Journal: bioRxiv

Article Title: The Hedgehog receptors PTCH1 and PTCH2 exist as active homomeric and heteromeric complexes

doi: 10.1101/2023.08.08.549832

Figure Lengend Snippet: A. Schematic representation of the FRAP assay to detect direct protein-protein interactions within live cells. Two PTCH1 proteins, one containing a C-terminal mCherry and the other a C-terminal eGFP are co-expressed. If the two proteins directly interact, some emission energy from the eGFP is absorbed by mCherry. Photo-bleaching of mCherry prevents this excitation and the eGFP emission energy previously absorbed by mCherry can be detected as additional post-bleach eGFP emission intensity. Image created with BioRender.com. B. HEK 293 cells transiently co-expressing PTCH1-eGFP and PTCH1-mCherry, were subjected to confocal imaging with the FRAP interaction assay. Change in eGFP fluorescence intensity (expressed as % of initial fluorescence intensity) was measured across 8 acquisitions, with mCherry photo-bleached (green trace) or not bleached (grey trace) after the third image in the series. Statistical significance of indicated image number compared to image 3: **** P<0.0001 (n=23). C. Change in GFP fluorescence intensity between images 3 and 4 in HEK 293 cells in which mCherry was bleached or not (from ). D. Competition-based FRAP measurements in HEK 293 cells co-expressing PTCH1-eGFP and PTCH1-mCherry, when co-transfected with 4X mass excess of empty pcDNA 3.1 + vector (green trace) or vector encoding PTCH1-His (black trace). Statistical significance of indicated image number compared to image 3 of the same group: ** P<0.01; **** P<0.0001 (n=27). E. Net change in GFP fluorescence intensity immediately post-bleach, corresponding to image 4 in the series, in PTCH1-eGFP and PTCH1-mCherry expressing HEK 293 cells with 4X excess of empty vector (green symbols) or 4X PTCH1-His (black symbols). F . Representative co-immunoprecipitation of HA-tagged PTCH1 with myc-tagged PTCH1 after co-expression in HEK 293 cells. WCL: whole cell lysate.

Article Snippet: HEK 293 human embryonic kidney epithelial cells and NIH 3T3 murine embryonic fibroblast cells were obtained from American Type Culture Collection (ATCC).

Techniques: FRAP Assay, Expressing, Imaging, Fluorescence, Transfection, Plasmid Preparation, Immunoprecipitation

Journal: bioRxiv

Article Title: The Hedgehog receptors PTCH1 and PTCH2 exist as active homomeric and heteromeric complexes

doi: 10.1101/2023.08.08.549832

Figure Lengend Snippet: A. Alignment of NPC1 (PDB ID: 5U73) shown in orange, against the secondary sequence of PTCH1, threaded through NPC1 (PDB ID: 5U73) shown in blue. Residues selected for mutational investigation are shown with side chains in magenta. Left: bottom-up view of protein alignments, with the hydrophobic pocket of NPC1 indicated by a red circle. Right: restricted side view, depicting residues selected for mutational investigation. B. Western blot confirming comparable expression of wild type PTCH1 and the following mutants: P490A, AAT/LIN, L504A, F1109A in HEK 293 cells. C. Relative Gli-luciferase activity (Firefly/Renilla) in Ptch1 −/− MEFs, co-transfected with empty vector (pcDNA3.1), PTCH1 or the indicated single or triple mutants in the absence (black) or the presence of Shh (grey). ns, not significant; *** P<0.001 (n=4). D. Competition-based FRAP of homomeric PTCH1-eGFP/PTCH1-mCherry interaction with 4X mass excess empty plasmid (pcDNA3.1), WT PTCH1 or the indicated mutants. E. Scatter graph of post-bleach GFP emission intensity, corresponding to image 4 in . ****P<0.0001, n=28-43. F. Relative Gli-luciferase activity in Ptc1 −/− MEFs, co-transfected with empty vector (pcDNA3.1), wild type PTCH1 or PTCH1-VLW mutant (n=3 biological repeats). ns, not significant; ** P<0.001. G. HEK 293 cells were transiently transfected with PTCH1-eGFP and PTCH1-mCherry (green trace), PTCH1-eGFP and PTCH1-VLW -mCherry (yellow trace) or PTCH1-VLW-eGFP and PTCH1-VLW-mCherry (purple trace) were subjected to the FRAP-based interaction assay. Change in eGFP fluorescence intensity (expressed as % of initial fluorescence intensity) was measured across 8 acquisitions, with mCherry photo-bleached after the third image in the series. Statistical significance of indicated image number compared to image 3: ***P<0.001; **** P<0.0001 (n=30-41). H. Change in GFP fluorescence intensity with the indicated FRET-pairs photobleached or not (related to ). ***P<0.001; ****P<0.0001 (n=30-41).

Article Snippet: HEK 293 human embryonic kidney epithelial cells and NIH 3T3 murine embryonic fibroblast cells were obtained from American Type Culture Collection (ATCC).

Techniques: Sequencing, Western Blot, Expressing, Luciferase, Activity Assay, Transfection, Plasmid Preparation, Mutagenesis, Fluorescence

Journal: bioRxiv

Article Title: The Hedgehog receptors PTCH1 and PTCH2 exist as active homomeric and heteromeric complexes

doi: 10.1101/2023.08.08.549832

Figure Lengend Snippet: A. HEK 293 cells transiently expressing PTCH2-eGFP and PTCH2-mCherry, were subjected to confocal imaging with the FRAP interaction assay. Change in eGFP fluorescence intensity (expressed as % of initial fluorescence intensity) was measured across 8 acquisitions, with mCherry photo-bleached (blue trace) or not bleached (grey trace) after the third image in the series. Statistical significance of indicated image number compared to image 3 bleached: ** P<0.005 (n=22). B. Change in GFP fluorescence intensity between images 3 and 4 in HEK 293 cells in which mCherry was bleached or not (from ) **P<0.005; ****P<0.0001 (n=22). C. Representative co-immunoprecipitation of FLAG-tagged PTCH2 with HA-tagged PTCH2 after co-expression in HEK 293 cells. WCL: whole cell lysate. D. Side and top view of the 3D structure of PTCH1 (PDB ID: 6MG8) shown in blue, with the threaded PTCH2 (UniProtKB: Q9Y6C5) sequence in turquoise. The putative sterol channel is indicated by a black ring, with zoom views of the hydrophobic tunnel residues in PTCH1 (top left) and predicted equivalent residues in PTCH2 (top right), in magenta. Cholesterol-like densities are displayed in yellow. The top zoom windows display original residues and their side chains, whilst the windows beneath display the predicted mutational alterations to side chains. Structural analysis and alterations were performed in Chimera . E. Relative Gli-luciferase activity in Ptch1 −/− MEFs co-transfected with empty vector (pcDNA3.1), wild type PTCH2 or PTCH2-LLW mutant (n=3 biological repeats). ns, not significant; *** P<0.005. F. HEK 293 cells were transiently transfected with PTCH2-eGFP and PTCH2-mCherry (blue trace) or PTCH2-eGFP and PTCH2-LLW-mCherry (dark blue trace) were subjected to the FRAP-based interaction assay. Change in eGFP fluorescence intensity (expressed as % of initial fluorescence intensity) was measured across 8 acquisitions, with mCherry photo-bleached after the third image in the series. Grey traces show change in GFP fluorescence in unbleached adjacent ROIs. Statistical significance of indicated image number compared to bleached image 3: *P<0.05; **P<0.005; *** P<0.0001 (n=24-32). G. Change in GFP fluorescence intensity in the indicated FRET pairs photobleached or not (from ), * P<0.05; ** P<0.005.

Article Snippet: HEK 293 human embryonic kidney epithelial cells and NIH 3T3 murine embryonic fibroblast cells were obtained from American Type Culture Collection (ATCC).

Techniques: Expressing, Imaging, Fluorescence, Immunoprecipitation, Sequencing, Luciferase, Activity Assay, Transfection, Plasmid Preparation, Mutagenesis

Journal: bioRxiv

Article Title: The Hedgehog receptors PTCH1 and PTCH2 exist as active homomeric and heteromeric complexes

doi: 10.1101/2023.08.08.549832

Figure Lengend Snippet: A. Two-way co-immunoprecipitation of myc-PTCH1 and FLAG-PTCH2 after co-expression in HEK 293 cells. IP: immunoprecipitation; WCL: whole cell lysate. B. Schematic representation of the FRAP assay to detect interaction between PTCH1 and PTCH2, using either of the proteins as donors or acceptors, created with BioRender.com. C. Change in GFP fluorescence intensity in HEK 293 cells co-expression PTCH2-eGFP and PTCH1-mCherry upon photobleaching of mCherry after the third image (blue trace) or in control unbleached regions (grey trace). * P<0.05; ** P<0.01 (n=15-16). D. Change in GFP fluorescence intensity between image 3 and 4 in regions subjected to photobleaching (blue symbols) or not (grey symbols) (from ), * P<0.05; ** P<0.001 (n=15-16). E. Change in GFP fluorescence intensity in HEK 293 cells co-expressing PTCH1-eGFP and PTCH2-mCherry upon photobleaching of mCherry after the third image (green trace) or in control unbleached regions (grey trace). ** P<0.005; *** P<0.001 (n=15-17). F. Change in GFP fluorescence intensity between image 3 and 4 in regions subjected to photobleaching (green symbols) or not (grey symbols) (from ), ** P<0.005; *** P<0.001 (n=15-17). G. Competition-based FRAP of homomeric PTCH1-eGFP/PTCH1-mCherry interaction with 4X mass excess empty plasmid (pcDNA3.1; green trace) or PTCH2-FLAG (blue trace). * P<0.05; ** P<0.005; ***P<0.0005 (n=14-20). H. Change in GFP fluorescence intensity in HEK 293 cells co-expressing PTCH1-eGFP and PTCH1-mCherry upon photobleaching of mCherry after the third image with a 4X excess of empty vector (pcDNA3.1, green symbols) or a 4X excess of PTCH2-FLAG (blue symbols). * P<0.05; *** P<0.001 (n=15-17).

Article Snippet: HEK 293 human embryonic kidney epithelial cells and NIH 3T3 murine embryonic fibroblast cells were obtained from American Type Culture Collection (ATCC).

Techniques: Immunoprecipitation, Expressing, FRAP Assay, Fluorescence, Plasmid Preparation

Journal: bioRxiv

Article Title: The Hedgehog receptors PTCH1 and PTCH2 exist as active homomeric and heteromeric complexes

doi: 10.1101/2023.08.08.549832

Figure Lengend Snippet: PTCH2-eGFP was transfected into Ptch1 −/− MEFs (top) or HEK 293 cells. After 24 h serum starvation to induce ciliogenesis, cells were fixed and stained with Arl13b (ciliary marker) and GFP, and then mounted with DAPI to counterstain nuclei. Scale bar = 10 μm.

Article Snippet: HEK 293 human embryonic kidney epithelial cells and NIH 3T3 murine embryonic fibroblast cells were obtained from American Type Culture Collection (ATCC).

Techniques: Transfection, Staining, Marker

Journal: bioRxiv

Article Title: The Hedgehog receptors PTCH1 and PTCH2 exist as active homomeric and heteromeric complexes

doi: 10.1101/2023.08.08.549832

Figure Lengend Snippet: A . Free cholesterol staining with filipin in HEK 293 cells transfected with empty vector (pcDNA) or with GFP-tagged constructs of wild type PTCH1, PTCH1 (VLW), wild type PTCH2 or PTCH2 (LLW). Numbers indicate filipin intensity compared to pcDNA control. B . Gli-luciferase activity of the same PTCH variants alone and in combination transfected in the indicated percent amounts into Ptch1 −/− MEFs. *** P<0.0005; ****P<0.0001 (n=3-5 independent experiments). C. Comparison of the predicted position of PTCH2-D469 to that of PTCH1-D499. The Aspartic acid in question and two known interaction partners are shown with side chains in magenta. PTCH1 (PDB ID: 6MG8) is shown in blue. PTCH2, shown in turquoise, represents the secondary structure, threaded into the template structure of PTCH1. The protein structures are depicted from a bottom view. D. Gli-luciferase activity of PTCH2, PTCH2 D469A, PTCH1 and PTCH1 D513Y in Ptch1 −/− MEFs. Ns, not significant; * P<0.05; *** P<0.0005 (n=3 independent experiments). E. Gli-luciferase activity of the indicated PTCH1 and PTCH2 variants transfected alone and in combination in the indicated percent amounts into Ptch1 −/− MEFs. *** P=0.0001; ****P<0.0001 (n=4 independent experiments).

Article Snippet: HEK 293 human embryonic kidney epithelial cells and NIH 3T3 murine embryonic fibroblast cells were obtained from American Type Culture Collection (ATCC).

Techniques: Staining, Transfection, Plasmid Preparation, Construct, Luciferase, Activity Assay

Journal: Viruses

Article Title: The Insertion of an Evolutionary Lost Four-Amino-Acid Cytoplasmic Tail Peptide into a Syncytin-1 Vaccine Increases T- and B-Cell Responses in Mice

doi: 10.3390/v15081686

Figure Lengend Snippet: Characterisation of non-fusogenic mutants of HERV-W Env. ( A ) Schematic overview of plasmids encoding an assembled HERV-W Gag (W-Gag) sequence and three different versions of the HERV-W envelope (W-Env), Syncytin-1, sequences: wildtype Env (green, HERV-W WT ), Env with LQMV insertion (purple, HERV-W LQMV ), and Env with a C>A amino acid substitution (blue, HERV-W C>A ). The Gag and Env antigens are separated by a self-cleavable P2A peptide sequence, and the expression of the antigens is controlled by a CMV promoter (CMV p.). The illustration also depicts the location of the R-peptide, the CX 6 CC and CXXC disulfide motifs, and the different domains of the Env: the surface subunit (SU) and the transmembrane subunit (TU), where the TU contains the ectodomain (Ecto), the transmembrane domain (TM), and the cytoplasmic tail (CT). The scissor illustrates the cleavage site between SU and TU. ( B ) Transmission electron microscopy (TEM) pictures of human T24 cells transfected with plasmids encoding GFP, HERV-W WT , HERV-W LQMV , or HERV-W C>A . The top row shows an overview of several cells, and the bottom row shows individual cells at a higher magnification. ( C ) ( right ) Graph showing the geometric mean fluorescent intensity (MFI) of the HERV-W Env surface expression of two technical repeats of transfected HEK293 cells, as indicated by bullets, and ( left ) histograms showing the distribution of one representative sample of each of the HERV-W plasmids and a negative control without transfection (Neg. ctrl). ( D ) The same as ( C ) but depicting results for T24 cells. ( E ) The MFI of the HERV-W Env surface expression of A549 cells 24 h after transduction with the hAd19a/64 vaccines encoding either HERV-W WT , HERV-W LQMV , or the empty expression cassette (Neg. ctrl vaccine). Bullets illustrate the three technical replicates per condition. ( F ) The Western blot shows HERV-W Gag expression in A549 cells 24 h after transduction with either the HERV-W WT or HERV-W LQMV vaccine. The right graph shows the difference in the expression of the HERV-W Gag in the Western blot between the two vaccines, relative to the expression of the housekeeping protein GAPDH.

Article Snippet: The human T24 urinary bladder carcinoma cell line (HTB-4; ATCC, Manassas, VA, USA) and the human HEK293 epithelial kidney cell line (CRL-1573; ATCC, Manassas, VA, USA) were cultured in DMEM GlutaMAX.

Techniques: Sequencing, Expressing, Transmission Assay, Electron Microscopy, Transfection, Negative Control, Transduction, Western Blot

Journal: Journal of Biological Engineering

Article Title: Development of a universal, oriented antibody immobilization method to functionalize vascular prostheses for enhanced endothelialization for potential clinical application

doi: 10.1186/s13036-023-00356-6

Figure Lengend Snippet: Cell binding testing using mixed pre-stained KG1a and HEK293 cells. Calcein-AM stained KG1a cells (green) and CMTMR stained HEK293 cells (yellow) are shown in panels a and b , respectively. CD34 antibody-coated substrates were incubated with the mixed pre-stained KG1a and HEK293 cells, and as shown in panel c , numerous KG1a cells bound the substrate while HEK293 cells barely bound (panel d ). Penal e shows the merged image

Article Snippet: CD34 + KG1a human leukemic progenitor cells, HEK293 human kidney epithelial cells, human umbilical vein endothelial cells (HUVECs), Iscove’s Modified Dulbecco's Medium (IMDM), Eagle's Minimum Essential Medium (EMEM), Endothelial Basal Medium (EBM) and fetal bovine serum (FBS) were purchased from ATCC (Gaithersburg, MD, USA).

Techniques: Binding Assay, Staining, Incubation

Journal: Journal of Biological Engineering

Article Title: Development of a universal, oriented antibody immobilization method to functionalize vascular prostheses for enhanced endothelialization for potential clinical application

doi: 10.1186/s13036-023-00356-6

Figure Lengend Snippet: Cell binding analysis. CD34 antibody coated materials were incubated with KG1a or HEK293 cells, and the bound cells were demonstrated by nuclear staining with fluorescence dye Sytox Green. As shown in this figure, all CD34 antibody-coated materials bound CD34 + KG1a cells but not CD34- HEK293 cells as indicated. a coated CC disc; b coated CC stent; c coated ePTFE graft

Article Snippet: CD34 + KG1a human leukemic progenitor cells, HEK293 human kidney epithelial cells, human umbilical vein endothelial cells (HUVECs), Iscove’s Modified Dulbecco's Medium (IMDM), Eagle's Minimum Essential Medium (EMEM), Endothelial Basal Medium (EBM) and fetal bovine serum (FBS) were purchased from ATCC (Gaithersburg, MD, USA).

Techniques: Binding Assay, Incubation, Staining, Fluorescence

Journal: Drug Design, Development and Therapy

Article Title: 10 H -3,6-Diazaphenothiazine induces G 2 /M phase cell cycle arrest and caspase-dependent apoptosis and inhibits cell invasion of A2780 ovarian carcinoma cells through the regulation of NF-κB and (BIRC6-XIAP) complexes

doi: 10.2147/DDDT.S144415

Figure Lengend Snippet: Cell cytotoxicity activity of PTZ toward representative normal cell lines. Notes: PTZ expressed lesser cytotoxicity toward ( A ) HEK293 human embryonic kidney epithelial cells and ( B ) H9C2 rat embryonic heart myoblast cells by employing the same protocol as A2780 ovarian cancer cells, thus indicating PTZ is more potent and selective toward cancer cells. The results are expressed as mean percentage of living cells over untreated control, and the error bars indicate standard deviation. ***Each treated concentration is significantly different toward untreated control (0 µM) at p <0.05. Abbreviation: PTZ, 10 H -3,6-diazaphenothiazine.

Article Snippet: For normal cells, HEK293 human embryonic kidney epithelial cells and H9C2 rat embryonic heart myoblast cells were purchased from ATCC (catalog no CRL-1573 and CRL-1446) and cultured in DMEM + 10% FBS + 2% L-glutamine (Sigma-Aldrich Co.).

Techniques: Activity Assay, Standard Deviation, Concentration Assay

Journal: Oncology Research

Article Title: Long Noncoding RNA CAT104 Promotes Cell Viability, Migration, and Invasion in Gastric Carcinoma Cells Through Activation of MicroRNA-381-Inhibiting Zinc Finger E-box-Binding Homeobox 1 (ZEB1) Expression

doi: 10.3727/096504017X15144748428127

Figure Lengend Snippet: Relative expression of CAT104 was higher in several human gastric carcinoma cell lines. Quantitative real-time reverse transcriptase polymerase chain reaction (qRT-PCR) was performed to detect the expression of CAT104 in human gastric carcinoma cell lines NCI-N87, SGC7901, BGC823, BGC803, and AGS; human normal gastric cell line GES-1; human kidney epithelial cell line HEK293; human lung fibroblast CCL-153; and human endothelial cell line EC-304. * p < 0.05, ** p < 0.01.

Article Snippet: Human GC cell lines NCI-N87, SGC7901, BGC823, BGC803, and AGS; human normal gastric epithelium cell line GES-1; human kidney epithelial cell line HEK293; human lung fibroblast CCL-153; and human endothelial cell line EC-304 were all purchased from the American Type Culture Collection (ATCC; Manassas, VA, USA).

Techniques: Expressing, Polymerase Chain Reaction, Quantitative RT-PCR

Journal: BioMed Research International

Article Title: Acetylated Hyaluronic Acid: Enhanced Bioavailability and Biological Studies

doi: 10.1155/2014/921549

Figure Lengend Snippet: The IC 50 , expressed as μ mol/L, value is the concentration of compound that affords a 50% reduction in cell growth (after a 24 h incubation). J774.A1 = murine monocyte/macrophage cell lines. HEK-293 = human epithelial kidney cell lines. WEHI-164 = murine fibrosarcoma cell lines. 6-MP = 6-mercaptopurine.

Article Snippet: The murine monocyte/macrophage cell line (J774A.1), murine fibrosarcoma cells (WEHI-164), and human epithelial kidney cells (HEK-293) were obtained from American Tissue Culture Collection (ATCC).

Techniques: Concentration Assay, Incubation