Journal: Scientific Reports

Article Title: The G protein coupled receptor CXCR4 designed by the QTY code becomes more hydrophilic and retains cell signaling activity

doi: 10.1038/s41598-020-77659-x

Figure Lengend Snippet: Schematic representations of CXCR4 and the proposed CXCR4 QTY29 mutations. ( A ) Computational model of CXCR4 QTY29 . Hydrophobic residues are shown in yellow. The lipid-facing side faces the reader and the dimerization interface is on the other side. ( B ) Side view of CXCR4 (green) superimposed on the computational model of CXCR4 QTY29 (blue). The helices nearly overlap, suggesting that the proposed mutations do not significantly alter the structure of CXCR4. ( C ) Extracellular view of the image in ( B ). The similarity in structures suggests that receptor binding and signaling may not be significantly affected. ( D ) Full-length sequence of the native CXCR4 aligned with CXCR4 QTY29 . The changed residues, which comprise only ~ 8% of the protein, are highlighted in red. A C-terminal GSSG linker and rho1D4 purification tag are underlined. The models in ( A )–( C ) were visualized within the simulation software YASARA .

Article Snippet: The closeness of the fit, described by the statistical value χ 2 (Chi 2 ), was 1.78 for CXCR, and 13.4 for CXCR4 QTY29 , indicating that the model used is slightly more true to reality for CXCR4.

Techniques: Binding Assay, Sequencing, Purification, Software

Journal: Scientific Reports

Article Title: The G protein coupled receptor CXCR4 designed by the QTY code becomes more hydrophilic and retains cell signaling activity

doi: 10.1038/s41598-020-77659-x

Figure Lengend Snippet: Relative insoluble fractions of CXCR4 and CXCR4 QTY29 immediately after expression and 1 week later. ( A ) Insoluble fractions of CXCR4 and CXCR4 QTY29 immediately after expression in 0%, 0.01%, and 0.2% Brij-35 (w/v). All data was normalized to CXCR4 in 0% Brij-35. Error bars show ± SEM. For 0% and 0.01% Brij-35, P < 0.01 (Student’s t-test, n = 12). ( B ) Insoluble fractions of CXCR4 and CXCR4 QTY29 1 week after expression in 0%, 0.01%, and 0.2% Brij-35 (w/v). All data was normalized to CXCR4 in 0% Brij-35. Error bars show ± SEM. For 0% and 0.01% Brij-35, P < 0.01 (Student’s t-test, n = 12).

Article Snippet: The closeness of the fit, described by the statistical value χ 2 (Chi 2 ), was 1.78 for CXCR, and 13.4 for CXCR4 QTY29 , indicating that the model used is slightly more true to reality for CXCR4.

Techniques: Expressing

Journal: Scientific Reports

Article Title: The G protein coupled receptor CXCR4 designed by the QTY code becomes more hydrophilic and retains cell signaling activity

doi: 10.1038/s41598-020-77659-x

Figure Lengend Snippet: Western blot using a ρ1D4 monoclonal antibody, and silver stain of CXCR4 and CXCR4 QTY29 in 0.01% and 0.2% Brij-35. ( A ) Western blot of CXCR4 and CXCR4 QTY29 . Lane 1: CXCR4 in 0.01% Brij-35; Lane 2: CXCR4 QTY29 in 0.01% Brij-35; Lane 3: CXCR4 in 0.2% Brij-35; Lane 4: CXCR4 QTY29 in 0.2% Brij-35. Although dimers are visible in both CXCR4 and CXCR4 QTY29 , a higher percentage of the expressed CXCR4 dimerizes. ( B ) Silver stain of purified CXCR4 and CXCR4 QTY29 . Lane 1: Ladder; Lane 2: CXCR4 in 0.01% Brij-35; Lane 3: CXCR4 QTY29 in 0.01% Brij-35; Lane 4: CXCR4 in 0.2% Brij-35; Lane 5: CXCR4 QTY29 in 0.2% Brij-35.

Article Snippet: The closeness of the fit, described by the statistical value χ 2 (Chi 2 ), was 1.78 for CXCR, and 13.4 for CXCR4 QTY29 , indicating that the model used is slightly more true to reality for CXCR4.

Techniques: Western Blot, Silver Staining, Purification

Journal: Scientific Reports

Article Title: The G protein coupled receptor CXCR4 designed by the QTY code becomes more hydrophilic and retains cell signaling activity

doi: 10.1038/s41598-020-77659-x

Figure Lengend Snippet: Circular dichroism spectra of CXCR4 and CXCR4 QTY29 expressed in 0.01% or 0.2% Brij-35. All spectra have minima at ~ 208 nm and ~ 220 nm, which are characteristic for alpha-helical secondary structures. The spectra for receptors in 0.2% Brij-35 nearly overlap, suggesting nearly identical secondary structures. However, the spectra for the receptors in 0.01% Brij-35 are similar but distinct.

Article Snippet: The closeness of the fit, described by the statistical value χ 2 (Chi 2 ), was 1.78 for CXCR, and 13.4 for CXCR4 QTY29 , indicating that the model used is slightly more true to reality for CXCR4.

Techniques: Circular Dichroism

Journal: Scientific Reports

Article Title: The G protein coupled receptor CXCR4 designed by the QTY code becomes more hydrophilic and retains cell signaling activity

doi: 10.1038/s41598-020-77659-x

Figure Lengend Snippet: Surface Plasmon Resonance data of the interaction between SDF1α and ( A ) CXCR4 or ( B ), CXCR4 QTY29 . Colored lines represent the experimental data, while black lines represent fitted curves using the heterogeneous ligand-binding model in the BiaEvaluation program. Parameters from the fitting are shown in-line. The closeness of the fit, described by the statistical value χ 2 (Chi 2 ), was 1.78 for CXCR, and 13.4 for CXCR4 QTY29 , indicating that the model used is slightly more true to reality for CXCR4. The high-activity fractions of CXCR4 QTY29 and wild-type CXCR4 have similar affinities and binding kinetics. The low-activity fraction of CXCR4 QTY29 has a similar binding kinetic profile as CXCR4, but has a higher association rate constant yielding a lower affinity to the ligand. The active fractions of both receptors have comparable affinities to SDF1α. The K D values measured in this work are ~ 29 nM (CXCR4) and ~ 56 nM (CXCR4 QTY29 ), which are comparable to reported CXCR4 affinities ranging from 5 to 200 nM , .

Article Snippet: The closeness of the fit, described by the statistical value χ 2 (Chi 2 ), was 1.78 for CXCR, and 13.4 for CXCR4 QTY29 , indicating that the model used is slightly more true to reality for CXCR4.

Techniques: SPR Assay, Ligand Binding Assay, Activity Assay, Binding Assay

Journal: Scientific Reports

Article Title: The G protein coupled receptor CXCR4 designed by the QTY code becomes more hydrophilic and retains cell signaling activity

doi: 10.1038/s41598-020-77659-x

Figure Lengend Snippet: Laser-scanning confocal micrographs showing fluorescently-labeled cell surface expression of wild type CXCR4 and CXCR4 QTY29 . Left hand: fluorescence images (Ex. 488 nm/Em. 505 nm, LP filter). Right hand: transmission images. ( A ) Negative control, showing stable HEK293 cells not induced for CXCR4 expression. ( B ) HEK293 cells expressing CXCR4 wildtype. ( C ) HEK293 cells expressing CXCR4 QTY29 . Equal amounts of CXCR4 and CXCR4 QTY29 were transfected into HEK293 cells in parallel and under identical conditions. These images indicate that CXCR4 and CXCR4 QTY29 traffic to the cell membrane, but that the amount of membrane-bound CXCR4 QTY29 is less than that of CXCR4. The size bar is 10 mm.

Article Snippet: The closeness of the fit, described by the statistical value χ 2 (Chi 2 ), was 1.78 for CXCR, and 13.4 for CXCR4 QTY29 , indicating that the model used is slightly more true to reality for CXCR4.

Techniques: Labeling, Expressing, Fluorescence, Transmission Assay, Negative Control, Transfection, Membrane

Journal: Scientific Reports

Article Title: The G protein coupled receptor CXCR4 designed by the QTY code becomes more hydrophilic and retains cell signaling activity

doi: 10.1038/s41598-020-77659-x

Figure Lengend Snippet: Intracellular calcium signaling in CXCR4- or CXCR4 QTY29 -transfected HEK293 cells in response to the ligand SDF-1α. CXCR4 or CXCR4 QTY29 were co-transfected with the G-protein G aq into HEK293 cells, and the cytosolic Ca ++ concentration in response to SDF1α was monitored with FuraRed. ( A )–( C ) In CXCR4-transfected cells, the intracellular calcium response to application of 25 ng/ml (~ 3.2 nM) SDF1α and subsequent application of ATP (100 mM) as a viability control were recorded. ( D )–( F ) In CXCR4 QTY29 -transfected cells, the intracellular calcium response to application of 100 ng/ml (~ 13 nM) SDF1α and the subsequent application of ATP (100 mM) as a viability control were recorded. The fluorescence traces in ( A ) and ( D ) show the response of a single cell to both reagents. ( B ) and ( E ) show zoomed fields of transfected cells. ( C ) and ( F ) Show the frequency of individual Ca 2+ responses in populations of CXCR4- and CXCR4 QTY29 -transfected HEK293 cells in response to SDF1α. Circles highlight active cells. In both cases, PBS or mock-transfected cells were used as negative controls, and the fluorescence in response to SDF1α was indistinguishable from baseline, while ATP could still elicit a normal response.

Article Snippet: The closeness of the fit, described by the statistical value χ 2 (Chi 2 ), was 1.78 for CXCR, and 13.4 for CXCR4 QTY29 , indicating that the model used is slightly more true to reality for CXCR4.

Techniques: Transfection, Concentration Assay, Control, Fluorescence

Journal: Antioxidants

Article Title: Eukarion-134 Attenuates Endoplasmic Reticulum Stress-Induced Mitochondrial Dysfunction in Human Skeletal Muscle Cells

doi: 10.3390/antiox9080710

Figure Lengend Snippet: ER stress activation in tunicamycin-treated cells with or without EUK-134. ( A ) Relative fold changes in gene expression for ER stress markers. Data represent the mean fold change normalised to 18S housekeeper gene ± SEM of ΔCt values ( n = 3). ( B ) Cropped representative Western blot image and quantification of GRP94 protein levels relative to β-actin (loading control). Data represent the mean ± SEM ( n = 3). ( C ) Representative images of human skeletal muscle myoblast stained for GRP78 (green) and DAPI (blue). Images captured at 20× magnification. Scale bar = 100 μm. Brightness was adjusted equally in each image to enhance visualisation. ( D ) Quantification of total GRP78 fluorescence intensity normalised to nuclei number, relative to control (%). Data represent the mean ± SEM ( n = 26). * p ≤ 0.033, ** p < 0.002, and *** p < 0.001 against vehicle control or tunicamycin alone (#).

Article Snippet: We examined those changes and the impact of ROS generation in human skeletal muscle myoblasts using the ER stress inducer tunicamycin and a synthetic antioxidant, Eukarion (EUK)-134, which has both superoxide dismutase and catalase activity [ ].

Techniques: Activation Assay, Expressing, Western Blot, Staining, Fluorescence

Journal: Antioxidants

Article Title: Eukarion-134 Attenuates Endoplasmic Reticulum Stress-Induced Mitochondrial Dysfunction in Human Skeletal Muscle Cells

doi: 10.3390/antiox9080710

Figure Lengend Snippet: Cellular bioenergetics of tunicamycin-treated cells with or without EUK-134. ( A ) Bioenergetic status presented by basal oxygen consumption rate versus basal extracellular acidification rate, and real-time oxygen consumption rate measured under basal conditions followed by the sequential addition of oligomycin, FCCP, and rotenone/antimycin A mix. ( B – G ) Individual parameters of mitochondrial function; basal respiration, maximal respiration, non-mitochondrial respiration, spare respiratory capacity, ATP production, and proton leak, normalised to protein content. ( H ) Normalised respiratory flux control ratios, respiratory control ratio, phosphorylating respiration, and coupling efficiency (%), derived from the individual mitochondrial parameters. Data represent the mean ± SEM ( n = 5). * p ≤ 0.033, ** p < 0.002, and *** p < 0.001 against vehicle control or tunicamycin alone (#). ( I ) Relative fold change in mRNA expression of UCP3 . ( J ) Relative fold changes in mRNA expression of mitochondrial unfolded protein response markers, HSP60 and HSPA9 . Data represent the mean fold change normalised to 18S housekeeper gene ± SEM of ΔCt values, ( n = 3). * p ≤ 0.033, ** p < 0.002, and *** p < 0.001 against vehicle control or tunicamycin alone (#). OCR, oxygen consumption rate; ECAR, extracellular acidification rate; RCR, respiratory control ratio.

Article Snippet: We examined those changes and the impact of ROS generation in human skeletal muscle myoblasts using the ER stress inducer tunicamycin and a synthetic antioxidant, Eukarion (EUK)-134, which has both superoxide dismutase and catalase activity [ ].

Techniques: Derivative Assay, Expressing

Journal: Antioxidants

Article Title: Eukarion-134 Attenuates Endoplasmic Reticulum Stress-Induced Mitochondrial Dysfunction in Human Skeletal Muscle Cells

doi: 10.3390/antiox9080710

Figure Lengend Snippet: Mitochondrial membrane potential of tunicamycin-treated cells with or without EUK-134. Relative change (%) in ( A ) MitoTracker Red CMXRos fluorescence intensity ( n = 4), ( B ) JC-1 red fluorescence (polymers) intensity ( n = 8), ( C ) JC-1 red to green fluorescence (polymers/monomers) intensity ( n = 8), ( D ) TMRM fluorescence intensity ( n = 4), and ( E ) TMRM per MitoTracker Green (mitochondrial mass/volume) fluorescence intensity ( n = 4), normalised to protein content. Data represent the mean ± SEM, * p ≤ 0.033, ** p < 0.002, and *** p < 0.001 against vehicle control or tunicamycin alone (#).

Article Snippet: We examined those changes and the impact of ROS generation in human skeletal muscle myoblasts using the ER stress inducer tunicamycin and a synthetic antioxidant, Eukarion (EUK)-134, which has both superoxide dismutase and catalase activity [ ].

Techniques: Fluorescence

Journal: Antioxidants

Article Title: Eukarion-134 Attenuates Endoplasmic Reticulum Stress-Induced Mitochondrial Dysfunction in Human Skeletal Muscle Cells

doi: 10.3390/antiox9080710

Figure Lengend Snippet: Mitochondrial biogenesis of tunicamycin-treated cells with or without EUK-134. ( A ) Representative graph of mitochondrial mass/volume showing the relative change (%) in MitoTracker Green fluorescence intensity, normalised to protein content. Data represent the mean ± SEM ( n = 12). * p ≤ 0.033, ** p < 0.002, against vehicle control or tunicamycin alone (#). ( B ) Representative images of mitochondrial staining in human skeletal muscle myoblast using MitoTracker Green acquired under cell culture conditions. Images captured at 40× magnification. Scale bar = 75 μm. Brightness was adjusted equally in each image to enhance visualisation. ( C , D ) Relative fold changes in mRNA expression of Citrate Synthase and TFAM . Data represent the mean fold change normalised to 18S housekeeper gene ± SEM of ΔCt values, ( n = 3). * p ≤ 0.033, ** p < 0.002, and *** p < 0.001 against vehicle control or tunicamycin alone (#).

Article Snippet: We examined those changes and the impact of ROS generation in human skeletal muscle myoblasts using the ER stress inducer tunicamycin and a synthetic antioxidant, Eukarion (EUK)-134, which has both superoxide dismutase and catalase activity [ ].

Techniques: Fluorescence, Staining, Cell Culture, Expressing

Journal: Antioxidants

Article Title: Eukarion-134 Attenuates Endoplasmic Reticulum Stress-Induced Mitochondrial Dysfunction in Human Skeletal Muscle Cells

doi: 10.3390/antiox9080710

Figure Lengend Snippet: Mitochondrial morphological parameters of tunicamycin-treated cells with or without EUK-134. ( A ) Representative images of fixed human skeletal muscle myoblast stained with MitoTracker Red CMXRos. Images captured using 63×/1.4 oil immersion objective. Scale bar = 25 μm. ( B ) Mitochondrial content as % of cytoplasmic area, mitochondrial interconnectivity expressed by the ratio of mitochondrial area/perimeter, and mitochondrial elongation expressed by 1/circularity. ( C ) Mitochondrial fragmentation propensity described by mitochondrial area and perimeter. Data represent the mean ± SEM ( n = 100 cells per group). * p ≤ 0.033, ** p < 0.002, and *** p < 0.001 against vehicle control or tunicamycin alone (#).

Article Snippet: We examined those changes and the impact of ROS generation in human skeletal muscle myoblasts using the ER stress inducer tunicamycin and a synthetic antioxidant, Eukarion (EUK)-134, which has both superoxide dismutase and catalase activity [ ].

Techniques: Staining

Journal: Antioxidants

Article Title: Eukarion-134 Attenuates Endoplasmic Reticulum Stress-Induced Mitochondrial Dysfunction in Human Skeletal Muscle Cells

doi: 10.3390/antiox9080710

Figure Lengend Snippet: Mitochondrial dynamics of tunicamycin-treated cells with or without EUK-134. ( A ) Relative fold changes in mRNA expression of MFN2 fusion-associated gene. Data represent the mean fold change normalised to 18S housekeeper gene ± SEM of ΔCt values ( n = 3). * p ≤ 0.033, ** p < 0.002 against vehicle control or tunicamycin alone (#). ( B , C ) Cropped representative Western blot image and quantification of MFN2 protein levels relative to β-actin (loading control). Data represent the mean ± SEM ( n = 3). ( D , E ) Relative fold changes in mRNA expression of FIS1 and DRP1 fission-associated gene. Data represent the mean fold change normalised to 18S housekeeper gene ± SEM of ΔCt values ( n = 3). * p ≤ 0.033, ** p < 0.002, and *** p < 0.001 against vehicle control or tunicamycin alone (#).

Article Snippet: We examined those changes and the impact of ROS generation in human skeletal muscle myoblasts using the ER stress inducer tunicamycin and a synthetic antioxidant, Eukarion (EUK)-134, which has both superoxide dismutase and catalase activity [ ].

Techniques: Expressing, Western Blot

Journal: Antioxidants

Article Title: Eukarion-134 Attenuates Endoplasmic Reticulum Stress-Induced Mitochondrial Dysfunction in Human Skeletal Muscle Cells

doi: 10.3390/antiox9080710

Figure Lengend Snippet: Markers of ROS generation in tunicamycin-treated cells with or without EUK-134. Relative change (%) in ( A ) total cellular ROS levels (DCFH-DA, n = 8), ( B ) total cellular superoxide levels (DHE, n = 4), ( C ) mitochondrial superoxide levels (MitoSOX Red, n = 20), and ( D ) total thiol content ( n = 6), normalised to protein content. Data represent the mean ± SEM, * p ≤ 0.033, ** p < 0.002, and *** p < 0.001 against vehicle control or tunicamycin alone (#). ( E – G ) Cropped representative Western blot image and quantification of SOD1 and SOD2 protein levels relative to β-actin (loading control). Data represent the mean ± SEM ( n = 3–5).

Article Snippet: We examined those changes and the impact of ROS generation in human skeletal muscle myoblasts using the ER stress inducer tunicamycin and a synthetic antioxidant, Eukarion (EUK)-134, which has both superoxide dismutase and catalase activity [ ].

Techniques: Western Blot