Journal: PLoS Biology

Article Title: Smoothened transduces Hedgehog signals via activity-dependent sequestration of PKA catalytic subunits

doi: 10.1371/journal.pbio.3001191

Figure Lengend Snippet: ( A ) HEK293 cells transfected with SMO657-nanoluc and PKA-C-YFP were treated with SMO inverse agonist KAADcyc (1 μM) or agonist SAG21k (1 μM) for 1 hour prior to BRET measurements. ( B ) Images of HEK293 cells transfected with FLAG-tagged SMO674 (magenta) and YFP-tagged PKA-C (green) and treated with vehicle, KAADcyc (300 nM), or SAG21k (100 nM) alone or with the GRK2/3 inhibitor Cmpd101 (“101”, 30 μM). Scale bar = 10 μm. ( C ) Quantification of colocalization between SMO and PKA-C for the experiment in (B) (see “ ’). ( D ) HEK293 cells were transfected with SMO657-nanoluc and YFP-tagged versions of either NbSmo2 or PKA-C and treated with vehicle, KAADcyc (1 μM), or SAG21k (1 μM) for 1 hour or with Cmpd101 (30 μM) for 4 hours. ( E ) Effect of KAADcyc (1 μM) or Cmpd101 (30 μM) on SMO inhibition of the CREB reporter in HEK293 cells. For (E), CREB reporter was normalized to 100%, which reflects reporter activation from PKA-C-transfected cells treated with vehicle. Data in (A), (D), and (E): n = 3–6 biological replicates per condition. Error bars = SEM. Data in (C): n = 119–216 cells per condition pooled from 2 or more independent experiments. The underlying data for this figure can be found under . See for statistical analysis. BRET, bioluminescence resonance energy transfer; GRK, GPCR kinase; Cmpd101, Compound 101; CREB, cyclic AMP response element binding protein; KAADcyc, KAAD-cyclopamine; PKA-C, PKA catalytic subunits; SMO, Smoothened.

Article Snippet: Human DRD2 and bovine GRK2 were obtained from Addgene (catalog # 66269 and 14691, respectively), amplified by PCR, fused to nanoluc or GFP (respectively), and cloned into pVLAD6.

Techniques: Transfection, Inhibition, Activation Assay, Bioluminescence Resonance Energy Transfer, Binding Assay

Journal: PLoS Biology

Article Title: Smoothened transduces Hedgehog signals via activity-dependent sequestration of PKA catalytic subunits

doi: 10.1371/journal.pbio.3001191

Figure Lengend Snippet: ( A ) HEK293 cells expressing GRK2 and either SMO674 (lanes 1–4), SMO674Ala, which carries mutations in 7 GRK2/3 phosphorylation sites (lanes 5–8), or SMO566 (lanes 9–12). Following treatment with SMO modulators or Cmpd101 (4 hours), SMO was isolated via FLAG affinity chromatography, and total protein or phosphoprotein was visualized using Stain-Free imaging or Pro-Q Diamond staining, respectively. Although GRKs often phosphorylate GPCRs on the intracellular loops of their 7TM domains [ , ], we did not observe phosphorylation within this region of SMO via phosphoprotein staining (A) or MS . Molecular masses are in kDa. ( B ) Clusters of phosphorylated residues identified by MS are labeled above the sequence of mouse SMO. Orange indicates phosphorylation that depends on SMO and GRK2/3 activity, while yellow indicates non-GRK phosphorylation sites. Alignment with SMO from other species reveals sequence conservation (blue), particularly among vertebrates. Green indicates GRK phosphorylation sites previously mapped in Drosophila Smo . Vertical lines indicate breaks in sequence. See for complete alignment. ( C ) Targeted MS-based quantification of phosphorylation at each of the 3 activity- and GRK2/3-dependent clusters in the SMO pCT (left 3 graphs) and total SMO protein in each sample (right-most graph). “Intensity” is a measurement of the abundance of phosphorylation sites (left) or total protein (right), derived from model-based estimation in MSstats which combines individual peptide intensities (see “ ”). ( D ) BRET between PKA-C and wild-type SMO657 or SMO657Ala. Data in (C): n = 3 biological and 3 technical replicates per condition. Data in (D): n = 3–6 biological replicates per condition. Error bars = SEM. The underlying data for this figure can be found under . The uncropped protein gels are included in . See for statistical analysis. Cmpd101, Compound 101; GPCR, G protein–coupled receptor; GRK, GPCR kinase; KAADcyc, KAAD-cyclopamine; MS, mass spectrometry; pCT, proximal segment of the cytoplasmic tail; PKA-C, PKA catalytic subunits; SMO, Smoothened.

Article Snippet: Human DRD2 and bovine GRK2 were obtained from Addgene (catalog # 66269 and 14691, respectively), amplified by PCR, fused to nanoluc or GFP (respectively), and cloned into pVLAD6.

Techniques: Expressing, Phospho-proteomics, Isolation, Affinity Chromatography, Staining, Imaging, Labeling, Sequencing, Activity Assay, Derivative Assay, Mass Spectrometry

Journal: Human Gene Therapy

Article Title: Exosome-Mediated Transfer of Anti-miR-33a-5p from Transduced Endothelial Cells Enhances Macrophage and Vascular Smooth Muscle Cell Cholesterol Efflux

doi: 10.1089/hum.2019.245

Figure Lengend Snippet: BAEC-derived EV contain small RNA, including miR-33a-5p. Size distribution of total RNA from EC (A) and EC-derived EV (B) was assessed by bioanalyzer-generated digital gels and electropherograms. (A, B) Representative plots from one of three independent experiments. (C) Reverse transcriptase-mediated PCR was used to amplify miR-33a-5p from total RNA extracted from EC, EC-derived EV, BASMC, and THP-1 macrophages (a positive control). A no-template (negative) control for the PCR is included (NTC). Amplicons were not digested (–), digested with BsrDI (B; the gene encoding miR-33a-5p contains a single BsrDI site) or with NotI (N; the gene encoding miR-33a-5p does not contain an NotI site), and finally analyzed by gel electrophoresis. Gel image is representative of at least three independent experiments per cell type/purified EV. (D) EV preparations (harvested in five independent experiments) were: untreated; treated with RNase and protease; or treated with detergent, then with RNase, and protease. MiR-33a-5p expression was measured by reverse transcriptase-mediated quantitative PCR. Bars are group medians. (A, B) Peaks at 25 nucleotides are standards, used for calibration. BASMC, bovine aortic smooth muscle cells; NTC, non-template control; SMC, smooth muscle cells.

Article Snippet: Bovine aortic EC (BAEC) (Cell Applications, San Diego, CA) and bovine aortic SMC (BASMC) (Cell Applications) were maintained in high-glucose DMEM (GIBCO, Grand Island, NY), 10% FBS (VWR Life Science Seradigm, Radnor, PA), and 1% Pen-Strep (GIBCO) and they were used between passages 3 and 7.

Techniques: Derivative Assay, Generated, Reverse Transcription, Positive Control, Negative Control, Nucleic Acid Electrophoresis, Purification, Expressing, Real-time Polymerase Chain Reaction, Control

Journal: Human Gene Therapy

Article Title: Exosome-Mediated Transfer of Anti-miR-33a-5p from Transduced Endothelial Cells Enhances Macrophage and Vascular Smooth Muscle Cell Cholesterol Efflux

doi: 10.1089/hum.2019.245

Figure Lengend Snippet: EV-mediated transfer of anti-miR-33a-5p to macrophages and SMC decreases miR-33a-5p expression: effect of X-motif. (A, B) BAEC were transduced with an HDAd vector expressing anti-miR-33a-5p, either without (HDAdAntimiR33a5p) or with (HDAdXMoAntimiR33a5p) the X-motif in the anti-miR-33a transcript. Anti-miR-33a was measured in extracts of: (A) EC; or (B) EC-derived EV that were isolated by ultracentrifugation of serum-free EC-CM. Macrophages derived from THP-1 monocytes (C, D) and BASMC (E, F) were treated with EV-containing CM harvested from EC transduced with the two HDAds described earlier, which express anti-miR-33a-5p transcripts either without (–) or with (+) the X-motif. As a technical control, macrophages and SMC were also treated with medium that was conditioned by HDAdXMoAntimiR33a5p-transduced EC; then, the CM was ultracentrifuged to remove EV (EV–). Total RNA was extracted from EC, EV, macrophages, and SMC, and it was used as a template to measure anti-miR-33a-5p (A-C, E) and miR-33a-5p (D, F) with reverse transcriptase-mediated quantitative PCR. (A, B) Data points are from each of five independent experiments: (A) RNA was from single wells of EC; (B) RNA was from EV purified from CM of several dishes of EC, transduced in parallel with wells in (A). (C–F) Data points are from individual wells (biological replicates) and represent results of three independent experiments with two wells per condition in each experiment. Bars are group medians. AU, arbitrary units; HDAd, helper-dependent adenovirus.

Article Snippet: Bovine aortic EC (BAEC) (Cell Applications, San Diego, CA) and bovine aortic SMC (BASMC) (Cell Applications) were maintained in high-glucose DMEM (GIBCO, Grand Island, NY), 10% FBS (VWR Life Science Seradigm, Radnor, PA), and 1% Pen-Strep (GIBCO) and they were used between passages 3 and 7.

Techniques: Expressing, Transduction, Plasmid Preparation, Derivative Assay, Isolation, Control, Reverse Transcription, Real-time Polymerase Chain Reaction, Purification

Journal: Human Gene Therapy

Article Title: Exosome-Mediated Transfer of Anti-miR-33a-5p from Transduced Endothelial Cells Enhances Macrophage and Vascular Smooth Muscle Cell Cholesterol Efflux

doi: 10.1089/hum.2019.245

Figure Lengend Snippet: MiR-33a-5p transcripts in macrophages and SMC are decreased by treatment with EC-derived EV containing anti-miR-33a-5p. BAEC were transduced with HDAd vectors expressing either: (A) a non-targeted “scrambled” anti-miRNA transcript (Scr Anti-miR), or (B) an anti-miR-33a-5p transcript. Both transcripts contain the X-motif and were measured in extracts of EC and of EV that were isolated by ultracentrifugation. THP-1-derived macrophages (C) and BASMC (D) were treated with medium conditioned by EC transduced with HDAd expressing either the scrambled (Scr) or anti-miR-33a-5p (Anti) transcripts. Both cell types were also treated with EC-CM that was harvested from EC expressing the Anti-transcript; then, the CM was depleted of EV by ultracentrifugation (EV–). Total RNA was extracted from EC, EV, macrophages, and SMC, and miRNA were measured with reverse transcriptase-mediated quantitative PCR. (A, B) Data points are from three and five independent experiments, respectively. In each experiment, EC RNA was from a single well of EC and EV RNA was purified from CM of several dishes of EC, transduced in parallel. Data in (B) are from the same experiment illustrated in Fig. 3 (panels A and B); replotted and portrayed here for ease of comparison. (C, D) Data points are from individual wells (biological replicates) and represent results of three independent experiments, each with two wells per condition. Bars are group means. AU, arbitrary units.

Article Snippet: Bovine aortic EC (BAEC) (Cell Applications, San Diego, CA) and bovine aortic SMC (BASMC) (Cell Applications) were maintained in high-glucose DMEM (GIBCO, Grand Island, NY), 10% FBS (VWR Life Science Seradigm, Radnor, PA), and 1% Pen-Strep (GIBCO) and they were used between passages 3 and 7.

Techniques: Derivative Assay, Transduction, Expressing, Isolation, Reverse Transcription, Real-time Polymerase Chain Reaction, Purification, Comparison

Journal: Human Gene Therapy

Article Title: Exosome-Mediated Transfer of Anti-miR-33a-5p from Transduced Endothelial Cells Enhances Macrophage and Vascular Smooth Muscle Cell Cholesterol Efflux

doi: 10.1089/hum.2019.245

Figure Lengend Snippet: ABCA1 protein and apoAI-mediated cholesterol efflux are increased by treatment with EC-derived EV containing anti-miR-33a-5p. THP-1-derived macrophages (A, B, E) and BASMC (C, D, F) were treated with medium conditioned by EC transduced with HDAd vectors expressing either a non-targeted “scrambled” anti-miRNA transcript (Scr) or an anti-miR-33a-5p transcript (Anti). Both cell types were also treated with CM that was harvested from EC expressing the Anti transcript; then, the CM was depleted of EV by ultracentrifugation (EV–). Cell lysates were analyzed by SDS-PAGE and immunoblotting (A, C). ABCA1 protein (B, D) was quantified by densitometry of blots, including those in (A, C). The percentage of cellular [3H] cholesterol effluxed to exogenous apoAI was measured (E, F). Data points (B, D) are from individual wells (biological replicates) and represent results of three independent experiments, each with two wells per condition. Data points (E, F) are from individual wells (biological replicates) and represent results of three independent experiments, each with three wells per condition. (A, C) Size markers are in kDa; (B, D) All bars are group means. ABCA1, ATP-binding cassette subfamily A, member 1; apoAI, apolipoprotein AI; AU, arbitrary units.

Article Snippet: Bovine aortic EC (BAEC) (Cell Applications, San Diego, CA) and bovine aortic SMC (BASMC) (Cell Applications) were maintained in high-glucose DMEM (GIBCO, Grand Island, NY), 10% FBS (VWR Life Science Seradigm, Radnor, PA), and 1% Pen-Strep (GIBCO) and they were used between passages 3 and 7.

Techniques: Derivative Assay, Transduction, Expressing, SDS Page, Western Blot, Binding Assay

Journal: Nutrients

Article Title: JNK/p66Shc/ITCH Signaling Pathway Mediates Angiotensin II-induced Ferritin Degradation and Labile Iron Pool Increase

doi: 10.3390/nu12030668

Figure Lengend Snippet: Angiotensin II (Ang II) induces ferritin degradation. Immunoblotting analysis of ferritin H and L levels in lysates of ( a ) human umbilical vein endothelial cells (HUVEC), ( b ) HT22, and ( c ) CPAE cells treated with 0.4 μmol/L Ang II for ( a ) 4 h, ( b ) 1 h, and ( c ) 1 h. After probing with specific antibodies, the blots were stripped and reprobed with an anti-β-actin antibody to normalize for differences in protein loading. The results are representative of three independent experiments. ( d ) Immunoblotting analysis of ferritin L levels in a lysate of CPAE cells treated with 0.4 μmol/l Ang II for the indicated time periods. After probing with specific antibodies, the blots were stripped and reprobed with an anti-β-actin antibody to normalize for differences in protein loading. Data in the graph on the right are the mean ± SEM from three independent experiments, with values recalculated relative to the control; “a” indicates statistical significance at p < 0.05 (t-test).

Article Snippet: HT22 cells (a mouse hippocampal cell line) were a kind gift from Professor Tillman Grune (Friedrich Schiller University, Jena, Germany) and cultured in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% fetal bovine serum, 100 U/mL penicillin, and 100 μg/mL streptomycin B. CPAE cells (an endothelial cell line derived from the bovine stem pulmonary artery) were obtained from the American Type Culture Collection (ATCC CCL 209, Rockville, MD, USA) and grown in minimum essential Eagle’s medium supplemented with 20% fetal bovine serum, 100 U/mL penicillin, and 100 μg/mL streptomycin B.

Techniques: Western Blot, Control

Journal: Nutrients

Article Title: JNK/p66Shc/ITCH Signaling Pathway Mediates Angiotensin II-induced Ferritin Degradation and Labile Iron Pool Increase

doi: 10.3390/nu12030668

Figure Lengend Snippet: Ang II-induced reactive oxygen species (ROS) generation is associated with labile iron pool (LIP) increase. ( a ) Representative images of confocal microscopy analysis of calcein fluorescence of CPAE cells treated with 0.4 μmol/L Ang II. CPAE cells were pretreated with 25 μmol/L DFO for 1 h. The graph on the right shows the percentage of cells exhibiting quenched fluorescence. Data are shown as the mean + SEM from four independent experiments, recalculated relative to the control: “a” indicates statistical significance at p < 0.001 (one-way ANOVA followed by Tukey’s multiple comparison test); ( b ) Percent of control DCF fluorescence in CPAE cells treated for 1 h with 0.4 μmol/L Ang II. Data are presented as the mean + SE ( n = 3); “a” indicates statistical significance at p < 0.022 (t-test).

Article Snippet: HT22 cells (a mouse hippocampal cell line) were a kind gift from Professor Tillman Grune (Friedrich Schiller University, Jena, Germany) and cultured in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% fetal bovine serum, 100 U/mL penicillin, and 100 μg/mL streptomycin B. CPAE cells (an endothelial cell line derived from the bovine stem pulmonary artery) were obtained from the American Type Culture Collection (ATCC CCL 209, Rockville, MD, USA) and grown in minimum essential Eagle’s medium supplemented with 20% fetal bovine serum, 100 U/mL penicillin, and 100 μg/mL streptomycin B.

Techniques: Confocal Microscopy, Fluorescence, Control, Comparison

Journal: Nutrients

Article Title: JNK/p66Shc/ITCH Signaling Pathway Mediates Angiotensin II-induced Ferritin Degradation and Labile Iron Pool Increase

doi: 10.3390/nu12030668

Figure Lengend Snippet: Ang II-induced ferritin degradation is c-Jun terminal kinase (JNK) dependent. ( a ) Immunoblotting analysis of ferritin L levels in lysates of control CPAE cells or cells treated for 1 h with Ang II in the absence or presence of 20 μmol/L SP600125 (1 h pretreatment). After probing with specific antibodies, the blots were stripped and reprobed with an anti-β-actin antibody to normalize for differences in protein loading. Data in the graph on the right are the mean + SEM from three independent experiments, recalculated relative to the control; “a” indicates statistical significance at p < 0.05 (t-test). ( b ) Immunoblotting analysis of ferritin L levels in lysates of CPAE cells transiently transfected with an empty vector, or vectors encoding catalytically inactive variants of JNK1 or JNK2 following a 1-h treatment with 0.4 μmol/L Ang II. After probing with specific antibodies, the blots were stripped and reprobed with an anti-β-actin antibody to normalize for differences in protein loading. Data in the graphs at the bottom of the panel are the mean ± SEM from three independent experiments, recalculated relative to the control; “a” indicates statistical significance at p < 0.05 (t-test). ( c ) Immunoblotting detection of the HA-tag in lysates of CPAE cells transiently transfected with vectors encoding HA-tagged catalytically inactive variants of JNK1 or JNK2.

Article Snippet: HT22 cells (a mouse hippocampal cell line) were a kind gift from Professor Tillman Grune (Friedrich Schiller University, Jena, Germany) and cultured in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% fetal bovine serum, 100 U/mL penicillin, and 100 μg/mL streptomycin B. CPAE cells (an endothelial cell line derived from the bovine stem pulmonary artery) were obtained from the American Type Culture Collection (ATCC CCL 209, Rockville, MD, USA) and grown in minimum essential Eagle’s medium supplemented with 20% fetal bovine serum, 100 U/mL penicillin, and 100 μg/mL streptomycin B.

Techniques: Western Blot, Control, Transfection, Plasmid Preparation

Journal: Nutrients

Article Title: JNK/p66Shc/ITCH Signaling Pathway Mediates Angiotensin II-induced Ferritin Degradation and Labile Iron Pool Increase

doi: 10.3390/nu12030668

Figure Lengend Snippet: Ang II-induced ferritin degradation is ITCH-dependent. Immunoblotting analysis of ( a ) ferritin H and ( b ) L levels in lysates of CPAE cells transiently transfected with an empty vector or a vector encoding a catalytically inactive mutant of ITCH following a 1-h treatment with 0.4 μmol/L Ang II. After probing with specific antibodies, the blots were stripped and reprobed with an anti-β-actin antibody to normalize for differences in protein loading. Data in graphs at the bottom of the panel are the mean + SEM from three independent experiments, recalculated relative to the control. ( c ) Immunoblotting analysis of the ITCHlevels in lysates of CPAE cells transiently transfected with an empty vector or a vector encoding a catalytically inactive variant of ITCH. After probing with specific antibodies, the blots were stripped and reprobed with an anti-β-actin antibody to normalize for differences in protein loading.

Article Snippet: HT22 cells (a mouse hippocampal cell line) were a kind gift from Professor Tillman Grune (Friedrich Schiller University, Jena, Germany) and cultured in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% fetal bovine serum, 100 U/mL penicillin, and 100 μg/mL streptomycin B. CPAE cells (an endothelial cell line derived from the bovine stem pulmonary artery) were obtained from the American Type Culture Collection (ATCC CCL 209, Rockville, MD, USA) and grown in minimum essential Eagle’s medium supplemented with 20% fetal bovine serum, 100 U/mL penicillin, and 100 μg/mL streptomycin B.

Techniques: Western Blot, Transfection, Plasmid Preparation, Mutagenesis, Control, Variant Assay

Journal: Nutrients

Article Title: JNK/p66Shc/ITCH Signaling Pathway Mediates Angiotensin II-induced Ferritin Degradation and Labile Iron Pool Increase

doi: 10.3390/nu12030668

Figure Lengend Snippet: Ang II-induced ferritin degradation is p66Shc dependent. ( a ) Immunoblotting analysis of p66Shc and p-p66Shc levels in lysates of control CPAE cells or cell treated for 1 h with Ang II. After probing with specific antibodies, the blots were stripped and reprobed with an anti-β-actin antibody to normalize for differences in protein loading. Data in the graphs on the right are the mean + SEM from two independent experiments, recalculated relative to the control. ( b ) Immunoblotting analysis of p66Shc levels in lysates of CPAE cells transiently transfected with an empty vector or a vector encoding a dominant negative variant of p66Shc (p66ShcS36A). Immunoblotting analysis of ( c ) ferritin H and ( d ) ferritin L levels in lysates of CPAE cells transiently transfected with an empty vector or a vector encoding a dominant negative variant of p66Shc (p66ShcS36A) following a 1-h treatment with 0.4 μmol/L Ang II. After probing with specific antibodies, the blots were stripped and reprobed with an anti-β-actin antibody to normalize for differences in protein loading. Data in graphs at the bottom of the panel are the mean + SEM from three independent experiments, recalculated relative to the control.

Article Snippet: HT22 cells (a mouse hippocampal cell line) were a kind gift from Professor Tillman Grune (Friedrich Schiller University, Jena, Germany) and cultured in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% fetal bovine serum, 100 U/mL penicillin, and 100 μg/mL streptomycin B. CPAE cells (an endothelial cell line derived from the bovine stem pulmonary artery) were obtained from the American Type Culture Collection (ATCC CCL 209, Rockville, MD, USA) and grown in minimum essential Eagle’s medium supplemented with 20% fetal bovine serum, 100 U/mL penicillin, and 100 μg/mL streptomycin B.

Techniques: Western Blot, Control, Transfection, Plasmid Preparation, Dominant Negative Mutation, Variant Assay

Journal: Oncotarget

Article Title: Thrombospondin 1 promotes an aggressive phenotype through epithelial-to-mesenchymal transition in human melanoma

doi:

Figure Lengend Snippet: qRT-PCR analysis revealed expression of ( A ) E- and N-cadherins and ( B ) THBS1 in a panel of 54 melanoma cell lines. ( C ) Media from 4 mesenchymal- and 4 epithelial-like melanoma cells were collected and subjected to THBS1 ELISA. ANOVA analysis of the two sets of cell lines was significant (p<0.005).

Article Snippet: Cells were incubated with siRNA complex for 48 hours and then fixed with 4% paraformaldehyde, stained with anti-THBS1 antibody (A6.1, NB100-2059, Novus Biologicals, USA) was applied at 2.5μg/mL concentration overnight at 4°C and with Alexa flour 488 conjugated secondary antibody for 45 minutes at room temperature (Molecular probes, USA).

Techniques: Quantitative RT-PCR, Expressing, Enzyme-linked Immunosorbent Assay

Journal: Oncotarget

Article Title: Thrombospondin 1 promotes an aggressive phenotype through epithelial-to-mesenchymal transition in human melanoma

doi:

Figure Lengend Snippet: Melanoma cell line, LM-MEL-34 labelled with CM-DiI was imaged after ( A ) 2 days (scale bar = 50μm) or ( B ) 14 days (scale bar = 100μm), respectively. ( C ) Melanoma cells from a patient with malignant melanoma were labelled with CM-Dil and injected subcutaneously into the flank of three animals. After three weeks, tumors were harvested and embedded in paraffin, cut into sections and examined by fluorescent microscopy (scale bar = 500μm). ( D ) Example of a flow profile of LM-MEL-34 cells unstained, 1 hour after CM-Dil labeling, 12 days after labeling, and merged histogram overlay (from left to right). The red striped area represents the population of cells referred to as LRC. LM-MEL−28, −34, −42 and −44 were sorted into Dil bright and Dil dull cells 14 days after labeling with CM-Dil. RNA was subjected to genome-wide gene expression analysis using Illumina HT12 arrays. ( E ) Upregulated genes were employed to generate an evidence-based protein-interaction network in STRING and demonstrated connections between several ECM-related molecules with higher expression in the Dil bright cells. ( F ) Dil bright and Dil dull cells from the same 4 lines and 2 additional cell lines were separated and qPCR was performed to evaluate expression of THBS1 . Values are mean +/− SEM of three experiments in triplicate (* p<0.05, ** p<0.005).

Article Snippet: Cells were incubated with siRNA complex for 48 hours and then fixed with 4% paraformaldehyde, stained with anti-THBS1 antibody (A6.1, NB100-2059, Novus Biologicals, USA) was applied at 2.5μg/mL concentration overnight at 4°C and with Alexa flour 488 conjugated secondary antibody for 45 minutes at room temperature (Molecular probes, USA).

Techniques: Injection, Microscopy, Labeling, Genome Wide, Gene Expression, Expressing

Journal: Oncotarget

Article Title: Thrombospondin 1 promotes an aggressive phenotype through epithelial-to-mesenchymal transition in human melanoma

doi:

Figure Lengend Snippet: ( A ) Mesenchymal- and epithelial-like melanoma cells were subjected to invasion through matrigel-coated Boyden chambers. After 48 hours cells were fixed and images of invasive cells were taken (scale bar = 500μm) or stained using THBS1 antibody in conjunction with an Alexa-488 conjugated secondary antibody and visualized with a fluorescent microscope (scale bar = 100μm). ( B ) THBS1 protein expression in two clonally derived LM-MEL-34 and LM-MEL-62 cell lines and respective empty vector control was detected using immunostaining (scale bar = 100μm). ( C ) Invasiveness of epithelial-like cells with empty vector control or THBS-1 expression vector was determined (scale bar = 100μm). (D) The graphs show the total number of invasive cells counted. Values are mean +/− SEM of three experiments in triplicate (* p<0.05).

Article Snippet: Cells were incubated with siRNA complex for 48 hours and then fixed with 4% paraformaldehyde, stained with anti-THBS1 antibody (A6.1, NB100-2059, Novus Biologicals, USA) was applied at 2.5μg/mL concentration overnight at 4°C and with Alexa flour 488 conjugated secondary antibody for 45 minutes at room temperature (Molecular probes, USA).

Techniques: Staining, Microscopy, Expressing, Derivative Assay, Plasmid Preparation, Control, Immunostaining

Journal: Oncotarget

Article Title: Thrombospondin 1 promotes an aggressive phenotype through epithelial-to-mesenchymal transition in human melanoma

doi:

Figure Lengend Snippet: ( A ) Melanoma cells were plated out and transfected with either 20nM control siRNA or THBS1 specific siRNA. After 72 hours RNA was extracted and THBS1 qRT-PCR was performed. ( B ) Melanoma cells were transfected as described and representative images of invasion in LM-MEL-53 are shown after 24 hours (scale bar = 100μm). The graphs show the total number of invasive cells counted and cell numbers from control were set to 100% and compared to THBS1 siRNA transfected cells. ( C ) Invasiveness of melanoma cells after treatment with control anti-IgG or anti-THBS1 antibodies was tested, images captured (scale bar = 100μm) and quantified as above. Values are mean +/− SEM of four independent experiments in triplicate (* p<0.05, ** p<0.005, *** p<0.0005).

Article Snippet: Cells were incubated with siRNA complex for 48 hours and then fixed with 4% paraformaldehyde, stained with anti-THBS1 antibody (A6.1, NB100-2059, Novus Biologicals, USA) was applied at 2.5μg/mL concentration overnight at 4°C and with Alexa flour 488 conjugated secondary antibody for 45 minutes at room temperature (Molecular probes, USA).

Techniques: Transfection, Control, Quantitative RT-PCR

Journal: Oncotarget

Article Title: Thrombospondin 1 promotes an aggressive phenotype through epithelial-to-mesenchymal transition in human melanoma

doi:

Figure Lengend Snippet: Melanoma cells were treated with CM-DiO, transfected with the indicated constructs and cultured as hanging drops to encourage aggregate formation. Similar sized aggregates were introduced into the neural tube of developing chicken and re-incubated within the egg for 2 days. ( A ) Embryos were harvested and fluorescence pictures from whole-mounts taken (scale bar = 50μm). White dotted line shows the outline of the neural tube and the white arrows indicate melanoma cells that migrated out of the neural tube and into the surrounding tissue. ( B ) Analysis of cell numbers infiltrating the surrounding tissue from several independent experiments (n=4 for control siRNA and THBS1 siRNA) (* p<0.05). ( C ) Images from cross-sections of embryos. White arrows indicate melanoma cells and NT denotes neural tube, dorsal is to the top.

Article Snippet: Cells were incubated with siRNA complex for 48 hours and then fixed with 4% paraformaldehyde, stained with anti-THBS1 antibody (A6.1, NB100-2059, Novus Biologicals, USA) was applied at 2.5μg/mL concentration overnight at 4°C and with Alexa flour 488 conjugated secondary antibody for 45 minutes at room temperature (Molecular probes, USA).

Techniques: Transfection, Construct, Cell Culture, Incubation, Fluorescence, Control

Journal: Oncotarget

Article Title: Thrombospondin 1 promotes an aggressive phenotype through epithelial-to-mesenchymal transition in human melanoma

doi:

Figure Lengend Snippet: THBS1 expression by qRT-PCR ( A ) and secretion by ELISA ( B ) in PLX4720 resistant LM-MEL-28R1 and LM-MEL-64R3 melanoma cell lines relative to their sensitive parental lines was determined. ( C ) LM-MEL-28R1 and LM-MEL-64R3 melanoma cell lines were treated with control or THBS1 siRNA and knockdown was evaluated with qRT-PCR and ( D ) percentage survival was quantified from absorbance obtained from MTS measurement. Bars are mean values +/− SEM from three independent experiments in triplicate (* p<0.05, ** p<0.005, *** p<0.0005).

Article Snippet: Cells were incubated with siRNA complex for 48 hours and then fixed with 4% paraformaldehyde, stained with anti-THBS1 antibody (A6.1, NB100-2059, Novus Biologicals, USA) was applied at 2.5μg/mL concentration overnight at 4°C and with Alexa flour 488 conjugated secondary antibody for 45 minutes at room temperature (Molecular probes, USA).

Techniques: Expressing, Quantitative RT-PCR, Enzyme-linked Immunosorbent Assay, Control, Knockdown

Journal: Oncotarget

Article Title: Thrombospondin 1 promotes an aggressive phenotype through epithelial-to-mesenchymal transition in human melanoma

doi:

Figure Lengend Snippet: ( A ) Localization of THBS1 in metastatic melanoma tumor biopsies. Representative tumors show score staining intensities (scale bar = 50μm). ( B ) Graph shows number of tumors scored.

Article Snippet: Cells were incubated with siRNA complex for 48 hours and then fixed with 4% paraformaldehyde, stained with anti-THBS1 antibody (A6.1, NB100-2059, Novus Biologicals, USA) was applied at 2.5μg/mL concentration overnight at 4°C and with Alexa flour 488 conjugated secondary antibody for 45 minutes at room temperature (Molecular probes, USA).

Techniques: Staining

Journal: bioRxiv

Article Title: Rare variant in intracellular loop-2 of the ghrelin receptor reveals novel mechanisms of GPCR biased signaling and trafficking

doi: 10.1101/2025.03.11.642421

Figure Lengend Snippet: (a) hβarr1 recruitment to GHSR. (b) Constitutive, (c) hGhrelin C/R, and (d) hGhrelin E max (derived from Panel c) of hβarr1 recruitment to WT following 30-minute pretreatment (RT) with vehicle (grey), Go6983 (light blue; 3μM), Cmp101 (light green; 10μM), or Go6983+Cmp101 (light purple). (e) Constitutive, (f) hGhrelin C/R, and (g) hGhrelin E max (derived from Panel f ) hβarr1 recruitment to L149P following pretreatment with vehicle (light teal), Go6983 (light orange), Cmp101 (light yellow), or Go6983+Cmp101 (light red). (h) hβarr2 recruitment to GHSR. (i) Constitutive, (j) hGhrelin C/R, and (k) hGhrelin E max (derived from Panel j ) of hβarr2 recruitment to WT following pretreatment with vehicle (black), Go6983 (dark blue), Cmp10 (dark green), or Go6983+Cmp101 (dark purple). (l) Constitutive, (m) hGhrelin C/R, and (n) hGhrelin E max (derived from Panel m ) hβarr2 recruitment to L149P following pretreatment with vehicle (dark teal), Go6983 (dark orange), Cmp101 (dark yellow), or Go6983+Cmp101 (dark red). (o) hβarr1 recruitment to GHSR. (p) Constitutive, (q) hGhrelin C/R, (r) hGhrelin E max (derived from Panel q ) of hβarr1 recruitment to WT following co-expression/pretreatment with pcDNA-vehicle (control; grey), GRK2 K220R -vehicle (light red), or pcDNA-GRK2i (100μM, 30-minutes, RT). (s) Constitutive, (t) hGhrelin C/R, (u) hGhrelin E max (derived from Panel t ) of hβarr1 recruitment to L149P in pcDNA-vehicle (light teal), GRK2 K220R -vehicle (light purple), or pcDNA-GRK2i (light blue). (v) hβarr2 recruitment to GHSR. (w) Constitutive, (x) hGhrelin C/R, (y) hGhrelin E max (derived from Panel x ) of hβarr2 recruitment to WT in pcDNA-vehicle (black), GRK2 K220R -vehicle (dark red), or pcDNA-GRK2i (dark blue). (z) Constitutive, (aa) hGhrelin C/R, (bb) hGhrelin E max (derived from Panel aa ) of hβarr2 recruitment to L149P in pcDNA-vehicle (dark teal), GRK2 K220R -vehicle (dark purple), or pcDNA-GRK2i (dark blue). All constitutive and E max data were analyzed by one-way ANOVA. (cc) YFP-tagged WT-GRK2 (GRK2 WT ) recruitment to GHSR. (dd) Constitutive, (ee) hGhrelin C/R, (ff) hGhrelin E max (derived from Panel ee ) of GRK2 WT recruitment to WT-vehicle (black), WT-GRK2i (dark blue), L149P-vehicle (dark teal), and L149P-GRK2i (dark purple) following a 30-minute pretreatment (RT). Constitutive and E max data were analyzed by two-way ANOVA followed by Tukey’s post-hoc test. (gg) Constitutive, (hh) hGhrelin C/R, (ii) hGhrelin E max (derived from Panel hh ) of recruitment of GRK2 τιV661-ct to WT (grey) or L149P (light teal). Constitutive and E max data were analyzed by t -test. All data represent the pooled mean ± SEM from > 3 independent experiments in duplicate or triplicate.

Article Snippet: The dominant negative mutant, bovine GRK2-K220R, was a gift from Dr. Robert Lefkowitz (Addgene plasmid #35403).

Techniques: Derivative Assay, Expressing, Control

Journal: bioRxiv

Article Title: Rare variant in intracellular loop-2 of the ghrelin receptor reveals novel mechanisms of GPCR biased signaling and trafficking

doi: 10.1101/2025.03.11.642421

Figure Lengend Snippet: (a) hβarr1 recruitment to GHSR. (b) Constitutive, (c) hGhrelin-stimulated C/R, and (d) hGhrelin E max (derived from Panel c ) of hβarr1 recruitment to WT in parental (light grey), ΔGRK2/3/5/6 (light red), ΔGRK2/3 (light orange), and ΔGRK5/6 (light green). (e) Constitutive, (f) hGhrelin-stimulated C/R, and (g) hGhrelin E max (derived from Panel f ) of hβarr1 recruitment to L149P in parental (light teal), ΔGRK2/3/5/6 (light blue), ΔGRK2/3 (light purple), and ΔGRK5/6 (light maroon). (h) hβarr2 recruitment to GHSR. (i) Constitutive, (j) hGhrelin-stimulated C/R, and (k) hGhrelin E max (derived from Panel j ) of hβarr2 recruitment to WT in parental (dark grey), ΔGRK2/3/5/6 (dark red), ΔGRK2/3 (dark orange), and ΔGRK5/6 (dark green). (l) Constitutive, (m) hGhrelin-stimulated C/R, and (n) hGhrelin E max (derived from Panel m ) of hβarr2 recruitment to L149P in parental (dark teal), ΔGRK2/3/5/6 (dark blue), ΔGRK2/3 (dark purple), and ΔGRK5/6 (dark maroon). (o) GRK5/6 YFP coupling to GHSR. (p) Constitutive and (q) hGhrelin C/R-treated GRK5 YFP coupling, then (r) constitutive and (s) hGhrelin C/R-treated GRK6 YFP coupling over 30-minutes. Panel p , r were analyzed by Mann-Whitney nonparametric test; whereas, Panels q, s were analyzed by a mixed-model two-way ANOVA. All data represent the pooled mean ± SEM from 4 independent experiments in triplicate.

Article Snippet: The dominant negative mutant, bovine GRK2-K220R, was a gift from Dr. Robert Lefkowitz (Addgene plasmid #35403).

Techniques: Derivative Assay, MANN-WHITNEY

Journal: bioRxiv

Article Title: Rare variant in intracellular loop-2 of the ghrelin receptor reveals novel mechanisms of GPCR biased signaling and trafficking

doi: 10.1101/2025.03.11.642421

Figure Lengend Snippet: We hypothesize that the ultra-rare, natural L149P mutant (teal, right ), destabilizes the α-helical conformation of ICL2, thereby (i) reducing clustering within cholesterol/sphingolipid-enriched lipid rafts and (ii) blocking G protein activation. Consequently, L149P preferentially partitions into more disordered, non-lipid raft PM microdomains ( e.g. , unsaturated fatty acid-enriched) and exhibits poor agonist-directed endocytosis from lipid rafts. Under basal conditions, L149P expression is biased towards the PM over endosomes due to attenuated constitutive internalization/receptor cycling. However, upon agonist stimulation, L149P undergoes robust internalization and endosomal translocation via kinetics distinct from the WT-GHSR (black, left ), suggesting that L149P may utilize different endocytic route(s). The L149P mutant readily recruits βarr in a cholesterol-dependent manner, while simultaneously inhibiting Gα q via decreased guanine nucleotide exchange. Accordingly, L149P does not activate PKC or Gβγ-dependent GRK2 translocation and recruits βarr in G protein-independent manner, despite weak Gα i/o partial agonism. In contrast to the WT-GHSR, βarr recruitment to L149P requires both GRK2/3 and GRK5/6, with the latter associated with increased L149P-GRK6 proximity. Thus, GRK6 precoupling to L149P might ‘prime’ the receptor for βarr coupling/recruitment, desensitization, and endocytosis.

Article Snippet: The dominant negative mutant, bovine GRK2-K220R, was a gift from Dr. Robert Lefkowitz (Addgene plasmid #35403).

Techniques: Mutagenesis, Blocking Assay, Activation Assay, Expressing, Translocation Assay