Journal: Communications Biology

Article Title: Altered Signaling and Desensitization Responses in PTH1R Mutants Associated with Eiken Syndrome

doi: 10.1038/s42003-023-04966-0

Figure Lengend Snippet: a Map of the PTH1R with sites of three studied Eiken mutations (E35K, Y134S and R485X) shaded red; the site of the Jansen disease mutation (H223R) shaded orange, and sites of C-tail serine phosphorylation shaded blue. b 3-D view of the PTH1R in complex with LA-PTH {cryo-EM structural file PDB.6nBF, ref. } showing the extracellular domain (ECD) and upper portion of the transmembrane domain (TMD) regions of the complex with receptor shaded gray and ligand shaded blue. Sidechain atoms of E35 and Y134 in the PTH1R ECD, and Leu27, Arg19, and Ile5 in the ligand are displayed as spheres with oxygens colored red and nitrogens colored blue. Yellow dashed lines indicate measured distances between side chain oxygen of E35 and the proximal sidechain nitrogen of Arg19 (8.3 Å) and between the side chain oxygen of Y134 and the proximal sidechain carbon of Leu27 (7.9 A˚). c Surface expression levels of the HA-tagged PTH1R variants in transiently transfected Gs22a (HEK293/glosensor) cells measured by immunofluorescence flow cytometry. Mean total cell fluorescence levels are normalized to values in cells transfected with pCDNA3.1. Bar heights indicate means ± SEM of four to six experiments and data points indicate measurements from each separate experiment. P values indicate Student’s t test comparisons to PTH1R-WT. Gating strategies are as described in Supplemental Fig. . d Schematic of a possible mode of PTH1R complexing with PTH(1-34) or PTHrP(1-36) ligand and β-arrestin, which utilizes two key predicted sites of receptor contact involving (1) the N-terminal portion of β-arrestin and phosphorylated (P) serine or threonine residues in the PTH1R C-tail, and (2) the finger loop domain near the center of β-arrestin and the TMD core of the receptor. The inset depicts possible modes of interaction of the receptor with arrestin at the plasma membrane and in endosomes, as suggested by studies on other GPCRs , . The inset graphic was generated using BioRender.com.

Article Snippet: Gs22a cells are derived from HEK293 cells (ATCC CRL-1573) by stable transfection with the luciferase-based glosensor cAMP reporter plasmid pGlosensor-22F (Promega Corp.) .

Techniques: Mutagenesis, Cryo-EM Sample Prep, Expressing, Transfection, Immunofluorescence, Flow Cytometry, Fluorescence, Generated

Journal: Communications Biology

Article Title: Altered Signaling and Desensitization Responses in PTH1R Mutants Associated with Eiken Syndrome

doi: 10.1038/s42003-023-04966-0

Figure Lengend Snippet: a Basal intracellular cAMP generation assessed in Gs22a (HEK293/glosensor) cells transiently transfected with plasmids to express PTH1R-WT, PTH1R-R485X, PTH1R-E35K, PTH1R-Y134S, or PTH1R-H223R, or with pCDNA3.1; shown are changes in glosensor-derived luminescence over time after addition of luciferin ( t = 0). Signals are normalized to the peak signal observed with PTH1R-WT (100%). Peak signals attained for PTH1R-R485X, PTH1R-E35K, PTH1R-Y134S, and PTH1R-H223R, as per-cents of the PTH1R-WT peak, were 719 ± 72 ( P ≤ 0.0001), 163 ± 16 ( P = 0.0023), 111 ± 12 ( P = 0.39) and 756 ± 125 ( P ≤ 0.00036), respectively. b Basal cAMP signaling in Gs22A cells expressing PTH1R - PD (phosphorylation deficient), in which serines at position 489, 491, 492, 493, 494, 496, and 504 in the C-tail are replaced by alanine, and PTHR-E546X, which is truncated at position 546, normalized to PTH1R-WT (100%). c Basal cAMP signaling in Gs22A cells co-transfected with plasmids encoding a PTH1R (WT or mutant) and either pCDNA3.1 or β-arrestin2 YFP ; luminescence signals as counts per second (cps) are plotted vs. time after addition of luciferin (t = 0). The area-under-the-curve (AUC) of the responses for co-transfection with pCDNA3.1 vs. with β-arrestin2 YFP , normalized to the response for PTH1R-WT/pCDNA3.1, for PTH1R-1WT were 100 ± 0 vs. 84 ± 23, P = 0.5; for PTH1R-R485X were 602 ± 36 vs.150 ± 19, P = 0.0004, and for PTH1R-H223R were 466 ± 208 vs. 381 ± 170, P = 0.8 ( P values determined by Student’s t test). Data are means (±SEM) of six ( a ) or three independent experiments with three or more wells in each.

Article Snippet: Gs22a cells are derived from HEK293 cells (ATCC CRL-1573) by stable transfection with the luciferase-based glosensor cAMP reporter plasmid pGlosensor-22F (Promega Corp.) .

Techniques: Transfection, Derivative Assay, Expressing, Mutagenesis, Cotransfection

Journal: Communications Biology

Article Title: Altered Signaling and Desensitization Responses in PTH1R Mutants Associated with Eiken Syndrome

doi: 10.1038/s42003-023-04966-0

Figure Lengend Snippet: cAMP Signaling responses to PTH and PTHrP ligands were assessed in Gs22a (HEK293/glosensor) cells transiently transfected with PTH1R-WT or a PTH1R mutant. a Dose-responses for PTH(1-34) and PTHrP(1-36). For each ligand concentration on each receptor, the peak luminescence signal, which typically occurred ~15 min after ligand addition, was normalized to the maximum peak signal observed for that ligand on PTH1R-WT (100%) and plotted vs. ligand concentration. b Time courses of cAMP signaling following addition of PTH(1-34) or PTHrP(1-36) at concentrations of 3 nM. The luminescence signals are normalized to the peak signal observed for each ligand on PTH1R-WT (100%); the signals observed for PTHrP(1-36) on PTH1R-E35K at 12–16 min were higher than those on PTH1R-WT (* p < 0.05). c Time courses of cAMP signaling following washout to remove unbound ligand of the cells treated in b . The cAMP-dependent luminescence signals observed for each ligand after washout are normalized to the peak signal observed after washout on PTH1R-WT (100%). d Cells were pretreated for 30 min with Ile 5 -PTHrP(1-36) or PTHrP(1-36), each at a concentration of 10 nM, and then after washout, cAMP-dependent luminescence signals were recorded and normalized to the peak signal observed for each ligand after washout on PTH1R-WT (100%). Data are means (±SEM) of five ( a ), four ( b , c , e ) or three ( d ) separate experiments.

Article Snippet: Gs22a cells are derived from HEK293 cells (ATCC CRL-1573) by stable transfection with the luciferase-based glosensor cAMP reporter plasmid pGlosensor-22F (Promega Corp.) .

Techniques: Transfection, Mutagenesis, Concentration Assay

Journal: Communications Biology

Article Title: Altered Signaling and Desensitization Responses in PTH1R Mutants Associated with Eiken Syndrome

doi: 10.1038/s42003-023-04966-0

Figure Lengend Snippet: a GBR-24 (HEK293/β - arrestin2 YFP stable) cells transiently transfected to express the PTH1R-WT or a PTH1R mutant were treated with PTH(1-34) TMR (10 nM) for 30 min, then rinsed, fixed and imaged by fluorescence microscopy. The insets show 3X-expanded views of selected cell regions to highlight the co-localization of PTH(1-34) TMR (red) with β - arrestin2 YFP (green) into distinct clusters, interpreted as endosomes, in cells transfected with PTH1R-WT, PTH1R-E35K, PTH1R-Y134S, and PTH1R-H223R, and the absence of such co-localization with β - arrestin2 YFP into clusters with PTH1R-R485X. Cells that exhibit only diffuse green fluorescence for β-arrestin2 yfp and/or no TMR-PTH ligand red fluorescence are interpreted as cells not transfected with a PTH1R plasmid, which serve as internal negative controls , . Images are representative of three independent experiments. Scale bars indicate 10 μm. b ImageJ quantification of β - arrestin2 YFP clusters present in the fluorescent microscopy images; bar heights indicate the means ± SEM of counts from five cells shown by the individual data points; P values determined by Student’s t test are shown for significant differences vs. PTH1R-WT. c BRET analyses of β - arrestin2 recruitment in HEK293 cells transiently co-transfected with plasmids encoding a PTH1R variant, β-arrestin2-rLucII as BRET donor, and either the rGFP-CAAX (plasma-membrane) or rGFP-FYVE (early endosome) as BRET acceptor. BRET signals were measured upon addition of ligand with luciferin analog (prolume purple) and the maximum signal observed ~20 min after addition for each PTH1R variant was normalized to the signal observed in the absence of ligand. β - arrestin2 recruitment to the plasma membrane (CAAX) was not significantly different for each PTH1R mutant vs. PTH1R-WT ( p > 0.05), whereas recruitment to early endosomes (FYVE) was reduced for PTH1R-R485X (Emax as fold-increase from baseline = 1.3 ± 0.1 vs. 6.08 ± 1.2 for PTH1R-WT ( P = 0.02, Student’s t test). Data are means ± SEM from three (FYVE) or five (CAAX) independent experiments.

Article Snippet: Gs22a cells are derived from HEK293 cells (ATCC CRL-1573) by stable transfection with the luciferase-based glosensor cAMP reporter plasmid pGlosensor-22F (Promega Corp.) .

Techniques: Transfection, Mutagenesis, Fluorescence, Microscopy, Plasmid Preparation, Variant Assay

Journal: Communications Biology

Article Title: Altered Signaling and Desensitization Responses in PTH1R Mutants Associated with Eiken Syndrome

doi: 10.1038/s42003-023-04966-0

Figure Lengend Snippet: a GBR-24 (HEK293/ β- arrestin2 YFP stable) cells transiently transfected to express a PTH1R variant were treated with PTHrP(1-36) TMR or Ile 5 -PTHrP(1-36) TMR (30 nM) for 30 min, then rinsed, fixed and imaged by fluorescence microscopy. The insets show 3X-expanded views of selected cell regions to highlight co-localization of PTHrP(1-36) TMR (red) with β‐arrestin2 YFP (green) into distinct clusters (endosomes), with PTH1R-WT but not with PTH1R-E35K, PTH1R-Y134S or PTH1R-R485X. b Quantification of β- arrestin2 YFP -positive clusters in five cells positive for binding PTHrP(1-36) TMR for each PTH1R variant. c Cells treated with Ile 5 -PTHrP(1-36) TMR and imaged as in panel a . d Quantification of β- arrestin2 YFP -positive clusters in five cells positive for binding Ile 5 -PTHrP(1-36) TMR for each PTH1R variant. e and f) BRET analyses of β- arrestin2 recruitment to the plasma membrane (CAAX) and to early endosomes (FYVE) in HEK293 cells expressing PTH1R-E35K and BRET donor and acceptors (as used in Fig. ) upon treatment with PTHrP(1-36), PTH(1-34) or Ile 5 -PTHrP(1-36). Data are representative of three independent experiments (a-d); or means ± SEM of three or four independent experiments ( e , f ). Bar heights in graphs of panels b and d indicate the means ± SEM of counts from the five cells shown by the individual data points. Scale bars in a and c indicate 10 μm.

Article Snippet: Gs22a cells are derived from HEK293 cells (ATCC CRL-1573) by stable transfection with the luciferase-based glosensor cAMP reporter plasmid pGlosensor-22F (Promega Corp.) .

Techniques: Transfection, Variant Assay, Fluorescence, Microscopy, Binding Assay, Expressing

Journal: PLOS ONE

Article Title: Protein production from HEK293 cell line-derived stable pools with high protein quality and quantity to support discovery research

doi: 10.1371/journal.pone.0285971

Figure Lengend Snippet: Analytical SEC summary of proteins 1–11 expressed in different expression systems.

Article Snippet: HEK293S GnTI - adherent cells (cat. no. CRL-3022, ATCC) were adapted to suspension cultures internally and grown in FreeStyle 293 expression media (cat. no. 12338018, Thermo Fisher Scientific).

Techniques: Expressing

Journal: PLOS ONE

Article Title: Protein production from HEK293 cell line-derived stable pools with high protein quality and quantity to support discovery research

doi: 10.1371/journal.pone.0285971

Figure Lengend Snippet: (A) Workflow of HEK293S GnTI - and Expi293F GnTI - stable pool generation via the PB transposon system. (B) Expi293F GnTI - kill curves of un-transfected cells with different puromycin (upper panel) and hygromycin (lower panel) concentrations. The chosen antibiotic concentration for selection was the lowest concentration that killed 100% of un-transfected host cells but not transfected cells (red arrows). (C) Viability (left panel) and VCD (right panel) showed that HEK293S GnTI - and Expi293F GnTI - expressing proteins 5 or 6 recovered in about 14 days after antibiotic selection. Mock and Neg refer to empty vector transfected and un-transfected cells, respectively. (D) Production harvest cell viability (left panel) and VCD (right panel) for HEK293S GnTI - and Expi293F GnTI - stable pools expressing proteins 5–10.

Article Snippet: HEK293S GnTI - adherent cells (cat. no. CRL-3022, ATCC) were adapted to suspension cultures internally and grown in FreeStyle 293 expression media (cat. no. 12338018, Thermo Fisher Scientific).

Techniques: Transfection, Concentration Assay, Selection, Expressing, Plasmid Preparation

Journal: PLOS ONE

Article Title: Protein production from HEK293 cell line-derived stable pools with high protein quality and quantity to support discovery research

doi: 10.1371/journal.pone.0285971

Figure Lengend Snippet: (A) Expression titer and (B-C) purification yield for proteins 5 to 10 expressed in CHO-K1 stable pools (blue bars), HEK293S GnTI - stable pools (red bars) and Expi293F GnTI - stable pools (green bars).

Article Snippet: HEK293S GnTI - adherent cells (cat. no. CRL-3022, ATCC) were adapted to suspension cultures internally and grown in FreeStyle 293 expression media (cat. no. 12338018, Thermo Fisher Scientific).

Techniques: Expressing, Purification

Journal: PLOS ONE

Article Title: Protein production from HEK293 cell line-derived stable pools with high protein quality and quantity to support discovery research

doi: 10.1371/journal.pone.0285971

Figure Lengend Snippet: (A) C-terminally His6-tagged protein 5 expressed from CHO-K1, HEK293, HEK293S GnTI - and Expi293F GnTI - was analyzed by SDS-PAGE under non-reducing and reducing conditions and stained by Coomassie blue. CHO-K1, HEK293S GnTI - and Expi293F GnTI - expressed protein 5 were derived from stable expression pools. Protein 5 expressed from HEK293 was from a commercial source (R&D Systems). For each cell line, 0.5 μg (left) and 1 μg (right) of protein 5 was loaded. (B) As in (A), but with protein 6 with a C-terminal His6 tag expressed from CHO-K1, HEK293S GnTI - and Expi293F GnTI - stable pools. The gel was visualized by stain-free imaging. Protein 5 (C) and protein 6 (D) expressed from CHO-K1 stable pools (blue), HEK293S GnTI - stable pools (red) and Expi293F GnTI - stable pools (green) were analyzed by analytical SEC. Zoom-in analytical SEC traces of protein 6 expressed from different cell lines are also shown in (D). (E) As in (B), but with protein 7 and protein 8 with a C-terminal His6. (F-G) As in (D), but with (F) Protein 7 and (G) Protein 8 and their respective zoom-in analytical SEC traces.

Article Snippet: HEK293S GnTI - adherent cells (cat. no. CRL-3022, ATCC) were adapted to suspension cultures internally and grown in FreeStyle 293 expression media (cat. no. 12338018, Thermo Fisher Scientific).

Techniques: SDS Page, Staining, Derivative Assay, Expressing, Imaging

Journal: PLOS ONE

Article Title: Protein production from HEK293 cell line-derived stable pools with high protein quality and quantity to support discovery research

doi: 10.1371/journal.pone.0285971

Figure Lengend Snippet: (A) The disulfide-linked two-chain protein complexes with a C-terminal His6 tag fused to one chain (protein 9 and protein 10) were expressed from Expi293F GnTI - , HEK293S GnTI - and CHO-K1 and analyzed by SDS-PAGE under non-reducing and reducing conditions. The gel was visualized by stain-free imaging. Clipping was observed for the CHO-K1-produced protein. (B) As in (A) but with proteins 4, 9 and 10 expressed from CHO-K1 stable pools. The gel was visualized by stain-free imaging. * indicates clipped species of protein 4. (C) Protein 9 and (D) protein 10 expressed from CHO-K1 (blue), HEK293S GnTI - (red) and Expi293F GnTI - (green) was analyzed by analytical SEC. Zoom-in traces are also shown.

Article Snippet: HEK293S GnTI - adherent cells (cat. no. CRL-3022, ATCC) were adapted to suspension cultures internally and grown in FreeStyle 293 expression media (cat. no. 12338018, Thermo Fisher Scientific).

Techniques: SDS Page, Staining, Imaging, Produced

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