOV3 detected by real-time PCR. Error bars, SD. ( D ) Dual luciferase assay confirmed upregulation of E-cadherin expression at transcriptional level in SKOV3 cells after transfection (* P < 0.05). Error bars, SD.

Article Snippet: Two human ovarian cancer cell lines SKOV3 and OVCAR3 were purchased from the American Type Culture Collection (Manassas, VA).

Techniques: Over Expression, Expressing, Plasmid Preparation, Real-time Polymerase Chain Reaction, Transfection, Luciferase

Journal: PLoS ONE

Article Title: The BH3 only Bcl-2 family member BNIP3 regulates cellular proliferation

doi: 10.1371/journal.pone.0204792

Figure Lengend Snippet: HEK293 cells were engineered to stably co-express the Tet Repressor protein (TR) and tetracycline-inducible BNIP3 (TO-BNIP3) or beta-galactosidase (TO-β-gal) as described in the Materials & Methods. Multiple clones were established for each cell line. Addition of the tetracycline analog, doxycycline (dox), for 24 hours relieves TR repression and induces BNIP3 expression as detected by immunofluorescence (A), Western blot (B) and RT-PCR. In (A), nuclei are visualized by Hoescht dye (blue) and BNIP3 is seen in the cytoplasm (green fluorescence).

Article Snippet: MEF cells (obtained from Animal Care Services, University of Manitoba) and HEK293 cells (obtained from ATCC) were cultured in DMEM media (Thermofisher) consisting of 4mM L-glutamine, 4mM sodium pyruvate, 4.5 microg/mL glucose and supplemented with either 10% fetal bovine serum (MEFs) or 5% fetal bovine serum (HEK293), and 100 units/ml penicillin/streptomycin.

Techniques: Stable Transfection, Clone Assay, Expressing, Immunofluorescence, Western Blot, Reverse Transcription Polymerase Chain Reaction, Fluorescence

Journal: PLoS ONE

Article Title: The BH3 only Bcl-2 family member BNIP3 regulates cellular proliferation

doi: 10.1371/journal.pone.0204792

Figure Lengend Snippet: HEK293 cells were engineered to stably co-express the Tet Repressor protein (TR) and tetracycline-inducible BNIP3 (TO-BNIP3) or beta-galactosidase (TO-β-gal) as described in the Materials & Methods. Cells were untreated or treated with doxycycline (1 microg/mL) to induce gene expression. (A) Reactive oxygen species (ROS) production was increased upon BNIP3 (but not β-gal) induction. ROS were detected using the dye CM-H2DCFDA, which is oxidized to green fluorescent DCF (dichlorofluorescein) by hydrogen peroxide. (B) Autophagy was also increased upon BNIP3 (but not beta-gal) induction, as determined by GFP-LC3 localization. TO-cells were transiently transfected with GFP-LC3. 24 hours after transfection, cells were untreated or treated with doxycycline for an additional 24 hours. (C) Cell death was not induced upon BNIP3 or β-gal induction, as determined by the membrane permeability assay. (D) Cell growth was severely restricted upon BNIP3 (but not beta-gal) induction in HEK293 cells. For each cell type, 1.5x105 cells were seeded in one well of a 6-well culture dish, with or without doxycycline (gene induction). Three days later, the number of adherent cells in each well was determined using a Beckman Coulter Counter. The fold-increase represents the ratio of cells on day three compared to the number of cells originally seeded. Results represent the average of three independent experiments; error bars represent standard deviation. *p < .01.

Article Snippet: MEF cells (obtained from Animal Care Services, University of Manitoba) and HEK293 cells (obtained from ATCC) were cultured in DMEM media (Thermofisher) consisting of 4mM L-glutamine, 4mM sodium pyruvate, 4.5 microg/mL glucose and supplemented with either 10% fetal bovine serum (MEFs) or 5% fetal bovine serum (HEK293), and 100 units/ml penicillin/streptomycin.

Techniques: Stable Transfection, Gene Expression, Transfection, Membrane, Permeability, Standard Deviation

Journal: PLoS ONE

Article Title: The BH3 only Bcl-2 family member BNIP3 regulates cellular proliferation

doi: 10.1371/journal.pone.0204792

Figure Lengend Snippet: (A) HEK293 cells were transfected with either an empty control vector or nuclear BNIP3 expression vector. Total lysates from transfected cells were western blotted for BNIP3. The blot was stripped and reprobed with GAPDH as loading control. The results shown are representative of three independent experiments (three replicates each). (B) Proliferation of transfected cells was measured using the Click iT EdU cell proliferation assay. The results are an average of three independent experiments (three replicates each). The error bars show standard error of mean. Student’s t-test was used to calculate statistical significance (*p<0.0005). Results shown are representative of three independent experiments.

Article Snippet: MEF cells (obtained from Animal Care Services, University of Manitoba) and HEK293 cells (obtained from ATCC) were cultured in DMEM media (Thermofisher) consisting of 4mM L-glutamine, 4mM sodium pyruvate, 4.5 microg/mL glucose and supplemented with either 10% fetal bovine serum (MEFs) or 5% fetal bovine serum (HEK293), and 100 units/ml penicillin/streptomycin.

Techniques: Transfection, Control, Plasmid Preparation, Expressing, Western Blot, Proliferation Assay

Journal: Molecular Medicine Reports

Article Title: lncRNA FLVCR1-AS1 drives colorectal cancer progression via modulation of the miR-381/RAP2A axis

doi: 10.3892/mmr.2020.11778

Figure Lengend Snippet: FLVCR1-AS1 expression is upregulated in CRC. (A) RT-qPCR results show that FLVCR1-AS1 expression is higher in CRC tissues compared with normal tissues (n=26). (B) RT-qPCR was used to measure the expression of FLVCR1-AS1 in CRC cell lines (Caco-2, SW480, LoVo and SW1116) and the NCM460 normal colonic epithelial cell line. (C) RT-qPCR results showing the level of FLVCR1-AS1 in CRC tissues of different TNM stages. *P<0.05. (D) Kaplan-Meier analysis reveals the association of high FLVCR1-AS1 expression with poor overall survival in patients with CRC. FLVCR1-AS1, feline leukemia virus subgroup C receptor 1 antisense RNA 1; CRC, colorectal cancer; RT-qPCR, reverse transcription-quantitative polymerase chain reaction.

Article Snippet: Four human CRC cell lines, namely Caco-2, SW480, LoVo and SW1116, the NCM460 normal colonic epithelial cell line and 293T cells were purchased from the American Type Culture Collection.

Techniques: Expressing, Quantitative RT-PCR, Virus, Reverse Transcription, Real-time Polymerase Chain Reaction

Journal:

Article Title: Antitumor Activity and Prolonged Expression from a TRAIL-Expressing Adenoviral Vector

doi:

Figure Lengend Snippet: Induction of apoptosis in tumor cells by Ad.TRAIL-GFP vector. (A) Structure of Ad.TRAIL-GFP and Ad.GFP. The adenoviral sequences E1 and E3 are deleted. Ad.TRAIL-GFP vector expresses both TRAIL and GFP. (B) Western blot analysis of Ad.GFP-or Ad.TRAIL-GFP-transduced IMR90 cells or U87 glioma cells using an anti-TRAIL antibody (top), an anti-PARP antibody (middle), an antitubulin antibody (bottom), respectively. (C) Apoptotic index of cells (U87, T98, HUVEC, IMR90, NHA [MOI 100], and TA3 [MOI 50]) infected with Ad.TRAIL-GFP or Ad.GFP. Statistical significance between treatment was evaluated by Student's t test (asterisk: P<0.01). (D) Expression of four TRAIL receptors in a panel of cell lines, as determined by semiquantitative RT-PCR.

Article Snippet: The human umbilical vein endothelial cells (HUVECs), U87MG, T98 (human glioblastoma cells), 293 (human embryonic kidney cells), Colo-205 (human colon carcinoma), Ramos (Burkitt lymphoma), and IMR90 (human primary fibroblast cells) were purchased from American Type Culture Collection (ATCC, Rockville, MD).

Techniques: Plasmid Preparation, Western Blot, Infection, Expressing, Reverse Transcription Polymerase Chain Reaction

Journal: The Journal of Biological Chemistry

Article Title: Inclusion of an Arg-Gly-Asp receptor-recognition motif into the capsid protein of rabbit hemorrhagic disease virus enables culture of the virus in vitro

doi: 10.1074/jbc.M117.780924

Figure Lengend Snippet: The mRHDV mutant strain is safe to other animals. A, mRHDV has no ability to infect cells such as BHK-21 cells, 293T cells, Vero cells, MDCK cells, or DF-1 cells. The RK-13 cells were used as a positive control. B, mRHDV genome does not replicate in other species of cells such as BHK-21 cells, 293T cells, Vero cells, MDCK cells, or DF-1 cells. The β-actin gene was used as an internal control. C, mRHDV mutant strains have no ability to infect mouse. Histopathological examination of mouse tissues infected with mRHDV and wild-type RHDV (JX/CHA/97 strain) did not indicate symptoms of virus infection. Mouse tissues infected with PBS were used as a negative control. D, the RHDV and mRHDV genomes were not replicated in any mouse tissues. No VP60 gene was detected in mouse tissues using RT-PCR. Liver tissue of rabbits infected with RHDV JX/CHA/97 strain was used as a positive control, and the β-actin gene was used as an internal control; M, DNA ladder.

Article Snippet: Cells, virus, antibody, and plasmids Rabbit kidney cells (RK-13, ATCC CCL37), primary hepatocytes of rabbit (PriCells, Wuhan, China), 293T cells (ATCC, CRL-3216), Vero cells (ATCC, CCL-81), BHK-21 cells (ATCC, CCL-10), MDCK cells (ATCC, CCL-34), and CRFK cells (ATCC, CCL-94) were grown in minimal essential medium (Life Technologies) or Dulbecco's modified Eagle's medium (DMEM) (Life Technologies) supplemented with 10% fetal bovine serum (FBS) in 5% CO 2 at 37 °C, respectively.

Techniques: Mutagenesis, Positive Control, Control, Infection, Virus, Negative Control, Reverse Transcription Polymerase Chain Reaction

Journal: The Journal of Biological Chemistry

Article Title: Smad6 Inhibits the Transcriptional Activity of Tbx6 by Mediating Its Degradation *

doi: 10.1074/jbc.M109.007864

Figure Lengend Snippet: Smad6 mediates Tbx6 ubiquitination and proteasomal degradation. A, Smad6 induces Tbx6 degradation in a dose-dependent manner. Tbx6 expression construct (100 ng) was co-transfected with increasing amounts of Smad6 (0, 0.1, 0.2, and 0.4 μg/well in a 12-well plate; represented by the black triangle) expression plasmid into HEK 293T cells. In each transfection, an equal amount of green fluorescent protein (GFP) expression plasmid (50 ng) was included as an internal control. The protein levels of Tbx6 and Smad6 in whole cell lysates were detected by Western blotting (WB). β-Actin was used as a loading control. B, overexpression of Smad6 has no obvious effect on the mRNA level of Tbx6. TT-D6 cells transfected with control or Smad6 expression plasmids were subjected to RT-PCR analysis for related gene expression. The relative value for each target gene was normalized to the average intensity of the endogenous control gene, GAPDH. **, p < 0.01. C, Smad6 mediates Tbx6 ubiquitination and proteasomal degradation in a dose-dependent manner. 293T cells were transfected with expression constructs in the indicated combinations. After 24 h of transfection, cells were treated with 10 μm MG132 for 4 h before harvesting. Immunoprecipitation (IP) was performed using anti-Myc antibody and followed by Western blotting using anti-ubiquitin antibody. D, Tbx6 forms a ternary complex with Smad6 and Smurf1. Protein extracts from 293T cells transfected with the indicated plasmids were immunoprecipitated with anti-Myc or anti-FLAG antibodies and analyzed by Western blotting using the indicated antibodies. E, Smurf1 promotes Smad6-mediated Tbx6 ubiquitination. Tbx6 ubiquitination was assessed as described above. HA, hemagglutinin; Ub, ubiquitin. F, Smurf1 cooperates with Smad6 to dramatically reduce Tbx6-dependent transcriptional activity. TT-D6 cells were co-transfected with 4×Tbx-TK or 4×mTbx-TK reporter and additional expression constructs including Tbx6, Smad6, Smurf1, and its inactive mutant, in different combinations as indicated. *, p < 0.05; **, p < 0.01 using Student's t test.

Article Snippet: HEK 293T and COS-7 cells, obtained from ATCC (Manassas, VA), were cultured in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum at 37 °C under 5% CO 2 atmosphere.

Techniques: Ubiquitin Proteomics, Expressing, Construct, Transfection, Plasmid Preparation, Control, Western Blot, Over Expression, Reverse Transcription Polymerase Chain Reaction, Gene Expression, Immunoprecipitation, Activity Assay, Mutagenesis

Journal: The Journal of Biological Chemistry

Article Title: Smad6 Inhibits the Transcriptional Activity of Tbx6 by Mediating Its Degradation *

doi: 10.1074/jbc.M109.007864

Figure Lengend Snippet: Tbx6 interacts with Smad6. A, identification of Smad6 as a novel Tbx6-interacting protein. FLAG-tagged Smads 1–7 were individually co-transfected with Myc-tagged Tbx6 into HEK 293T cells as indicated. After transfection for 24 h, cells were lysed, immunoprecipitated (IP) with anti-FLAG antibody, and analyzed by Western blotting (WB) with anti-Myc antibody (top panels). Whole cell lysates were immunoblotted directly with anti-Myc or anti-FLAG antibodies to demonstrate the expression of Myc-Tbx6 and FLAG-Smads, respectively (bottom panels). B, reciprocal immunoprecipitation with anti-Myc was carried out. Only Smad6 was co-precipitated with Tbx6. C, Tbx6 directly binds to Smad6 in vitro. Bacterially expressed GST-Smad6 fusion protein was purified using glutathione-agarose beads and incubated with His6-Tbx6 fusion protein. Associated His6-Tbx6 protein was readily detected by immunoblotting (top panel). The asterisks denote degraded bands of GST-Smad6 fusion protein (bottom panel). An equimolar amount of GST was used as a negative control. D, purified His6-Tbx6 fusion protein was incubated with GST-Smad6 and precipitated with anti-His antibody. Associated GST-Smad6 protein was detected by Western blot. E, interaction between endogenous Tbx6 and Smad6 in TT-D6 cells. TT-D6 cell extracts were subjected to immunoprecipitation with anti-Smad6 polyclonal antibody. Normal rabbit IgG was used as a negative control. The immunocomplexes were analyzed by immunoblotting using anti-Tbx6 antibody. F, Tbx6 is co-localized with Smad6 in the nucleus. TT-D6 cells were fixed and stained with the indicated antibodies to determine the localization of Tbx6 (green) and Smad6 (red). Nuclei were stained with 4,6-diamidino-2-phenylindole. The bar represents 50 μm for each section. G, T-box proteins have distinct affinities for different Smad proteins. Top, schematic diagrams depict Tbx6 homologues in mouse and Xenopus. Their interactions with Smad1 or Smad6 from co-immunoprecipitation experiments are shown in the bottom panels. HA, hemagglutinin.

Article Snippet: HEK 293T and COS-7 cells, obtained from ATCC (Manassas, VA), were cultured in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum at 37 °C under 5% CO 2 atmosphere.

Techniques: Transfection, Immunoprecipitation, Western Blot, Expressing, In Vitro, Purification, Incubation, Negative Control, Staining

Journal: The Journal of Biological Chemistry

Article Title: Smad6 Inhibits the Transcriptional Activity of Tbx6 by Mediating Its Degradation *

doi: 10.1074/jbc.M109.007864

Figure Lengend Snippet: Determination of domains for mutual interaction between Smad6 and Tbx6. A, schematic diagram of Smad6 and its deletion mutants. Functional domains of Smad6 are characterized above the schematic diagram. The interaction with Tbx6 from co-immunoprecipitation experiments is summarized on the right. B, mapping of the domain in Smad6 for interaction with Tbx6. HEK 293T cells were co-transfected with Myc-tagged Tbx6 and FLAG-tagged Smad6 deletion mutants. Cell lysates were immunoprecipitated (IP) with anti-FLAG antibody and then immunoblotted (WB, Western blot) with anti-Myc (top panel). The total level of Tbx6 protein expressed in whole cell lysates is also shown (bottom panel). The MH2 domain of Smad6 mediates its interaction with Tbx6. C, schematic representation of wild-type and deletion mutants of Tbx6. The T-box domain of Tbx6 protein (residues 90–279) is illustrated above the schematic diagram, and relative positions of the remaining fragment(s) in each deletion mutant are numbered on the left. The interaction with Smad6 is summarized on the right. D, identification of Smad6 binding sites on Tbx6. The region of residues 90–180 in Tbx6 contributes to its interaction with Smad6. *, bands consistent with protein-protein interaction. HA, hemagglutinin.

Article Snippet: HEK 293T and COS-7 cells, obtained from ATCC (Manassas, VA), were cultured in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum at 37 °C under 5% CO 2 atmosphere.

Techniques: Functional Assay, Immunoprecipitation, Transfection, Western Blot, Mutagenesis, Binding Assay