mouse  (Santa Cruz Biotechnology)

Journal: Molecular pharmacology

Article Title: Dual nuclear receptor 4A1 (NR4A1/NR4A2) ligands inhibit glioblastoma growth and target TWIST1.

doi: 10.1016/j.molpha.2024.100009

Figure Lengend Snippet: Fig. 3. DIM-3,5-CI2 decreases TWIST1 mRNA and protein expression in GBM cells. Human (A) and mouse (B) GBM cell lines were treated with 15 mM DIM-3,5-CI2 for 24 hours, and whole cell lysates were analyzed by western blotting, as outlined in Materials and methods. Human (C) and mouse (D) GBM cells were treated with 15 mM DIM-3,5-CI2 for 18 hours, and mRNA extracts were analyzed for TWIST1 mRNA expression, as outlined in Materials and methods. Results are expressed as means ± SD for at least 3 replicate determinations and significantly (P < .05) decreased TWIST1 mRNA levels are indicated (*).

Article Snippet: Antibodies used in cell culture experiments include Sp1 (sc-17824), Sp4 NR4A1 (sc-365113X), NR4A2 (sc376984X and sc-81345), TWIST1 (sc-81417), IgG (sc-2025) from Santa Cruz Biotechnology; PARP (9532), CPARP (9541S), glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (5174S), secondary anti-rabbit horseradish peroxidase (707HS and 7076S) from Cell Signaling Technology; IgG (mouse) (AB18413), and NR4A1 (ab109180) antibodies from Abcam.

Techniques: Expressing, Western Blot

Journal: Molecular pharmacology

Article Title: Dual nuclear receptor 4A1 (NR4A1/NR4A2) ligands inhibit glioblastoma growth and target TWIST1.

doi: 10.1016/j.molpha.2024.100009

Figure Lengend Snippet: Fig. 4. TWIST1 is an NR4A1/2-regulated gene. U87MG and CT2A cells (A) were treated with DMSO or DIM-3,5-CI2 for 24 hours. U87MG (B) or CT2A (C) cells were transfected with oligonucleotides targeting NR4A1 or NR4A2, and whole cell lysates were analyzed by western blotting. (D) Knockdown of NR4A1 or NR4A2 in U87MG or CT2A cells was carried out, and TWIST1 mRNA expression (relative to control knockdown) was determined by reverse-transcription PCR. Results are means ± SD for at least 3 replicate determinations, and significant (P < .05) reduction of TWIST1 mRNA levels are indicated (*).

Article Snippet: Antibodies used in cell culture experiments include Sp1 (sc-17824), Sp4 NR4A1 (sc-365113X), NR4A2 (sc376984X and sc-81345), TWIST1 (sc-81417), IgG (sc-2025) from Santa Cruz Biotechnology; PARP (9532), CPARP (9541S), glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (5174S), secondary anti-rabbit horseradish peroxidase (707HS and 7076S) from Cell Signaling Technology; IgG (mouse) (AB18413), and NR4A1 (ab109180) antibodies from Abcam.

Techniques: Transfection, Western Blot, Knockdown, Expressing, Control, Reverse Transcription

Journal: Molecular pharmacology

Article Title: Dual nuclear receptor 4A1 (NR4A1/NR4A2) ligands inhibit glioblastoma growth and target TWIST1.

doi: 10.1016/j.molpha.2024.100009

Figure Lengend Snippet: Fig. 5. NR4A1/2/Sp regulates TWIST1 expression in GBM cells. U87MG (A) and CT2A (B) cells were transfected with a nonspecific (control) oligonucleotide or nucleotides targeting Sp1 or Sp4, and whole cell lysates were analyzed by western blotting. (C) Synthetic constructs from the TWIST1 human gene promoter. U87MG cells were transfected with TWwt or TWmt (wild type and mutant constructs, respectively) and treated with 10 or 15 mM DIM-3,5-CI2 (D) or transfected with oligonucleotides targeting NR4A1 or NR4A2 (E), and luciferase activity was determined as outlined in Materials and methods. (F) Cell lysates were immunoprecipitated with NR4A1 and NR4A2 antibodies, and western blot analysis was used to examine coimmunoprecipitation of Sp1 and Sp4 as outlined in Materials and methods. Results are expressed as means ± SD for at least 3 replicate determinations, and significantly (P < .05) decreased responses compared to control (DMSO) values are indicated (*).

Article Snippet: Antibodies used in cell culture experiments include Sp1 (sc-17824), Sp4 NR4A1 (sc-365113X), NR4A2 (sc376984X and sc-81345), TWIST1 (sc-81417), IgG (sc-2025) from Santa Cruz Biotechnology; PARP (9532), CPARP (9541S), glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (5174S), secondary anti-rabbit horseradish peroxidase (707HS and 7076S) from Cell Signaling Technology; IgG (mouse) (AB18413), and NR4A1 (ab109180) antibodies from Abcam.

Techniques: Expressing, Transfection, Control, Western Blot, Construct, Mutagenesis, Luciferase, Activity Assay, Immunoprecipitation

Journal: Molecular pharmacology

Article Title: Dual nuclear receptor 4A1 (NR4A1/NR4A2) ligands inhibit glioblastoma growth and target TWIST1.

doi: 10.1016/j.molpha.2024.100009

Figure Lengend Snippet: Fig. 6. Effects of mithramycin and ChIP assay on TWIST1 and in vivo studies. U87MG cells were treated with DMSO or mithramycin (200 nM) for 24 hours, and both whole cell lysates (A) and RNA (B) were isolated and analyzed by western blotting or mRNA quantification, respectively, as outlined in Materials and methods. A ChIP assay (C) was determined using primers targeted to the Sp binding region of the proximal promoter region of the TWIST1 gene as outlined in Materials and methods. Animals harboring Ha-Ras/shp57 (RP) tumors were treated with DIM-3,5-CI2 for the duration of the experiment. (D) Tumor images from treated (tumors 5 and 6) and untreated (1e4) mice; tumors 7e9 are derived from mice treated for 79 days. Tumor images were obtained using a fluorescence-capable Leica dissecting microscope. (E) Left: representative coronal section stained with hematoxylin and eosin shows a vascular tumor with multifocal growth (arrows). Right: high-power image shows invasive margin (dotted white lines). (F) DIM-3,5-CI2 treatment (7.5 mg/kg/day) significantly prolonged animal survival (log-rank P ¼ .0161). The experiment was terminated on day 79. In the treated group, among 3 euthanized animals, we only found microtumors in 2 animals. (G) Analysis of tumor lysates by western blotting from 3 control and 4 DIM-3,5-CI2 treated tumors. Experiments were carried out in triplicate, and significant (P < .05) differences from control values are indicated (*).

Article Snippet: Antibodies used in cell culture experiments include Sp1 (sc-17824), Sp4 NR4A1 (sc-365113X), NR4A2 (sc376984X and sc-81345), TWIST1 (sc-81417), IgG (sc-2025) from Santa Cruz Biotechnology; PARP (9532), CPARP (9541S), glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (5174S), secondary anti-rabbit horseradish peroxidase (707HS and 7076S) from Cell Signaling Technology; IgG (mouse) (AB18413), and NR4A1 (ab109180) antibodies from Abcam.

Techniques: In Vivo, Isolation, Western Blot, Binding Assay, Derivative Assay, Microscopy, Staining, Control

Journal: Molecular pharmacology

Article Title: Dual nuclear receptor 4A1 (NR4A1/NR4A2) ligands inhibit glioblastoma growth and target TWIST1.

doi: 10.1016/j.molpha.2024.100009

Figure Lengend Snippet: Fig. 7. Model of proposed mechanism of action of TWIST1 expression by NR4A1 and NR4A2 interacting with (GC-rich) promoter DNA and inactivation of this response by DIM-3,5 analogs binding both receptors. NR4A1 and NR4A2 regulate TWIST1 expres- sion through interactions with DNA-bound Sp1 and Sp4 transcription factors, and DIM-3,5 analogs bind both receptors to inactivate expression of TWIST1.

Article Snippet: Antibodies used in cell culture experiments include Sp1 (sc-17824), Sp4 NR4A1 (sc-365113X), NR4A2 (sc376984X and sc-81345), TWIST1 (sc-81417), IgG (sc-2025) from Santa Cruz Biotechnology; PARP (9532), CPARP (9541S), glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (5174S), secondary anti-rabbit horseradish peroxidase (707HS and 7076S) from Cell Signaling Technology; IgG (mouse) (AB18413), and NR4A1 (ab109180) antibodies from Abcam.

Techniques: Expressing, Binding Assay

Journal: EMBO Molecular Medicine

Article Title: Reciprocal inhibition of NOTCH and SOX2 shapes tumor cell plasticity and therapeutic escape in triple-negative breast cancer

doi: 10.1038/s44321-024-00161-8

Figure Lengend Snippet: Reagents and tools table

Article Snippet: Mouse monoclonal anti-TWIST (clone Twist2C1a)_WB: 1/500 , Santa Cruz , Cat#sc-81417; RRID:AB_1130910.

Techniques: Recombinant, Plasmid Preparation, Binding Assay, Polymer, SYBR Green Assay, Transfection, Protease Inhibitor, Reporter Assay, Gel Purification, Purification, Ligation, Software

Journal: Human Cell

Article Title: Adipose-derived stem cells modified by TWIST1 silencing accelerates rat sciatic nerve repair and functional recovery

doi: 10.1007/s13577-024-01087-6

Figure Lengend Snippet: The primer sequences used for the qRT-PCR analysis

Article Snippet: Immunoblots were visualized after incubation with primary antibodies: mouse monoclonal anti-rat TWIST1 antibody (ab50887, Abcam, Cambridge, UK), rabbit monoclonal anti-rat NT-3 antibody (ab263864, Abcam), rabbit monoclonal anti-rat BDNF antibody (ab108319, Abcam), rabbit monoclonal anti-rat NGF antibody (MA5-32067, eBioscience), and mouse monoclonal anti-rat GDNF antibody (sc-13147, Santa Cruz Biotechnology, CA, USA), followed by incubation with secondary antibodies.

Techniques: Sequencing

Journal: Human Cell

Article Title: Adipose-derived stem cells modified by TWIST1 silencing accelerates rat sciatic nerve repair and functional recovery

doi: 10.1007/s13577-024-01087-6

Figure Lengend Snippet: Characterization of ADSCs and their differentiation into Schwann cells. A, Phenotypic characterization of ADSCs by flow cytometric analysis. B, The qRT-PCR analysis of TWIST1 mRNA expression levels in ADSCs 2 weeks after LV transduction. C, Immunoblotting analysis of TWIST1 protein expression levels in ADSCs 2 weeks after LV transduction. * P < 0.05 compared to the scramble siRNA by unpaired t tests. D, Representative inverted microscope images of ADSCs under Schwann cell induction for 14 days. Without induction, ADSCs showed a mesh-like structure. After 14 days, cells adopted a spindle shape with reduced volume, fewer protrusions, and a spiral growth pattern, resembling Schwann cells. Notably, the sh-TWIST1 group exhibited more pronounced Schwann cell-like features than the scramble group

Article Snippet: Immunoblots were visualized after incubation with primary antibodies: mouse monoclonal anti-rat TWIST1 antibody (ab50887, Abcam, Cambridge, UK), rabbit monoclonal anti-rat NT-3 antibody (ab263864, Abcam), rabbit monoclonal anti-rat BDNF antibody (ab108319, Abcam), rabbit monoclonal anti-rat NGF antibody (MA5-32067, eBioscience), and mouse monoclonal anti-rat GDNF antibody (sc-13147, Santa Cruz Biotechnology, CA, USA), followed by incubation with secondary antibodies.

Techniques: Quantitative RT-PCR, Expressing, Transduction, Western Blot, Inverted Microscopy

Journal: Human Cell

Article Title: Adipose-derived stem cells modified by TWIST1 silencing accelerates rat sciatic nerve repair and functional recovery

doi: 10.1007/s13577-024-01087-6

Figure Lengend Snippet: The sciatic nerves of rats in non-lesioned, non-transplanted, LV-scramble-ADSC, and LV-sh-TWIST1-ADSC groups 8 weeks after transplantation of ADSCs were collected and subjected to histomorphological analysis. A, The sections of rat sciatic nerve stained with toluidine blue; the non-lesioned rats showed homogeneously distributed fibers, uniform myelin sheath, and normal axon diameter; the non-transplanted rats showed clear signs of nerve fiber degeneration with presence of myelin degradation, indicated by white arrows; the LV-scramble-ADSC rats showed some signs of regenerated nerve fibers but presence of degenerated axons and decreased myelin sheath thickness; the LV-sh-TWIST1-ADSC rats showed clear signs of regenerated nerve fibers surrounded by newly formed myelin sheaths. B, Morphometric analysis of axion diameter and myelin sheath thickness. * P < 0.05 compared to the non-transplanted group and # P < 0.05 compared to the LV-scramble-ADSC group by one-way ANOVA followed by Tukey’s multiple comparisons test

Article Snippet: Immunoblots were visualized after incubation with primary antibodies: mouse monoclonal anti-rat TWIST1 antibody (ab50887, Abcam, Cambridge, UK), rabbit monoclonal anti-rat NT-3 antibody (ab263864, Abcam), rabbit monoclonal anti-rat BDNF antibody (ab108319, Abcam), rabbit monoclonal anti-rat NGF antibody (MA5-32067, eBioscience), and mouse monoclonal anti-rat GDNF antibody (sc-13147, Santa Cruz Biotechnology, CA, USA), followed by incubation with secondary antibodies.

Techniques: Transplantation Assay, Staining

Journal: Human Cell

Article Title: Adipose-derived stem cells modified by TWIST1 silencing accelerates rat sciatic nerve repair and functional recovery

doi: 10.1007/s13577-024-01087-6

Figure Lengend Snippet: The mRNA expressions of TWIST1, NT-3, BDNF, NGF, and GDNF in the sciatic nerves of rats after sciatic repair in non-lesioned, non-transplanted, LV-scramble-ADSC, and LV-sh-TWIST1-ADSC groups 8 weeks after transplantation of ADSCs were determined by the qRT-PCR analysis. * P < 0.05 compared to the non-transplanted group and # P < 0.05 compared to the LV-scramble-ADSC group by one-way ANOVA followed by Tukey’s multiple comparisons test

Article Snippet: Immunoblots were visualized after incubation with primary antibodies: mouse monoclonal anti-rat TWIST1 antibody (ab50887, Abcam, Cambridge, UK), rabbit monoclonal anti-rat NT-3 antibody (ab263864, Abcam), rabbit monoclonal anti-rat BDNF antibody (ab108319, Abcam), rabbit monoclonal anti-rat NGF antibody (MA5-32067, eBioscience), and mouse monoclonal anti-rat GDNF antibody (sc-13147, Santa Cruz Biotechnology, CA, USA), followed by incubation with secondary antibodies.

Techniques: Transplantation Assay, Quantitative RT-PCR

Journal: Human Cell

Article Title: Adipose-derived stem cells modified by TWIST1 silencing accelerates rat sciatic nerve repair and functional recovery

doi: 10.1007/s13577-024-01087-6

Figure Lengend Snippet: The protein expressions of TWIST1, NT-3, BDNF, NGF, and GDNF in the sciatic nerves of rats after sciatic repair in non-lesioned, non-transplanted, LV-scramble-ADSC, and LV-sh-TWIST1-ADSC groups 8 weeks after transplantation of ADSCs were determined by the immunoblotting analysis. * P < 0.05 compared to the non-transplanted group and # P < 0.05 compared to the LV-scramble-ADSC group by one-way ANOVA followed by Tukey’s multiple comparisons test

Article Snippet: Immunoblots were visualized after incubation with primary antibodies: mouse monoclonal anti-rat TWIST1 antibody (ab50887, Abcam, Cambridge, UK), rabbit monoclonal anti-rat NT-3 antibody (ab263864, Abcam), rabbit monoclonal anti-rat BDNF antibody (ab108319, Abcam), rabbit monoclonal anti-rat NGF antibody (MA5-32067, eBioscience), and mouse monoclonal anti-rat GDNF antibody (sc-13147, Santa Cruz Biotechnology, CA, USA), followed by incubation with secondary antibodies.

Techniques: Transplantation Assay, Western Blot