Journal: bioRxiv

Article Title: Structural basis for EROS binding to human phagocyte NADPH oxidase NOX2

doi: 10.1101/2023.09.11.557130

Figure Lengend Snippet: A. Images from confocal fluorescent microscopy showing the expression of EROS (FITC, green fluorescence) and NOX2 (PE, red fluorescence) on the surface of differentiated HL-60 cells (dHL-60). Colocalization of the two membrane proteins (yellow, merged image) was shown in the lower right panel. Scale bar: 5 μm. B. Colocalization of EROS and NOX2 in transiently transfected COS-7 cells. The cells were cotransfected with NOX2-N-Clover (green) and EROS-C-mRubby2 (red). Confocal microscopy images were taken 24 h after transfection. C. Verification of cell surface expression of EROS and NOX2 in dHL-60 by flow cytometry, using the primary antibodies anti-EROS-FITC and anti-NOX2 (7D5) plus PE-labeled goat anti-mouse secondary antibody for 1-h incubation. D. Size-exclusion chromatography of the EROS-NOX2-p22 phox -7G5 Fab complex on Superose 6. The two major peaks were collected and subjected to SDS-PAGE. E. NOX2 was detected at an expected molecular weight of ∼91 kDa, indicating that the EROS-associated NOX2 is in mature form.

Article Snippet: The solved cryo-EM structure of the EROS-NOX2-p22 phox complex does not contain FAD or NADPH.

Techniques: Microscopy, Expressing, Fluorescence, Membrane, Transfection, Confocal Microscopy, Flow Cytometry, Labeling, Incubation, Size-exclusion Chromatography, SDS Page, Molecular Weight

Journal: bioRxiv

Article Title: Structural basis for EROS binding to human phagocyte NADPH oxidase NOX2

doi: 10.1101/2023.09.11.557130

Figure Lengend Snippet: A. The cryo-EM map of the EROS-NOX2-p22 phox -7G5 complex. EROS (blue) and p22 phox (purple) are opposite to each other and both associated with NOX2 (green). There is no direct interaction between EROS and p22 phox . For clarity, the 7G5-Fab dimers are colored differently in pink and gray. The left panel shows a side view of the cryo-EM map, and the middle panel depicts a side view at a 90° rotation to the left panel. The right panel is a bottom view of the complex from an intracellular perspective. The inner heme (orange) is visible in this view. B. Cartoon representation of the EROS structure. There are four helices (H1-H4) and six β strands, with the N terminus buried inside and the C terminal fragment (C165-S187) is disordered (not shown). H1 (I21-Y40) and H2 (W47-Q61) are connected by Loop 1; H3 (L85-F93) is nearly perpendicular to H1 and H2. The C terminal H4 (R147-L161) is tilted relative to plasma membrane and does not form a transmembrane helix. The two longest β-strands are anti-parallel and connected by Loop 2 (V117-G121). C. Topological model of EROS (blue), NOX2 (green) and p22 phox (purple) in plasma membrane. The yellow hexagon labels indicate three glycosylation sites on NOX2. LA-LE corresponds to Loop A to Loop E. DH denotes the dehydrogenase domain of NOX2. PH represents the Pleckstrin Homology domain separated by H1 and H2. L1 and L2 refer to Loop 1 and Loop 2. ECL and ICL stand for extracellular and intracellular loops, respectively.

Article Snippet: The solved cryo-EM structure of the EROS-NOX2-p22 phox complex does not contain FAD or NADPH.

Techniques: Cryo-EM Sample Prep, Membrane

Journal: bioRxiv

Article Title: Structural basis for EROS binding to human phagocyte NADPH oxidase NOX2

doi: 10.1101/2023.09.11.557130

Figure Lengend Snippet: Sequence alignment of EROS and the YCF4 gene product EROS is an ortholog of the plant protein encoded by YCF4 , which is necessary for the expression of proteins belonging to the photosynthetic photosystem I complex. The Homo sapiens CYBC1 (UniProtKB: Q9BQA9) and Arabidopsis thaliana YCF4 (UniProtKB: P56788) sequences were aligned. Conserved residues are highlighted in pink and gray (pink: fully conserved, gray: highly conserved). Secondary structures are depicted as light blue cylinders (α helices), arrows (β sheets), and lines (loops). Dashed lines indicate unmodeled residues. The residues involved in NOX2 interactions are colored in blue.

Article Snippet: The solved cryo-EM structure of the EROS-NOX2-p22 phox complex does not contain FAD or NADPH.

Techniques: Sequencing, Expressing

Journal: bioRxiv

Article Title: Structural basis for EROS binding to human phagocyte NADPH oxidase NOX2

doi: 10.1101/2023.09.11.557130

Figure Lengend Snippet: A. The 3-D reconstruction of the EROS-NOX2-p22 phox complex, with NOX2 in green, EROS in blue and p22 phox in purple. The heme groups are marked in maroon. B. TM2 of NOX2 in the presence of EROS (green) vs. in its absence (gray, PDB ID: 8GZ3), showing a nearly 79° upward rotational shift (red arrow) of a.a. 67-83 of TM2 (dark green) from its position in the resting state (silver). The atom-to-atom distance of the shifted R80 is 18.6Å. TM6 and EROS are removed from this panel for clarity. C. A 45° horizontal rotation of B showing a 48° backward rotation of a.a. 265-292 (dark green) of TM6 when NOX2 is bound to EROS. TM6 in the resting state (PDB ID: 8GZ3) is depicted in gray, and the same fragment in silver. The distance between the shifted Q292 is 41.9Å, along with a movement of the N terminus of NOX2 towards TM2. D. Top view (extracellular view, left) and bottom view (intracellular view, right) of the superimposed NOX2 structures in EROS-bound (green) and resting (gray) states, with emphasis on the dislocated TM2 and TM6 of NOX2. EROS is marked in blue.

Article Snippet: The solved cryo-EM structure of the EROS-NOX2-p22 phox complex does not contain FAD or NADPH.

Techniques:

Journal: bioRxiv

Article Title: Structural basis for EROS binding to human phagocyte NADPH oxidase NOX2

doi: 10.1101/2023.09.11.557130

Figure Lengend Snippet: A. Interactions between H1 and H2 of EROS and TM2 and TM6 of NOX2. Of note, EROS residues R22 and N62 form hydrogen bonds with NOX2 residues F78 and R80, respectively. Additionally, the EROS residue Y51 is involved in hydrogen bonding with NOX2 residue W270. Residues at this interface (EROS: R22, L26, A37, S41, p43, F50, Y51, F57, N62; NOX2: L76, F78, L79, R80, M268, W270, I273, Y280). The residues of EROS and NOX2 are depicted as blue and green sticks, respectively; the hydrogen bonds are represented by dark dash lines. B. Interaction of the inner heme with EROS. Loop 2 protrudes from EROS and forms a hydrogen bond between Y119 and the lower heme (maroon). Two other EROS residues, E115 and Q140, form hydrogen bonds with NOX2 residues C86 and C85 on Loop B, respectively. In addition, there are hydrophobic interactions between R118/Y119 of EROS and W206/H210 of NOX2 in TM5. C. The TM domains are removed to show more clearly pocket-like interactions between NOX2 and three clusters of EROS including four polar interactions (R22, N62, E115, Q140 on EROS). D. The EROS-NOX2 interface in the FAD-binding domain of NOX2. A total of 20 amino acids, 10 each from EROS and NOX2, participate in this interaction and form a tight zipper that prevents FAD access to the DH domain of NOX2. The hydrogen bonds are shown as dark dashed lines. E and F. Cartoon and surface representation of FAD binding to NOX2 (green) in the presence of EROS (blue in E ) and in its absence ( F , NOX2 is shown in gray). G. The EROS-NOX2 interface in the NADPH-binding domain of NOX2. Ten residues, five from EROS (R108, R129, A131, T132, G133) and the other five from NOX2 (G412, P415, G538, E568, F570) are involved in this interaction. H. Schematic representation of the electron transfer path showing the relative positions and distances between FAD and the two hemes in the absence (FAD in orange) and presence (FAD in blue) of EROS. The edge-to-edge distance between the inner heme and FAD in resting NOX2 (6.1 Å) is shorter than that in EROS-NOX2 (26.4 Å). The ferric ions are shown as orange spheres.

Article Snippet: The solved cryo-EM structure of the EROS-NOX2-p22 phox complex does not contain FAD or NADPH.

Techniques: Residue, Binding Assay

Journal: bioRxiv

Article Title: Structural basis for EROS binding to human phagocyte NADPH oxidase NOX2

doi: 10.1101/2023.09.11.557130

Figure Lengend Snippet: A. Schematic representation of NanoLuc complementation assay. The two components, LgBiT and SmBiT, were fused to EROS and NOX2 respectively, as detailed in Methods . These engineered constructs (EROS-N-LgBiT, NOX2-C-SmBiT) were cotransfected into COS-7 cells together with the p47 phox and p67 phox expression plasmids. The changes in luminescence intensity were measured after addition of the substrate coelenterazine H (10 μM) and PMA (200 ng/ml), with a 1 min interval recording at 460 nm. Hypothetical dissociation of EROS from NOX2 is marked by an arrowhead. B. Data from 10 to 60 min after PMA addition are shown and quantified in the right panel. C. Superoxide production in reconstituted COS-7 cells (COS 91/22 ) cotransfected with expression plasmids of p47 phox , p67 phox , with or without an EROS expressing plasmid. The PMA-induced superoxide production was measured in real time for 60 min using isoluminol, and data were quantified and presented in the right panel. Data shown are mean ± SEM based on multiple independent experiments. *, p < 0.05, ***, p <0.001.

Article Snippet: The solved cryo-EM structure of the EROS-NOX2-p22 phox complex does not contain FAD or NADPH.

Techniques: Construct, Expressing, Plasmid Preparation

Journal: bioRxiv

Article Title: Structural basis for EROS binding to human phagocyte NADPH oxidase NOX2

doi: 10.1101/2023.09.11.557130

Figure Lengend Snippet: A. NOX2 in the inactive state. The association of EROS protects nascent NOX2 against proteosomal degradation and, at the same time, prevents FAD and NADPH from binding to the DH domain. B. NOX2 in the primed state. Priming signals induce dissociation of EROS from NOX2, allowing FAD access to NOX2. C. NOX2 in the active state. Docking of the cytosolic NOX activators and Rac-GTP changes the conformation of NOX2, permitting NADPH binding and electron transfer.

Article Snippet: The solved cryo-EM structure of the EROS-NOX2-p22 phox complex does not contain FAD or NADPH.

Techniques: Binding Assay

Journal: bioRxiv

Article Title: Structural basis for EROS binding to human phagocyte NADPH oxidase NOX2

doi: 10.1101/2023.09.11.557130

Figure Lengend Snippet: A. Images from confocal fluorescent microscopy showing the expression of EROS (FITC, green fluorescence) and NOX2 (PE, red fluorescence) on the surface of differentiated HL-60 cells (dHL-60). Colocalization of the two membrane proteins (yellow, merged image) was shown in the lower right panel. Scale bar: 5 μm. B. Colocalization of EROS and NOX2 in transiently transfected COS-7 cells. The cells were cotransfected with NOX2-N-Clover (green) and EROS-C-mRubby2 (red). Confocal microscopy images were taken 24 h after transfection. C. Verification of cell surface expression of EROS and NOX2 in dHL-60 by flow cytometry, using the primary antibodies anti-EROS-FITC and anti-NOX2 (7D5) plus PE-labeled goat anti-mouse secondary antibody for 1-h incubation. D. Size-exclusion chromatography of the EROS-NOX2-p22 phox -7G5 Fab complex on Superose 6. The two major peaks were collected and subjected to SDS-PAGE. E. NOX2 was detected at an expected molecular weight of ∼91 kDa, indicating that the EROS-associated NOX2 is in mature form.

Article Snippet: COS 91/22 was prepared using the original protocol of Dinauer and coworkers [ ] except that the p22 phox cDNA was expressed in pcDNA3.1-neuromycin vector (Thermo Fisher Scientific).

Techniques: Microscopy, Expressing, Fluorescence, Membrane, Transfection, Confocal Microscopy, Flow Cytometry, Labeling, Incubation, Size-exclusion Chromatography, SDS Page, Molecular Weight

Journal: bioRxiv

Article Title: Structural basis for EROS binding to human phagocyte NADPH oxidase NOX2

doi: 10.1101/2023.09.11.557130

Figure Lengend Snippet: A. The cryo-EM map of the EROS-NOX2-p22 phox -7G5 complex. EROS (blue) and p22 phox (purple) are opposite to each other and both associated with NOX2 (green). There is no direct interaction between EROS and p22 phox . For clarity, the 7G5-Fab dimers are colored differently in pink and gray. The left panel shows a side view of the cryo-EM map, and the middle panel depicts a side view at a 90° rotation to the left panel. The right panel is a bottom view of the complex from an intracellular perspective. The inner heme (orange) is visible in this view. B. Cartoon representation of the EROS structure. There are four helices (H1-H4) and six β strands, with the N terminus buried inside and the C terminal fragment (C165-S187) is disordered (not shown). H1 (I21-Y40) and H2 (W47-Q61) are connected by Loop 1; H3 (L85-F93) is nearly perpendicular to H1 and H2. The C terminal H4 (R147-L161) is tilted relative to plasma membrane and does not form a transmembrane helix. The two longest β-strands are anti-parallel and connected by Loop 2 (V117-G121). C. Topological model of EROS (blue), NOX2 (green) and p22 phox (purple) in plasma membrane. The yellow hexagon labels indicate three glycosylation sites on NOX2. LA-LE corresponds to Loop A to Loop E. DH denotes the dehydrogenase domain of NOX2. PH represents the Pleckstrin Homology domain separated by H1 and H2. L1 and L2 refer to Loop 1 and Loop 2. ECL and ICL stand for extracellular and intracellular loops, respectively.

Article Snippet: COS 91/22 was prepared using the original protocol of Dinauer and coworkers [ ] except that the p22 phox cDNA was expressed in pcDNA3.1-neuromycin vector (Thermo Fisher Scientific).

Techniques: Cryo-EM Sample Prep, Membrane

Journal: bioRxiv

Article Title: Structural basis for EROS binding to human phagocyte NADPH oxidase NOX2

doi: 10.1101/2023.09.11.557130

Figure Lengend Snippet: A. The 3-D reconstruction of the EROS-NOX2-p22 phox complex, with NOX2 in green, EROS in blue and p22 phox in purple. The heme groups are marked in maroon. B. TM2 of NOX2 in the presence of EROS (green) vs. in its absence (gray, PDB ID: 8GZ3), showing a nearly 79° upward rotational shift (red arrow) of a.a. 67-83 of TM2 (dark green) from its position in the resting state (silver). The atom-to-atom distance of the shifted R80 is 18.6Å. TM6 and EROS are removed from this panel for clarity. C. A 45° horizontal rotation of B showing a 48° backward rotation of a.a. 265-292 (dark green) of TM6 when NOX2 is bound to EROS. TM6 in the resting state (PDB ID: 8GZ3) is depicted in gray, and the same fragment in silver. The distance between the shifted Q292 is 41.9Å, along with a movement of the N terminus of NOX2 towards TM2. D. Top view (extracellular view, left) and bottom view (intracellular view, right) of the superimposed NOX2 structures in EROS-bound (green) and resting (gray) states, with emphasis on the dislocated TM2 and TM6 of NOX2. EROS is marked in blue.

Article Snippet: COS 91/22 was prepared using the original protocol of Dinauer and coworkers [ ] except that the p22 phox cDNA was expressed in pcDNA3.1-neuromycin vector (Thermo Fisher Scientific).

Techniques:

Journal: bioRxiv

Article Title: Structural basis for EROS binding to human phagocyte NADPH oxidase NOX2

doi: 10.1101/2023.09.11.557130

Figure Lengend Snippet: A. Images from confocal fluorescent microscopy showing the expression of EROS (FITC, green fluorescence) and NOX2 (PE, red fluorescence) on the surface of differentiated HL-60 cells (dHL-60). Colocalization of the two membrane proteins (yellow, merged image) was shown in the lower right panel. Scale bar: 5 μm. B. Colocalization of EROS and NOX2 in transiently transfected COS-7 cells. The cells were cotransfected with NOX2-N-Clover (green) and EROS-C-mRubby2 (red). Confocal microscopy images were taken 24 h after transfection. C. Verification of cell surface expression of EROS and NOX2 in dHL-60 by flow cytometry, using the primary antibodies anti-EROS-FITC and anti-NOX2 (7D5) plus PE-labeled goat anti-mouse secondary antibody for 1-h incubation. D. Size-exclusion chromatography of the EROS-NOX2-p22 phox -7G5 Fab complex on Superose 6. The two major peaks were collected and subjected to SDS-PAGE. E. NOX2 was detected at an expected molecular weight of ∼91 kDa, indicating that the EROS-associated NOX2 is in mature form.

Article Snippet: In this study, we present the first high-resolution structure of EROS bound to NOX2-p22 phox , based on cryo-EM electron density maps.

Techniques: Microscopy, Expressing, Fluorescence, Membrane, Transfection, Confocal Microscopy, Flow Cytometry, Labeling, Incubation, Size-exclusion Chromatography, SDS Page, Molecular Weight

Journal: bioRxiv

Article Title: Structural basis for EROS binding to human phagocyte NADPH oxidase NOX2

doi: 10.1101/2023.09.11.557130

Figure Lengend Snippet: A. The cryo-EM map of the EROS-NOX2-p22 phox -7G5 complex. EROS (blue) and p22 phox (purple) are opposite to each other and both associated with NOX2 (green). There is no direct interaction between EROS and p22 phox . For clarity, the 7G5-Fab dimers are colored differently in pink and gray. The left panel shows a side view of the cryo-EM map, and the middle panel depicts a side view at a 90° rotation to the left panel. The right panel is a bottom view of the complex from an intracellular perspective. The inner heme (orange) is visible in this view. B. Cartoon representation of the EROS structure. There are four helices (H1-H4) and six β strands, with the N terminus buried inside and the C terminal fragment (C165-S187) is disordered (not shown). H1 (I21-Y40) and H2 (W47-Q61) are connected by Loop 1; H3 (L85-F93) is nearly perpendicular to H1 and H2. The C terminal H4 (R147-L161) is tilted relative to plasma membrane and does not form a transmembrane helix. The two longest β-strands are anti-parallel and connected by Loop 2 (V117-G121). C. Topological model of EROS (blue), NOX2 (green) and p22 phox (purple) in plasma membrane. The yellow hexagon labels indicate three glycosylation sites on NOX2. LA-LE corresponds to Loop A to Loop E. DH denotes the dehydrogenase domain of NOX2. PH represents the Pleckstrin Homology domain separated by H1 and H2. L1 and L2 refer to Loop 1 and Loop 2. ECL and ICL stand for extracellular and intracellular loops, respectively.

Article Snippet: In this study, we present the first high-resolution structure of EROS bound to NOX2-p22 phox , based on cryo-EM electron density maps.

Techniques: Cryo-EM Sample Prep, Membrane

Journal: bioRxiv

Article Title: Structural basis for EROS binding to human phagocyte NADPH oxidase NOX2

doi: 10.1101/2023.09.11.557130

Figure Lengend Snippet: Sequence alignment of EROS and the YCF4 gene product EROS is an ortholog of the plant protein encoded by YCF4 , which is necessary for the expression of proteins belonging to the photosynthetic photosystem I complex. The Homo sapiens CYBC1 (UniProtKB: Q9BQA9) and Arabidopsis thaliana YCF4 (UniProtKB: P56788) sequences were aligned. Conserved residues are highlighted in pink and gray (pink: fully conserved, gray: highly conserved). Secondary structures are depicted as light blue cylinders (α helices), arrows (β sheets), and lines (loops). Dashed lines indicate unmodeled residues. The residues involved in NOX2 interactions are colored in blue.

Article Snippet: In this study, we present the first high-resolution structure of EROS bound to NOX2-p22 phox , based on cryo-EM electron density maps.

Techniques: Sequencing, Expressing

Journal: bioRxiv

Article Title: Structural basis for EROS binding to human phagocyte NADPH oxidase NOX2

doi: 10.1101/2023.09.11.557130

Figure Lengend Snippet: A. The 3-D reconstruction of the EROS-NOX2-p22 phox complex, with NOX2 in green, EROS in blue and p22 phox in purple. The heme groups are marked in maroon. B. TM2 of NOX2 in the presence of EROS (green) vs. in its absence (gray, PDB ID: 8GZ3), showing a nearly 79° upward rotational shift (red arrow) of a.a. 67-83 of TM2 (dark green) from its position in the resting state (silver). The atom-to-atom distance of the shifted R80 is 18.6Å. TM6 and EROS are removed from this panel for clarity. C. A 45° horizontal rotation of B showing a 48° backward rotation of a.a. 265-292 (dark green) of TM6 when NOX2 is bound to EROS. TM6 in the resting state (PDB ID: 8GZ3) is depicted in gray, and the same fragment in silver. The distance between the shifted Q292 is 41.9Å, along with a movement of the N terminus of NOX2 towards TM2. D. Top view (extracellular view, left) and bottom view (intracellular view, right) of the superimposed NOX2 structures in EROS-bound (green) and resting (gray) states, with emphasis on the dislocated TM2 and TM6 of NOX2. EROS is marked in blue.

Article Snippet: In this study, we present the first high-resolution structure of EROS bound to NOX2-p22 phox , based on cryo-EM electron density maps.

Techniques:

Journal: bioRxiv

Article Title: Structural basis for EROS binding to human phagocyte NADPH oxidase NOX2

doi: 10.1101/2023.09.11.557130

Figure Lengend Snippet: A. Interactions between H1 and H2 of EROS and TM2 and TM6 of NOX2. Of note, EROS residues R22 and N62 form hydrogen bonds with NOX2 residues F78 and R80, respectively. Additionally, the EROS residue Y51 is involved in hydrogen bonding with NOX2 residue W270. Residues at this interface (EROS: R22, L26, A37, S41, p43, F50, Y51, F57, N62; NOX2: L76, F78, L79, R80, M268, W270, I273, Y280). The residues of EROS and NOX2 are depicted as blue and green sticks, respectively; the hydrogen bonds are represented by dark dash lines. B. Interaction of the inner heme with EROS. Loop 2 protrudes from EROS and forms a hydrogen bond between Y119 and the lower heme (maroon). Two other EROS residues, E115 and Q140, form hydrogen bonds with NOX2 residues C86 and C85 on Loop B, respectively. In addition, there are hydrophobic interactions between R118/Y119 of EROS and W206/H210 of NOX2 in TM5. C. The TM domains are removed to show more clearly pocket-like interactions between NOX2 and three clusters of EROS including four polar interactions (R22, N62, E115, Q140 on EROS). D. The EROS-NOX2 interface in the FAD-binding domain of NOX2. A total of 20 amino acids, 10 each from EROS and NOX2, participate in this interaction and form a tight zipper that prevents FAD access to the DH domain of NOX2. The hydrogen bonds are shown as dark dashed lines. E and F. Cartoon and surface representation of FAD binding to NOX2 (green) in the presence of EROS (blue in E ) and in its absence ( F , NOX2 is shown in gray). G. The EROS-NOX2 interface in the NADPH-binding domain of NOX2. Ten residues, five from EROS (R108, R129, A131, T132, G133) and the other five from NOX2 (G412, P415, G538, E568, F570) are involved in this interaction. H. Schematic representation of the electron transfer path showing the relative positions and distances between FAD and the two hemes in the absence (FAD in orange) and presence (FAD in blue) of EROS. The edge-to-edge distance between the inner heme and FAD in resting NOX2 (6.1 Å) is shorter than that in EROS-NOX2 (26.4 Å). The ferric ions are shown as orange spheres.

Article Snippet: In this study, we present the first high-resolution structure of EROS bound to NOX2-p22 phox , based on cryo-EM electron density maps.

Techniques: Residue, Binding Assay

Journal: bioRxiv

Article Title: Structural basis for EROS binding to human phagocyte NADPH oxidase NOX2

doi: 10.1101/2023.09.11.557130

Figure Lengend Snippet: A. Schematic representation of NanoLuc complementation assay. The two components, LgBiT and SmBiT, were fused to EROS and NOX2 respectively, as detailed in Methods . These engineered constructs (EROS-N-LgBiT, NOX2-C-SmBiT) were cotransfected into COS-7 cells together with the p47 phox and p67 phox expression plasmids. The changes in luminescence intensity were measured after addition of the substrate coelenterazine H (10 μM) and PMA (200 ng/ml), with a 1 min interval recording at 460 nm. Hypothetical dissociation of EROS from NOX2 is marked by an arrowhead. B. Data from 10 to 60 min after PMA addition are shown and quantified in the right panel. C. Superoxide production in reconstituted COS-7 cells (COS 91/22 ) cotransfected with expression plasmids of p47 phox , p67 phox , with or without an EROS expressing plasmid. The PMA-induced superoxide production was measured in real time for 60 min using isoluminol, and data were quantified and presented in the right panel. Data shown are mean ± SEM based on multiple independent experiments. *, p < 0.05, ***, p <0.001.

Article Snippet: In this study, we present the first high-resolution structure of EROS bound to NOX2-p22 phox , based on cryo-EM electron density maps.

Techniques: Construct, Expressing, Plasmid Preparation

Journal: bioRxiv

Article Title: Structural basis for EROS binding to human phagocyte NADPH oxidase NOX2

doi: 10.1101/2023.09.11.557130

Figure Lengend Snippet: A. NOX2 in the inactive state. The association of EROS protects nascent NOX2 against proteosomal degradation and, at the same time, prevents FAD and NADPH from binding to the DH domain. B. NOX2 in the primed state. Priming signals induce dissociation of EROS from NOX2, allowing FAD access to NOX2. C. NOX2 in the active state. Docking of the cytosolic NOX activators and Rac-GTP changes the conformation of NOX2, permitting NADPH binding and electron transfer.

Article Snippet: In this study, we present the first high-resolution structure of EROS bound to NOX2-p22 phox , based on cryo-EM electron density maps.

Techniques: Binding Assay

Journal: bioRxiv

Article Title: Structural basis for EROS binding to human phagocyte NADPH oxidase NOX2

doi: 10.1101/2023.09.11.557130

Figure Lengend Snippet: A. Images from confocal fluorescent microscopy showing the expression of EROS (FITC, green fluorescence) and NOX2 (PE, red fluorescence) on the surface of differentiated HL-60 cells (dHL-60). Colocalization of the two membrane proteins (yellow, merged image) was shown in the lower right panel. Scale bar: 5 μm. B. Colocalization of EROS and NOX2 in transiently transfected COS-7 cells. The cells were cotransfected with NOX2-N-Clover (green) and EROS-C-mRubby2 (red). Confocal microscopy images were taken 24 h after transfection. C. Verification of cell surface expression of EROS and NOX2 in dHL-60 by flow cytometry, using the primary antibodies anti-EROS-FITC and anti-NOX2 (7D5) plus PE-labeled goat anti-mouse secondary antibody for 1-h incubation. D. Size-exclusion chromatography of the EROS-NOX2-p22 phox -7G5 Fab complex on Superose 6. The two major peaks were collected and subjected to SDS-PAGE. E. NOX2 was detected at an expected molecular weight of ∼91 kDa, indicating that the EROS-associated NOX2 is in mature form.

Article Snippet: In our study, mild detergents including LMNG and CHS were used in order to obtain the EROS-NOX2-p22 phox heterotrimeric complex for cryo-EM analysis.

Techniques: Microscopy, Expressing, Fluorescence, Membrane, Transfection, Confocal Microscopy, Flow Cytometry, Labeling, Incubation, Size-exclusion Chromatography, SDS Page, Molecular Weight

Journal: bioRxiv

Article Title: Structural basis for EROS binding to human phagocyte NADPH oxidase NOX2

doi: 10.1101/2023.09.11.557130

Figure Lengend Snippet: A. The cryo-EM map of the EROS-NOX2-p22 phox -7G5 complex. EROS (blue) and p22 phox (purple) are opposite to each other and both associated with NOX2 (green). There is no direct interaction between EROS and p22 phox . For clarity, the 7G5-Fab dimers are colored differently in pink and gray. The left panel shows a side view of the cryo-EM map, and the middle panel depicts a side view at a 90° rotation to the left panel. The right panel is a bottom view of the complex from an intracellular perspective. The inner heme (orange) is visible in this view. B. Cartoon representation of the EROS structure. There are four helices (H1-H4) and six β strands, with the N terminus buried inside and the C terminal fragment (C165-S187) is disordered (not shown). H1 (I21-Y40) and H2 (W47-Q61) are connected by Loop 1; H3 (L85-F93) is nearly perpendicular to H1 and H2. The C terminal H4 (R147-L161) is tilted relative to plasma membrane and does not form a transmembrane helix. The two longest β-strands are anti-parallel and connected by Loop 2 (V117-G121). C. Topological model of EROS (blue), NOX2 (green) and p22 phox (purple) in plasma membrane. The yellow hexagon labels indicate three glycosylation sites on NOX2. LA-LE corresponds to Loop A to Loop E. DH denotes the dehydrogenase domain of NOX2. PH represents the Pleckstrin Homology domain separated by H1 and H2. L1 and L2 refer to Loop 1 and Loop 2. ECL and ICL stand for extracellular and intracellular loops, respectively.

Article Snippet: In our study, mild detergents including LMNG and CHS were used in order to obtain the EROS-NOX2-p22 phox heterotrimeric complex for cryo-EM analysis.

Techniques: Cryo-EM Sample Prep, Membrane

Journal: bioRxiv

Article Title: Structural basis for EROS binding to human phagocyte NADPH oxidase NOX2

doi: 10.1101/2023.09.11.557130

Figure Lengend Snippet: Sequence alignment of EROS and the YCF4 gene product EROS is an ortholog of the plant protein encoded by YCF4 , which is necessary for the expression of proteins belonging to the photosynthetic photosystem I complex. The Homo sapiens CYBC1 (UniProtKB: Q9BQA9) and Arabidopsis thaliana YCF4 (UniProtKB: P56788) sequences were aligned. Conserved residues are highlighted in pink and gray (pink: fully conserved, gray: highly conserved). Secondary structures are depicted as light blue cylinders (α helices), arrows (β sheets), and lines (loops). Dashed lines indicate unmodeled residues. The residues involved in NOX2 interactions are colored in blue.

Article Snippet: In our study, mild detergents including LMNG and CHS were used in order to obtain the EROS-NOX2-p22 phox heterotrimeric complex for cryo-EM analysis.

Techniques: Sequencing, Expressing

Journal: bioRxiv

Article Title: Structural basis for EROS binding to human phagocyte NADPH oxidase NOX2

doi: 10.1101/2023.09.11.557130

Figure Lengend Snippet: A. The 3-D reconstruction of the EROS-NOX2-p22 phox complex, with NOX2 in green, EROS in blue and p22 phox in purple. The heme groups are marked in maroon. B. TM2 of NOX2 in the presence of EROS (green) vs. in its absence (gray, PDB ID: 8GZ3), showing a nearly 79° upward rotational shift (red arrow) of a.a. 67-83 of TM2 (dark green) from its position in the resting state (silver). The atom-to-atom distance of the shifted R80 is 18.6Å. TM6 and EROS are removed from this panel for clarity. C. A 45° horizontal rotation of B showing a 48° backward rotation of a.a. 265-292 (dark green) of TM6 when NOX2 is bound to EROS. TM6 in the resting state (PDB ID: 8GZ3) is depicted in gray, and the same fragment in silver. The distance between the shifted Q292 is 41.9Å, along with a movement of the N terminus of NOX2 towards TM2. D. Top view (extracellular view, left) and bottom view (intracellular view, right) of the superimposed NOX2 structures in EROS-bound (green) and resting (gray) states, with emphasis on the dislocated TM2 and TM6 of NOX2. EROS is marked in blue.

Article Snippet: In our study, mild detergents including LMNG and CHS were used in order to obtain the EROS-NOX2-p22 phox heterotrimeric complex for cryo-EM analysis.

Techniques:

Journal: bioRxiv

Article Title: Structural basis for EROS binding to human phagocyte NADPH oxidase NOX2

doi: 10.1101/2023.09.11.557130

Figure Lengend Snippet: A. Interactions between H1 and H2 of EROS and TM2 and TM6 of NOX2. Of note, EROS residues R22 and N62 form hydrogen bonds with NOX2 residues F78 and R80, respectively. Additionally, the EROS residue Y51 is involved in hydrogen bonding with NOX2 residue W270. Residues at this interface (EROS: R22, L26, A37, S41, p43, F50, Y51, F57, N62; NOX2: L76, F78, L79, R80, M268, W270, I273, Y280). The residues of EROS and NOX2 are depicted as blue and green sticks, respectively; the hydrogen bonds are represented by dark dash lines. B. Interaction of the inner heme with EROS. Loop 2 protrudes from EROS and forms a hydrogen bond between Y119 and the lower heme (maroon). Two other EROS residues, E115 and Q140, form hydrogen bonds with NOX2 residues C86 and C85 on Loop B, respectively. In addition, there are hydrophobic interactions between R118/Y119 of EROS and W206/H210 of NOX2 in TM5. C. The TM domains are removed to show more clearly pocket-like interactions between NOX2 and three clusters of EROS including four polar interactions (R22, N62, E115, Q140 on EROS). D. The EROS-NOX2 interface in the FAD-binding domain of NOX2. A total of 20 amino acids, 10 each from EROS and NOX2, participate in this interaction and form a tight zipper that prevents FAD access to the DH domain of NOX2. The hydrogen bonds are shown as dark dashed lines. E and F. Cartoon and surface representation of FAD binding to NOX2 (green) in the presence of EROS (blue in E ) and in its absence ( F , NOX2 is shown in gray). G. The EROS-NOX2 interface in the NADPH-binding domain of NOX2. Ten residues, five from EROS (R108, R129, A131, T132, G133) and the other five from NOX2 (G412, P415, G538, E568, F570) are involved in this interaction. H. Schematic representation of the electron transfer path showing the relative positions and distances between FAD and the two hemes in the absence (FAD in orange) and presence (FAD in blue) of EROS. The edge-to-edge distance between the inner heme and FAD in resting NOX2 (6.1 Å) is shorter than that in EROS-NOX2 (26.4 Å). The ferric ions are shown as orange spheres.

Article Snippet: In our study, mild detergents including LMNG and CHS were used in order to obtain the EROS-NOX2-p22 phox heterotrimeric complex for cryo-EM analysis.

Techniques: Residue, Binding Assay

Journal: bioRxiv

Article Title: Structural basis for EROS binding to human phagocyte NADPH oxidase NOX2

doi: 10.1101/2023.09.11.557130

Figure Lengend Snippet: A. Schematic representation of NanoLuc complementation assay. The two components, LgBiT and SmBiT, were fused to EROS and NOX2 respectively, as detailed in Methods . These engineered constructs (EROS-N-LgBiT, NOX2-C-SmBiT) were cotransfected into COS-7 cells together with the p47 phox and p67 phox expression plasmids. The changes in luminescence intensity were measured after addition of the substrate coelenterazine H (10 μM) and PMA (200 ng/ml), with a 1 min interval recording at 460 nm. Hypothetical dissociation of EROS from NOX2 is marked by an arrowhead. B. Data from 10 to 60 min after PMA addition are shown and quantified in the right panel. C. Superoxide production in reconstituted COS-7 cells (COS 91/22 ) cotransfected with expression plasmids of p47 phox , p67 phox , with or without an EROS expressing plasmid. The PMA-induced superoxide production was measured in real time for 60 min using isoluminol, and data were quantified and presented in the right panel. Data shown are mean ± SEM based on multiple independent experiments. *, p < 0.05, ***, p <0.001.

Article Snippet: In our study, mild detergents including LMNG and CHS were used in order to obtain the EROS-NOX2-p22 phox heterotrimeric complex for cryo-EM analysis.

Techniques: Construct, Expressing, Plasmid Preparation

Journal: bioRxiv

Article Title: Structural basis for EROS binding to human phagocyte NADPH oxidase NOX2

doi: 10.1101/2023.09.11.557130

Figure Lengend Snippet: A. NOX2 in the inactive state. The association of EROS protects nascent NOX2 against proteosomal degradation and, at the same time, prevents FAD and NADPH from binding to the DH domain. B. NOX2 in the primed state. Priming signals induce dissociation of EROS from NOX2, allowing FAD access to NOX2. C. NOX2 in the active state. Docking of the cytosolic NOX activators and Rac-GTP changes the conformation of NOX2, permitting NADPH binding and electron transfer.

Article Snippet: In our study, mild detergents including LMNG and CHS were used in order to obtain the EROS-NOX2-p22 phox heterotrimeric complex for cryo-EM analysis.

Techniques: Binding Assay

Journal: The EMBO Journal

Article Title: The EGFR phosphatase RPTPγ is a redox‐regulated suppressor of promigratory signaling

doi: 10.15252/embj.2022111806

Figure Lengend Snippet: A Left: Western blot showing endogenous p22 phox (top row) and GAPDH (loading control; bottom row) in cell lysates obtained from different clones of MCF7 cells subjected to CRISPR‐Cas9‐mediated p22 phox ‐KO (lanes 1–9) and MCF7 WT cells (last lane). Middle: Western blot showing Akt and phospho‐Akt (pS473) in lysates of MCF7 WT cells (lane 1) and selected p22 phox ‐KO cell lines (lanes 2–5) treated for 5′ with 160 ng/ml EGF. Right: Same as middle for Erk and phospho‐Erk (pT202 and pY204). B Western blot showing endogenous RPTPγ (top row) and Na + /K + ATPase (loading control; bottom row) in membrane protein extracts of MCF7 WT cells (first lane) and different clones of MCF7 cells subjected to CRISPR‐Cas9‐mediated RPTPγ‐KO (lanes 2–6). C, D (C) Representative western blots showing EGFR (left), Akt (middle) and Erk (right) in the top rows with corresponding phosphorylation (middle row: EGFR: pY1068, Akt: pS473, Erk: pT202 and pY204) in WT (lanes 1–4) and p22 phox ‐KO (lanes 5–8) MCF7 cells, without EGF and upon 5′ stimulation with EGF‐Alexa647 (20, 80 and 160 ng/ml). Bottom row: GAPDH (loading control). (D) Same arrangement as (C) for WT and RPTPγ‐KO MCF7 cells. E Top panel: Representative western blot showing phosphorylated EGFR at tyrosine 1068 (pY1068) in MCF7 WT and HT29 cells, without EGF and upon 5′ stimulation with EGF‐Alexa647 (20, 80 and 160 ng/ml). Bottom graph: Quantification with mean ± SD, N = 3 biological replicates, P : unpaired two‐tailed t ‐test. F Top panel: Representative confocal micrographs of immunostained endogenous EGFR (left image), phosphorylated EGFR at tyrosine 1068 (middle image: pY1068) and ectopically expressed RPTPγ‐mTFP (right image) in HT29 cells in absence of EGF‐stimulus. Scale bar: 10 μm. Bottom panel: Quantification of phosphorylated (pY1068) over total EGFR staining in cells without (blue) and with (yellow) RPTPγ‐mTFP expression. Individual cells with mean ± SD, N = 3, n > 75 cells per condition, P : unpaired two‐tailed t ‐test. G Representative western blots showing EGFR (left), Akt (middle) and Erk (right) in the top rows with corresponding phosphorylation (lower row: EGFR: pY1068, Akt: pS473, Erk: pT202 and pY204) in WT (lanes 2–5) and RPTPγ‐KO (lanes 6–9) MCF7 cells treated with 10 μM of EGFR‐inhibitor gefitinib for 1 h, without EGF and upon 5′ stimulation with EGF‐Alexa647 (20, 80 and 160 ng/ml). Lane 1: WT MCF7 cells treated with 80 ng/ml EGF in the absence of gefitinib. H Representative fluorescence micrographs of EGF‐Alexa647 (green) bound to EGFR in WT MCF7 cells at the corresponding, indicated concentrations applied for 5′. Blue: Hoechst33342, scale bar 10 μm. Insets: Individually contrast‐stretched fluorescence micrographs.

Article Snippet: WT, RPTPγ‐KO, and p22 phox ‐KO MCF7 cells were seeded on Lab‐Tek™ chambered cover glass slides (ThermoFisher), transfected with EGFR‐mCitrine or BFP‐tkRas and incubated in cell culture medium with 0.5% FBS for at least 6 h before the experiment.

Techniques: Western Blot, Clone Assay, CRISPR, Two Tailed Test, Staining, Expressing, Fluorescence

Journal: The EMBO Journal

Article Title: The EGFR phosphatase RPTPγ is a redox‐regulated suppressor of promigratory signaling

doi: 10.15252/embj.2022111806

Figure Lengend Snippet: EGFR (pY1068, left), Akt (pS473, middle), and Erk (pT202 and pY204, right) phosphorylation response in WT (red) compared to p22 phox ‐KO (green) MCF7 cells as function of EGF concentration (ng/ml; nM) upon 5′ stimulation with different doses of EGF‐Alexa647 quantified from Western blot analysis. N = 4 biological replicates with mean ± SD, P: unpaired two‐tailed t ‐test. Same as (A) comparing WT (red) to RPTPγ‐KO (blue) MCF7 cells. N = 3 biological replicates with mean ± SD, P: unpaired two‐tailed t ‐test. Quantitative Western blot analysis as in (A) comparing WT (red) and RPTPγ‐KO (blue) MCF7 cells after EGF stimulus (20, 80, 160 ng/ml from (B), left column: w/o Gefitinib) to the cells from the corresponding cell line treated with 10 μM of the EGFR‐inhibitor Gefitinib for 1 h and the indicated EGF concentration for the last 5′ (ng/ml). N = 3 biological replicates with mean ± SD, P: unpaired two‐tailed t ‐test. Quantification of live cell fluorescence anisotropy microscopy measurements of EGFR‐QG‐mCitrine dimerization level in WT (red) and RPTPγ‐KO (blue) MCF7 cells before and after 160 ng/ml EGF‐Alexa647 stimulus for 15′. mean ± SEM, N = 3 biological replicates, n = 31 cells, P: paired two‐tailed t ‐test, against respective unstimulated cases. Comparison of normalized EGFR-phosphorylation (pY1068/EGFR total ) as a function of EGF concentration ( N = 10, from Figs , and , red) to EGF‐Alexa647 bound to WT MCF7 cells at the corresponding, indicated concentrations normalized to the 160 ng/ml, measured by fluorescence microscopy. N = 5 biological replicates, n = 16–19 fields of view, mean ± SD, P: One‐way ANOVA with Tukey's multiple comparison test. Source data are available online for this figure.

Article Snippet: WT, RPTPγ‐KO, and p22 phox ‐KO MCF7 cells were seeded on Lab‐Tek™ chambered cover glass slides (ThermoFisher), transfected with EGFR‐mCitrine or BFP‐tkRas and incubated in cell culture medium with 0.5% FBS for at least 6 h before the experiment.

Techniques: Concentration Assay, Western Blot, Two Tailed Test, Fluorescence, Microscopy

Journal: The EMBO Journal

Article Title: The EGFR phosphatase RPTPγ is a redox‐regulated suppressor of promigratory signaling

doi: 10.15252/embj.2022111806

Figure Lengend Snippet: Representative fluorescence micrographs of in cell EGF‐Alexa647 (0–320 ng/ml) dose–response imaging of EGFR phosphorylation in EmCit_MCF7 cells. Concentrations of EGF‐Alexa647 were increased at 1.5′ time interval and are shown as cumulative dose in ng/ml and corresponding relative receptor occupancies (α L ), obtained by normalizing the ratiometric fluorescence of EGF‐Alexa647/EGFR‐mCitrine to that at saturating EGF‐Alexa647 dose. First row: EGF‐Alexa647; Second row: EGFR‐mCitrine; Third row: phosphorylated EGFR‐mCitrine fraction (α p ); Scale bar: 10 μm. Left: Peak normalized photon intensity distribution histograms as a function of their time of arrival obtained from time‐correlated single photon counting measurements of EGFR‐mCitrine (with PTB‐mCherry as FRET‐acceptor; Fig ) at different cumulative EGF‐Alexa647 doses (A) (color code in inset). Right: Average fluorescence lifetime of mCitrine (τ avg, ns) obtained by integrating the area under individual normalized decay curves as a function of cumulative EGF‐Alexa647 dose. Left: fraction of EGF‐Alexa647 binding to EGFR‐mCitrine (receptor occupancy α L ) upon each administered dose (cumulative doses 2.5–640 ng/ml), middle: fraction of phosphorylated EGFR‐mCitrine (α p ) derived from FLIM measurments as a function of administered EGF‐Alexa647 dose, right: α p plotted against α L . Colored thin lines: individual cell profiles; Solid red line with shaded bounds: moving median with median absolute deviation. Same as (A) for RPTPγ‐KO EmCit_MCF7 cells. Same as (A) for p22 phox ‐KO EmCit_MCF7 cells. Top row: Left: Representative western blot showing EGFR (top) and corresponding phosphorylation response at Y1068 (bottom) in lysates of MCF7 WT cells as a function of indicated EGF‐Alexa647 stimulus for 5′. Lysate from cells treated for 5′ with 0.33 mM of PTP‐inhibitor pervanadate (PV, last lane) was used as a positive control for EGFR‐phosphorylation. Middle: Same for Akt (top) and phosphorylation at pS473 (bottom). Right: Same for Erk (top) and phosphorylation at pT202 and pY204 (bottom). Bottom row: Quantification of phosphorylated Akt (pS473/Akt total ; left) and (pErk/Erk total ; right) as a function of the receptor occupancy α L (C) corresponding to the applied doses of EGF‐Alexa647. N = 4 biological replicates, mean (red symbols) ± SD and fit to the hill equation (solid black line). Inserts: Hill coefficient (HC) and EC50 of the fitted hill equation (95% confidence interval). Left: RPTPγ‐mTFP/EGFR‐mCitrine fluorescence ratio of individual EmCit_MCF7 RPTPγ‐KO cells with RPTPγ‐mTFP ectopic expression plotted against Hill coefficient (HC) obtained from fitting the hill equation to the corresponding in cell EGF‐dose response (compare Fig , N = 3 biological replicates, n = 23 cells) with colored lines encircling data points of the three clusters; Middle/Right: HC versus RPTPγ‐mTFP/EGFR‐mCitrine range of the three clusters; mean ± SD ( y ‐axis) and full range of values ( x ‐axis), P : unpaired two‐tailed t ‐test. Representative western blot showing RPTPγ (top row) and Na + /K + ATPase as a loading control (bottom row) in membrane protein extract lysates of WT MCF7 and MCF7‐RPTPγ‐KO cells stably expressing RPTPγ‐mCitrine. 17 (left and middle) and 28 (right) times more total protein was loaded for WT cells compared to MCF7‐RPTPγ‐KO cells expressing RPTPγ‐mCitrine to maintain band intensity within the dynamic range of the fluorescence detector of the scanner. Western blot showing endogenous TCPTP expression (top row) and GAPDH (loading control; bottom row) in cell lysates obtained from WT with (lane1) and without (lane2) ectopic TCPTP expression and different clones of MCF7 cells subjected to CRISPR‐Cas9 mediated TCPTP‐KO (3–7 lanes). Same as (A) for TCPTP‐KO EmCit_MCF7 cells. Same as (A) for TCPTP‐KO EmCit_MCF7 cells with TCPTP‐mTFP (fourth row) ectopic expression. Data information: All scale bars: 10 μm.

Article Snippet: WT, RPTPγ‐KO, and p22 phox ‐KO MCF7 cells were seeded on Lab‐Tek™ chambered cover glass slides (ThermoFisher), transfected with EGFR‐mCitrine or BFP‐tkRas and incubated in cell culture medium with 0.5% FBS for at least 6 h before the experiment.

Techniques: Fluorescence, Imaging, Binding Assay, Derivative Assay, Western Blot, Positive Control, Expressing, Two Tailed Test, Stable Transfection, Clone Assay, CRISPR

Journal: The EMBO Journal

Article Title: The EGFR phosphatase RPTPγ is a redox‐regulated suppressor of promigratory signaling

doi: 10.15252/embj.2022111806

Figure Lengend Snippet: Left panel: comparison of normalized EGF‐Alexa647 (160 ng/ml; 5′) fluorescence intensity bound to individual endogenous EGFR expressing MCF10A (yellow), to exogenous EGFR‐mCitrine expressing EmCit_MCF7 cells (black) and WT MCF7 cells (red); Right panel: normalized EGF‐Alexa647 fluorescence plotted against normalized EGFR‐mCitrine fluorescence intensity in WT (red) and EmCit_MCF7 (black, with 2 nd order polynomal fit: gray line) cells. N = 3 biological replicates, n > 75 cells, mean ± SD. Quantitative imaging of EGFR phosphorylation: Right: Binding of PTB‐mCherry (acceptor) to phosphorylated EGFR‐mCitrine (donor) causes FRET between donor and acceptor resulting in a reduced excited state lifetime (τ DA ) of the donor (mCitrine) in the EGFR‐mCitrine/PTB‐mCherry complex. Left: Unphosphorylated EGFR‐mCitrine exhibits a discrete fluorescence lifetime (τ D ) that is distinct from τ DA . The spatially invariant τ DA and τ D are shared global parameters for all pixels that enable the mapping of the local fraction of phosphorylated EGFR‐mCitrine (α p , local parameter) within living cells by global analysis. Representative fluorescence micrographs of in cell EGF‐Alexa647 (0–320 ng/ml) dose–response imaging for EGFR phosphorylation in RPTPγ‐KO EmCit_MCF7 cells expressing RPTPγ‐mTFP. Concentrations of EGF‐Alexa647 were increased at 1.5′ time interval and are shown as cumulative dose in ng/mL and corresponding relative receptor occupancies (α L ), obtained by normalizing the ratiometric fluorescence of EGF‐Alexa647/EGFR‐mCitrine to that at saturating EGF‐Alexa647 dose. First row: EGF‐Alexa647; Second row: EGFR‐mCitrine; Third row: phosphorylated EGFR‐mCitrine fraction (α p ); Fourth row: RPTPγ‐mTFP; Scale bar: 10 μm. Gray: α L upon each administered dose for individual EmCit_MCF7 cells to cumulative doses of EGF‐Alexa647 (2.5–640 ng/ml). N = 3 biological replicates, n = 13 cells. Black: EGF‐Alexa647 bound to WT MCF7 cells at the indicated concentrations normalized to the mean fluorescence intensity at 160 ng/ml EGF‐Alexa647 (Fig ; mean ± SD, N = 5 biological replicates, n = 16–19 fields of view). Top: Relative fraction of PM‐localized fraction of EGFR during the course of in cell dose–response experiments in EmCit_MCF7 cells. N = 3 biological replicates, n = 10 cells. Bottom: EGFR‐mCitrine phosphorylation (α p ) plotted as a function of EGF‐receptor occupancy (α L ) at the PM to incremental EGF‐Alexa647 doses in p22 phox ‐KO (green, N = 3 biological replicates, n = 12 cells), RPTPγ‐KO (blue, N = 3 biological replicates, n = 14 cells), RPTPγ‐KO with RPTPγ‐mTFP ectopic expression (yellow, N = 4 biological replicates, n = 13 cells) and WT (red, N = 3 biological replicates, n = 13 cells) EmCit_MCF7 cells. Solid lines: moving medians from single cell profiles; shaded bounds: median absolute deviations. Left: EGFR‐ (pY1068‐) phosphorylation response in WT MCF7 cells obtained from western blots normalized to maximal phosphorylation obtained by inhibiting all phosphatases by 0.33 mM pervanadate ( N = 6; red symbols with mean ± SD and fit to the hill equation (solid line)) at 0 (plotted as 0.001 to fit the logarithmic x ‐axis), 0.5, 1, 2, 5, 10, 20, 40, and 80 ng/ml plotted against corresponding α L (obtained from in cell dose response experiments in EmCit_MCF7 cells (D)). Correspondig molecular RPTPγ/EGFR‐ratio (see G, H, Fig ) is depicted above the graphs; Inserted into each graph are the values of Hill coefficient (HC) and EC50 of the fitted hill equation (95% confidence interval). 2 nd graph: Same as left graph with α p plotted vs α L both obtained from in cell dose response experiments in EmCit_MCF7 cells. 3 rd –5 th graph: Same as 2 nd graph for EmCit_MCF7 RPTPγ‐KO with RPTPγ‐mTFP ectopic expression clustered by RPTPγ/EGFR‐expression ratio and HC (Fig ). Number of molecules obtained by normalizing background‐subtracted fluorescence intensities of individual transfected cells against the mean background intensity of untransfected cells, yielding relative expressions levels independent on the fluorophore . This value was then set into proportion to the known mean number of EGFR per MCF10A cell to yield absolute molecule count/cell. 1 st column: EGFR‐mCitrine in EmCit_MCF7 cells ( N = 3 biological replicates, n = 102 cells); 2 nd column EGFR‐mCitrine expressed in EmCit_MCF7 RPTPγ‐KO expressing RPTPγ‐mTFP ( N = 3 biological replicates, n = 26 cells); 3 rd column: RPTPγ‐mCitrine in MCF7 cells expressing additionally EGFR‐mCherry ( N = 3 biological replicates, n = 253 cells); 4 th column: RPTPγ‐mTFP expressed in EmCit_MCF7 RPTPγ‐KO ( N = 3 biological replicates, n = 26 cells; individual cells with mean + SD). Color code in 2 nd and 4 th column is attribution to respective cluster (H and Fig ). Number of RPTPγ‐mTFP and EGFR‐mCitrine molecules in EmCit_MCF7 RPTPγ‐KO expressing RPTPγ‐mTFP plotted for the three HC‐RPTPγ/EGFR‐ratio clusters identified in Fig together with RPTPγ over EGFR molecular ratio (top; mean ± SD). EGFR‐mCitrine phosphorylation (α p ) plotted as a function of EGF‐receptor occupancy (α L ) at the PM to incremental EGF‐Alexa647 doses in WT (red, N = 3 biological replicates, n = 13 cells), TCPTP‐KO (blue, N = 3 biological replicates, n = 14 cells) and TCPTP‐KO with TCPTP‐mTFP ectopic expression (yellow, N = 3 biological replicates, n = 13 cells) EmCit_MCF7 cells. Solid lines: moving medians from single cell profiles; shaded bounds: median absolute deviations. Source data are available online for this figure.

Article Snippet: WT, RPTPγ‐KO, and p22 phox ‐KO MCF7 cells were seeded on Lab‐Tek™ chambered cover glass slides (ThermoFisher), transfected with EGFR‐mCitrine or BFP‐tkRas and incubated in cell culture medium with 0.5% FBS for at least 6 h before the experiment.

Techniques: Fluorescence, Expressing, Imaging, Binding Assay, Western Blot, Transfection

Journal: The EMBO Journal

Article Title: The EGFR phosphatase RPTPγ is a redox‐regulated suppressor of promigratory signaling

doi: 10.15252/embj.2022111806

Figure Lengend Snippet: A Reaction schematic of EGFR‐dependent PTP‐oxidation: Phosphorylated EGFR (red circles) activates PI3K, which results in the activation of Rac‐GTPase and the cytosolic components of NOX‐assembly like p40 phox , p47 phox and p67 phox . Recruitment of these components to the PM‐based major NOX‐unit and p22 phox subunit, mediates the transfer of electrons from the cytosolic NADPH to extracellular oxygen (O 2 ) leading to the formation of superoxide anion (O 2 − ) that dismutates to hydrogen peroxide (H 2 O 2 ). Diffusion of H 2 O 2 through the PM causes the cysteine oxidation of the PM‐vicinal PTPs, from thiol (SH) to sulfenic acid (SOH) state. B Schematic of FLIM assay for the quantitative imaging of PTP‐oxidation in live cells: Binding of DyTo (atto590, acceptor) to oxidized cysteines (S‐OH) of PTP‐mCitrine (donor) results in FRET between donor and acceptor reducing the excited state lifetime of the donor (τ DA ). Spatial invariance of τ DA and τ D enable the mapping of the fraction of oxidized PTP‐mCitrine (α ox , local parameter) by global analysis. C–E (C) In cell EGF‐dose response imaging for RPTPγ‐mCitrine oxidation. Left panel: Representative confocal micrographs of RPTPγ‐mCitrine in EmTFP_MCF7 cells (top row) together with its oxidized fraction estimated using DyTo‐FLIM (α ox , bottom row), upon 10′ stimulation with EGF‐Alexa647 (0–160 ng/ml) including 5′ together with 0.5 mM DyTo. Scale bar: 10 μm. Right panel: Quantification depicting the PM‐proximal (orange) and PM‐distal (blue) oxidized fractions as functions of receptor occupancy (α L ) and corresponding EGF‐Alexa647, or H 2 O 2 concentration in EmTFP MCF7 cells expressing RPTPγ‐mCitrine (WT) or RPTPγ C1060S ‐mCitrine (C1060S) as well as WT cells treated with 0.5 mM atto590 instead of DyTo (atto590). Mean of individual cells (symbols) with mean ± SD (black lines), N = 3 biological replicates, n = 13–15 cells per EGF dose. P: unpaired two‐tailed t ‐test, between PM (serpentine peripheral structures) and endosomal (vesicular structures) fractions. (D) Same as in (C), for RPTPγ‐mCitrine oxidation in p22 phox ‐KO cells. N = 3 biological replicates, n = 14–26 cells per EGF dose. (E) Same as in (C), for TCPTP‐mCitrine or TCPTP C216S ‐mCitrine (C216S) oxidation in EmTFP_MCF7 cells. N = 3 biological replicates, n = 18–21 cells per EGF dose.

Article Snippet: WT, RPTPγ‐KO, and p22 phox ‐KO MCF7 cells were seeded on Lab‐Tek™ chambered cover glass slides (ThermoFisher), transfected with EGFR‐mCitrine or BFP‐tkRas and incubated in cell culture medium with 0.5% FBS for at least 6 h before the experiment.

Techniques: Activation Assay, Diffusion-based Assay, Imaging, Binding Assay, Concentration Assay, Expressing, Two Tailed Test

Journal: The EMBO Journal

Article Title: The EGFR phosphatase RPTPγ is a redox‐regulated suppressor of promigratory signaling

doi: 10.15252/embj.2022111806

Figure Lengend Snippet: A Representative confocal micrographs of MCF7 WT cells showing the co‐localization of RPTPγ‐mCitrine (1 st column: green; 3 rd column: blue) and EGFR‐mCherry (2 nd column: green; 3 rd column: yellow) with recycling‐endosome defined by immunostaining against Rab11a (magenta), without (top row) or after 30' EGF‐DyLight405 stimulus (160 ng/ml; bottom row). Scale bar: 10 μm. B Fraction of RPTPγ‐mCitrine (cyan) or EGFR‐mCherry (green) that spatially overlaps with Rab11a (top left, N = 3, n = 23–26 cells per timepoint), PM (bottom left, N = 3 biological replicates, n = 15–17 cells), EEA1‐positive EEs (top right, N = 3 biological replicates, n = 25–28 cells) or Rab7‐positive LEs (bottom right, N = 3 biological replicates, n = 23–27 cells) in MCF7 cells as function of time after 160 ng/ml EGF‐stimulus. Orange symbols/dotted line: same for EGFR‐mCitrine in RPTPγ‐KO cells ( N = 3 biological replicates, n = 17–21 cells). P: unpaired two‐tailed t ‐test; colored P values compare to the respective species before stimulation, black in between species. C Left: Representative confocal micrographs of MCF7 cells depicting the steady state localization of expressed RPTPγ‐mCitrine (cyan), without (top) or with co‐expression of BFP‐Rab11a (yellow, bottom). Top right: Quantification of PM‐localized fraction of RPTPγ‐mCitrine without (WT) and with co‐expression of BFP‐Rab11a. Bottom right: Fraction of RPTPγ‐mCitrine localized to the PM in individual cells as a function of BFP‐Rab11a expression level, measured by mean BFP‐fluorescence intensity. Scale bar: 10 μm. N = 2 biological replicates, n > 40 per condition, mean ± SD; P : unpaired two‐tailed t ‐test. D Fraction of fluorescent paGFP‐RPTPγ at the PM over time after photoactivation of paGFP exclusively in the perinuclear region, in cells with (orange) or without (pink) co‐expression of BFP‐Rab11a. mean ± SD, N = 3 biological replicates, n = 4–7 cells. E Upper panel: Dual‐color widefield images (1 st column), SRRF reconstructions (2 nd column) with magnifications of boxed areas (3 rd column) of Alexa647‐SNAP‐EGFR (green) and RPTPγ‐mCitrine (magenta) of cryo‐arrested MCF7 cells, unstimulated (top row) or stimulated with 100 ng/ml EGF (bottom row) for 15′. Scale bar: 10 μm. Lower panel: corresponding Manders colocalization coefficients for Alexa647‐Snap‐EGFR/RPTPγ‐mCitrine from SRRF reconstructions on intracellular compartments or PM area for unstimulated ( n = 12–18) and 15' EGF‐stimulated ( n = 13–14) cells. mean ± SD, P : unpaired two‐tailed t ‐test. F Left: Representative IP‐western blot showing co‐IP of EGFR (2 nd row) upon RPTPγ‐mCitrine (1 st row: lanes 1–6) or RPTPγ C1060S ‐mCitrine (lane 7) pull‐down by anti‐GFP antibody from lysates of MCF7 cells co‐transfected with EGFR and RPTPγ‐mCitrine or RPTPγ C1060S ‐mCitrine: without stimulus (0 ng/ml), upon 10′ stimulus with EGF‐Alexa647 (5–320 ng/ml, also displayed as corresponding receptor‐occupancy α L , Fig ) or 8 mM of H 2 O 2 . 3 rd and 4 th row: total protein concentrations of RPTPγ‐mCitrine and EGFR in the lysate measured by western blot as input control for the Co‐IP. Right: corresponding ratiometric quantification of co‐immunoprecipitated EGFR over pulled down RPTPγ-mCitrine or RPTPγ C1060S ‐mCitrine protein bands (mean ± SD, N = 4 biological replicates, P : unpaired two‐tailed t ‐test). G Oxidized fraction of RPTPγ‐mCitrine (α ox ) at the PM of live EmTFP_MCF7 cells estimated using DyTo‐FLIM at indicated timepoints upon receptor sub‐saturating (20 ng/ml, magenta, N = 3 biological replicates, n = 21–25 cells) or saturating (160 ng/ml, green, N = 3 biological replicates, n = 23–26 cells) sustained EGF‐Alexa647 stimulus. mean ± SD, P : unpaired two‐tailed t ‐test between 20 ng/ml and 160 ng/ml treatment ( P < 0.001 from 15′ to 60′). H, I (H) Average spatial–temporal maps constructed from confocal micrographs obtained at 1′ interval from live MCF7 cells showing the distributions of EGFR‐mCherry (left), RPTPγ‐mCitrine (middle) and RPTPγ‐mCitrine/EGFR‐mCherry (right) as a function of their normalized and binned radial distance (r) between PM and nuclear membranes (NM) and time (0–120′), upon sustained treatment with receptor‐saturatig (α L = 0.96 ± 0.05) dose of 160 ng/ml EGF‐Alexa647. N = 3 biological replicates, n = 14 cells. (I) Same as (H) for a receptor‐subsaturating (α L = 0.19 ± 0.04) dose of 20 ng/ml EGF‐Alexa647. N = 3 biological replicates, n = 13 cells.

Article Snippet: WT, RPTPγ‐KO, and p22 phox ‐KO MCF7 cells were seeded on Lab‐Tek™ chambered cover glass slides (ThermoFisher), transfected with EGFR‐mCitrine or BFP‐tkRas and incubated in cell culture medium with 0.5% FBS for at least 6 h before the experiment.

Techniques: Immunostaining, Two Tailed Test, Expressing, Fluorescence, Western Blot, Co-Immunoprecipitation Assay, Transfection, Immunoprecipitation, Construct

Journal: The EMBO Journal

Article Title: The EGFR phosphatase RPTPγ is a redox‐regulated suppressor of promigratory signaling

doi: 10.15252/embj.2022111806

Figure Lengend Snippet: A, B (A) Representative confocal micrographs of MCF7 WT cells showing RPTPγ‐mCitrine (1 st column: green; 3 rd column: blue) and EGFR‐mCherry (2 nd column: green; 3 rd column: yellow) with early‐endosomes marked by immunostaining against EEA1 (magenta), without (top row) or after 15' EGF‐DyLight405 stimulus (160 ng/ml; bottom row). Scale bar: 10 μm. (B) Same as (A) with late‐endosomes marked by immunostaining against Rab7 (magenta) without (top row) or after 60' EGF‐DyLight405 stimulus (160 ng/ml; bottom row). C Top panel: Representative confocal micrographs comparing the steady state co‐localized fraction of EGFR‐mCitrine (magenta) with the ER‐marker TCPTP‐mTFP (yellow) in WT (top row) to RPTPγ‐KO (bottom row) MCF7 cells. Bottom panel: Quantification ( N = 3, n > 50 per cell type) of the fraction of EGFR‐mCitrine fluorescence co‐localizing with TCPTP‐mTFP fluorescence for WT and RPTPγ‐KO cells. Individual cells with mean ± SD, P : unpaired two‐tailed t ‐test. D, E (D) Representative confocal time lapse images of the fluorescence photoactivation of RPTPγ‐paGFP (top row) on the RE (white‐rimmed region) in MCF7 cells with co‐expressed RPTPγ‐mCherry (middle row) and BFP‐Rab11a (last row) at indicated times after photoactivation. Gamma correction for all channels: 0.18. (E) Same as (D) without BFP‐Rab11a co‐expression. F, G (F) Representative confocal micrographs of RPTPγ‐mCitrine (top row) and its oxidized fraction (α ox , bottom row, color‐code lower right), obtained at the indicated times upon receptor‐saturating (160 ng/ml), sustained EGF‐Alexa647 stimulus in live MCF7 WT cells expressing EGFR‐mTFP and RPTPγ‐mCitrine. (G) Same as (F) with non‐saturating (20 ng/ml) EGF‐Alexa647 stimulus. H Temporal profile of EGFR‐mCitrine phosphorylation (α p ) determined by FLIM in EmCit_MCF7 cells co‐expressing PTB‐mCherry, after pulsed stimulation from 0 to 5′ with saturating EGF‐Alexa647 (160 ng/ml). mean ± SD, N = 3 biological replicates, n = 24 cells. Data information: All scale bars: 10 μm.

Article Snippet: WT, RPTPγ‐KO, and p22 phox ‐KO MCF7 cells were seeded on Lab‐Tek™ chambered cover glass slides (ThermoFisher), transfected with EGFR‐mCitrine or BFP‐tkRas and incubated in cell culture medium with 0.5% FBS for at least 6 h before the experiment.

Techniques: Immunostaining, Marker, Fluorescence, Two Tailed Test, Expressing

Journal: The EMBO Journal

Article Title: The EGFR phosphatase RPTPγ is a redox‐regulated suppressor of promigratory signaling

doi: 10.15252/embj.2022111806

Figure Lengend Snippet: Full RPTPγ‐EGFR‐TCPTP network architecture depicting the chemical conversions (black arrows; p: phosphorylation on EGFR, Ox: oxidized catalytic cysteine on RPTPγ, A: active RPTPγ with reduced catalytic cysteine) and all possible regulatory interactions (colored arrows: causal links; ε 1 − ε 4 corresponding catalytic, α 1 − α 4 autocatalytic rate constant for EGFR phosphorylation (Fig )); γ 1 and γ 3 – second‐order RPTP γ ‐specific, γ 2 and γ 4 – second‐order TCPTP‐dependent dephosphorylation; β – second‐order EGFR‐dependent oxidation of RPTPγ; κ 2 and κ 1 – intrinsic PTP deactivation and activation rate. Rate constants ( ε 1 − ε 4 , α 1 − α 4 , γ 1 − γ 4 , β ) are color coded as in (B–F) and Fig . Ordinary differential equations (ODEs) that describe the dynamics of the coupled reactants EGFRp, EGF‐EGFRp and RPTPγ A in the general symmetric autocatalytic toggle switch model. EGFR p / T , RPTPγ A / T and EGF − EGFR p / T describe the fractions of active (phosphorylated) proteins, relative to the respective total protein concentration. EGF R np / T and PT P Ox / T describe the fractions of inactive (non‐phosphorylated or oxidized) proteins, EGF‐EGFR depicts EGFR molecules liganded by EGF. Γ 1 , Γ 2 , Γ 3 , Γ 4 , Β : fitted relative kinetic parameter groups color coded to their corresponding rate constants. Catalytic ( ε 1 − ε 4 ) and autocatalytic ( α 1 − α 4 ) rate constants obtained from iterative global fitting the ODEs in (B) solved for steady state (dEGFR p/T /dt = dEGF‐EGFR p/T /dt = dRPTPγ A/T /dt = 0) to EGF‐dose response ( a L − a P ) data from all EGFR and PTP expression conditions (Fig ). EGFRp, EGF‐EGFRp: product of the corresponding reactions. Relative catalytic ( E 1 − E 4 ) and autocatalytic ( A 1 − A 4 ) EGFR phosphorylation rates at steady state as a function of receptor occupancy a L . Steady state reaction rates were calculated by multiplication of the rate constants ( ε 1 − ε 4 ; α 1 − α 4 ) by the relative abundance of the corresponding reactants and catalysts (EGFR, EGFRp, EGF‐EGFR, EGF‐EGFRp) obtained by the global fit. Inset: Calculation of the initiation of the signal by catalytic reactions ( E 1 − E 4 ) at a P = 0 , calculated by multiplication of rate constants ( ε 1 − ε 4 ) by the relative abundance of reactants (EGFR = 1 − a L ; EGF‐EGFR = a L ). Maximal dephosphorylation rates by RPTPγ (Γ 1,3 = γ 1,3 . RPTPγ/EGFR T ; dark blue) or TCPTP (Γ 2,4 = γ 2,4 .TCPTP/EGFR T ; light blue) of ligandless EGFRp (Γ 1 , Γ 2 ) or liganded EGF‐EGFRp (Γ 3 , Γ 4 ) obtained from iterative global fitting the ODEs in (B) solved for steady state (dEGFR p/T /dt = dEGF‐EGFR p/T /dt = dRPTPγ A/T /dt = 0) to EGF‐dose response ( a L − a P ) data of MCF7 WT cells (Fig ). Change of the free parameter groups Γ 1 = γ 1 RPTPγ / EGFR T and Β = β EGFR T / k 1 in EmCit_MCF7 RPTPγ‐KO (blue), EmCit_MCF7 (red), EmCit_MCF7 RPTPγ‐KO expressing RPTPγ‐mTFP splitted in three clusters with increasing RPTPγ‐mTFP/EGFR‐mCitrine ratio (yellow, green, purple; Figs and ) and WT MCF7 cells (black). RPTPγ‐EGFR‐TCPTP network architecture depicting the chemical conversions and regulatory interactions that are relevant for the EGFR‐phosphorylation response at physiological ( a L < 0.1 ) EGF‐concentrations. EGFR phosphorylation is mainly driven by autocatalysis among unliganded EGFR (α 1 ; see (D)). EGFRp oxidatively inactivates RPTPγ via ROS (β). RPTPγ counteracts this autocatalysis by dephosphorylation of EGFRp (γ 1 ). The autocatalytic activation needs to be triggered by a sufficient amount of EGFRp in the system that must come from (ε 2 ) and/or from (ε 3, ε 4 ), which produce EGF‐EGFRp that can generate EGFRp via α 2 . TCPTP (γ 2,4 ) has a comparably weaker, but constitutive modulatory dephosphorylation activity. Experimentally reconstructed 3D‐bifurcation diagrams showing the dependence of steady‐state EGFR phosphorylation (α p ) on Γ 1 ( = γ 1 .RPTPγ/EGFR) and EGF‐receptor occupancy (α L ) for EmCit_MCF7 cells (left, 2 nd row) with derivated p22phox‐, TCPTP‐ and RPTPγ‐KO and corresponding TCPTP‐ and RPTPγ‐rescue cells, indicated by the black arrows. Last row: MCF7 WT cells (left) with a numerical knockout of TCPTP, (TCPTP associated rates Γ 2 and Γ 4 = 0). Molecular ratio of RPTPγ/EGFR are depicted on top of the corresponding diagram; red line: fit to the experimentally derived dose response trajectory.

Article Snippet: WT, RPTPγ‐KO, and p22 phox ‐KO MCF7 cells were seeded on Lab‐Tek™ chambered cover glass slides (ThermoFisher), transfected with EGFR‐mCitrine or BFP‐tkRas and incubated in cell culture medium with 0.5% FBS for at least 6 h before the experiment.

Techniques: De-Phosphorylation Assay, Activation Assay, Protein Concentration, Expressing, Activity Assay, Knock-Out, Derivative Assay

Journal: The EMBO Journal

Article Title: The EGFR phosphatase RPTPγ is a redox‐regulated suppressor of promigratory signaling

doi: 10.15252/embj.2022111806

Figure Lengend Snippet: Representative images of a clonogenic assay of WT (top), RPTPγ‐KO (middle) and p22 phox ‐KO (bottom) MCF7 cells plated in medium containing 20 ng/ml EGF and 0.5% FCS at an initial density of 100, 200 and 300 cells/well, fixed stained with crystal violet and imaged on the 7 th day post plating. Same representation as (A), for cells plated in complete serum growth medium containing 10% FCS. Representative transmitted light micrographs of WT (top row), RPTPγ‐KO (middle row) and p22 phox ‐KO (bottom row) MCF7 cells during stimulation with EGF‐Alexa647 (160 ng/ml) at the indicated times (0, 12 h) after removal of a migration barrier in confluent cell layers. Scale bars: 100 μm. Insets left of the images: Average cell number ( N = 4–5) distributed in six spatial bins (position of the bins schematized in Fig ) around the initial cell front measured every 10′, color‐coded (upper right) by time after barrier removal.

Article Snippet: WT, RPTPγ‐KO, and p22 phox ‐KO MCF7 cells were seeded on Lab‐Tek™ chambered cover glass slides (ThermoFisher), transfected with EGFR‐mCitrine or BFP‐tkRas and incubated in cell culture medium with 0.5% FBS for at least 6 h before the experiment.

Techniques: Clonogenic Assay, Staining, Migration

Journal: The EMBO Journal

Article Title: The EGFR phosphatase RPTPγ is a redox‐regulated suppressor of promigratory signaling

doi: 10.15252/embj.2022111806

Figure Lengend Snippet: Chemical equations for all possible catalytic (green with corresponding rate constants ε 1–4 ) and autocatalytic (red, α 1–4 ) reactions among liganded (EGF=) and unliganded EGFR that yield phosphorylated EGFR (EGFRp, EGF‐EGFRp), sorted by their corresponding reaction intermediate (transient EGFR‐dimers with 0 (ligandless), 1 (extracellular domain asymmetric: ExAsym) or 2 (extracellular domain symmetric: ExSym) EGF molecules). Catalytic (green, ε 1–4 ) and autocatalytic (red, α 1–4 ) rate constants as well as kinetic parameter groups (Γ1–4, Β, κ21) obtained from iteratively fitting of the ODEs depicted in Fig considering all possible interactions in the EGFR‐PTP system (Fig ) to all EGFR, RPTPγ and TCPTP‐expression conditions, as depicted on the y‐axis. EmCit_MCF7: MCF7 cells ectopically expressing ~2 × 10 5 EGFR‐mCitrine; RPTPγ‐KO/TCPTP‐KO: CRISPR‐Cas9 meditated knock out in EmCit_MCF7 cells; R/E: RPTPγ/EGFR molecular ratio (Fig ); RPTPγ‐KO rescue/TCPTP‐KO rescue: Protein expression rescued by ectopic overexpression of PTP‐mTFP; x: shared parameters that were linked during the global fit and therefore have the same value as shown for EmCit_MCF7. Overlay of the resulting fit (solid line) yielding the parameters shown in (B) to the experimental data (blue circles) of the fraction of phosphorylated EGFR (α p ) versus the fraction of EGF‐bound EGFR (α L ), for all EGFR, RPTPγ, NOX and TCPTP‐expression perturbations as well as for endogenous expression in WT MCF7 cells (see B). Fraction of phosphorylated unliganded (EGFRp/EGFR T ) and liganded (EGF‐EGFRp/EGFR T ) in MCF7 WT cells plotted against the fraction of EGF‐bound EGFR (α L ). Fractions were obtained from iterative global fitting of the ODEs in (Fig ) solved for steady state (dEGFR p/T /dt = dEGF‐EGFR p/T /dt = dRPTPγ A/T /dt = 0) to EGF‐dose response ( a L − a P ) data surface (B). Sum: total fraction of phosphorylated EGFR ((EGFRp + EGF‐EGFRp)/EGFR T ). Maximal dephosphorylation rates by RPTPγ (Γ 1,3 = γ 1,3 . RPTPγ/EGFR T ; dark blue) or constitutive dephosphorylation rates by TCPTP (Γ 2,4 = γ 2,4 .TCPTP/EGFR T ; light blue) of ligandless EGFRp (Γ 1 , Γ 2 ) or liganded EGF‐EGFRp (Γ 3 , Γ 4 ) of EmCit_MCF7 WT cells obtained from iterative global fitting of the ODEs in (Fig ) solved for steady state (dEGFR p/T /dt = dEGF‐EGFR p/T /dt = dRPTPγ A/T /dt = 0) to EGF‐dose response ( a L − a P ) data surface (B). 3D‐bifurcation diagram showing the dependence of EGFR phosphorylation (α p ) on Γ 1 (=γ 1 .RPTPγ/EGFR) and Γ 2 ( = γ 2 .TCPTP/EGFR) for α L = 0 using the kinetic parameters obtained for WT MCF7 cells. red dot: Steady‐state poising of the system at endogenous RPTPγ/TCPTP expression.

Article Snippet: WT, RPTPγ‐KO, and p22 phox ‐KO MCF7 cells were seeded on Lab‐Tek™ chambered cover glass slides (ThermoFisher), transfected with EGFR‐mCitrine or BFP‐tkRas and incubated in cell culture medium with 0.5% FBS for at least 6 h before the experiment.

Techniques: Expressing, CRISPR, Knock-Out, Over Expression, De-Phosphorylation Assay

Journal: The EMBO Journal

Article Title: The EGFR phosphatase RPTPγ is a redox‐regulated suppressor of promigratory signaling

doi: 10.15252/embj.2022111806

Figure Lengend Snippet: Representative cell contour maps showing the temporal changes (color bar: time (min), bottom right) in cell morphology for WT (upper row), RPTPγ‐KO (middle row) and p22 phox ‐KO (bottom row) MCF7 cells, expressing PM‐marker BFP‐tkRas imaged every 2′ over 60′, without (1 st column) or with 1 ng/ml (2 nd column) or 160 ng/ml (3 rd column) EGF‐Alexa647. Scale bar: 10 μm. Morphometric quantification by the ratio of the perimeter of an equiareal circle to the actual perimeter of all cells ( N = 3 biological replicates, n = 9–20 cells) at all timepoints (P circle /P cell ). 1 st row: WT, 2 nd row: RPTPγ‐KO, 3 rd row: p22phox‐KO MCF7 cells. P : one‐way ANOVA with Šídák's multiple comparisons. Top: Representative Western blot against Erk and phosphorylated Erk (pT202 and pY204) in WT (red) compared to p22 phox ‐KO (green) MCF7 cells after the indicated times of sustained stimulation with 20 ng/ml EGF‐Alexa647. Bottom: Corresponding quantification of the fraction of phosphorylated ERK as a function of stimulation time. Mean ± SD, N = 3 biological replicates, P : unpaired two‐tailed t ‐test. Quantification of cell proliferation using retinoblastoma (Rb) protein phosphorylation detected by immunofluorescence, for WT (red), RPTPγ‐KO (blue) and p22 phox ‐KO (green) MCF7 cells without or post 24 h of EGF‐Alexa647 treatment (1, 20, 160 ng/ml). Mean ± SEM, N = 3 biological replicates, n > 2,000 cells per EGF stimulus per cell line, P : two‐way ANOVA with Tukey multiple comparisons. Quantification of the culture‐well area (%) occupied by proliferating cell‐colonies, obtained from clonogenic assays of WT, RPTPγ‐KO and p22 phox ‐KO MCF7 cells, plated either in medium containing 20 ng/ml EGF and 0.5% FCS (left: orange bars, N = 3–4 biological replicates, 11–12 wells each) or complete serum growth medium containing 10% FCS (right: pink bars, N = 4 biological replicates, 12 wells each). Mean ± SEM, P : unpaired two‐tailed t ‐test with Welch's correction. Representative transmitted light micrographs of WT (top row), RPTPγ‐KO (middle row) and p22 phox ‐KO (bottom row) MCF7 cells, without (1 st column) and during stimulation with, H 2 O 2 (0.5 mM, 2 nd column) or EGF‐Alexa647 (1 ng/ml, 3 rd column) at the indicated times (0, 12 h) after removal of a migration barrier. Scale bar: 100 μm. Insets left of the images: Temporal maps (color‐code lower right) depicting the average cell number (over N = 4–5 biological replicates) distributed in six spatial bins around the initial cell front measured every 10′ (schematic in second column: location of the lateral bins in the migration chamber). Left panel: Exemplary images of RPTPγ‐KO (top row) and p22 phox ‐KO (bottom row) MCF7 cells stimulated with 1 ng/ml EGF‐Alexa647, at 16 h after removal of a migration barrier together with Hoechst 33342 (2 nd column) and 5‐Ethinyl‐2'‐Desoxyuridin (EdU 10 μM, 1 h; 3rd column) staining obtained after 17 h. Right Graph: Quantification of the fraction of dividing (EdU + ) cells between 16 th and 17 th hour. N = 4 biological replicates, Mean ± SD.

Article Snippet: WT, RPTPγ‐KO, and p22 phox ‐KO MCF7 cells were seeded on Lab‐Tek™ chambered cover glass slides (ThermoFisher), transfected with EGFR‐mCitrine or BFP‐tkRas and incubated in cell culture medium with 0.5% FBS for at least 6 h before the experiment.

Techniques: Expressing, Marker, Western Blot, Two Tailed Test, Immunofluorescence, Migration, Staining

Journal: The EMBO Journal

Article Title: The EGFR phosphatase RPTPγ is a redox‐regulated suppressor of promigratory signaling

doi: 10.15252/embj.2022111806

Figure Lengend Snippet: Left: The continuous recycling (orange circular arrows) of interacting RPTPγ‐EGFR monomers through the reducing environment of the cytoplasm maintains the catalytic cysteine of RPTPγ in the reduced (SH) state, such that it continuously dephosphorylates spontaneously phosphorylated EGFRp monomers at the PM. Upon receptor sub‐saturating EGF stimulus (curved orange arrow), transient EGF‐EGFR 2 dimers catalytically trigger (orange straight arrow) the autocatalytic phosphorylation reaction that generates EGFRp monomers at the PM (black circular arrow). EGFRp activate NOX complexes (black arrow to NOX‐p22 phox ) that produce H 2 O 2 (purple cloud and dashed arrow) at and near the PM that oxidatively inactivates the inhibitory phosphatase activity of RPTPγ (oxidated catalytic cysteine: SOH) on ligandless phosphorylated EGFRp monomers. These signal and activate promigratory effectors at the PM. The toggle switch causality resulting from the EGFRp‐mediated oxidative inhibition of RPTPγ and RPTPγ's dephosphorylating activity on EGFRp is represented by the mutual inhibitory arrows between interacting EGFR and RPTPγ. On the other hand, the constitutive dephosphorylation of EGFRp by the PM‐proximal pool of endoplasmic reticulum associated TCPTP (green) maintains reversibility in the ultrasensitive EGFR phosphorylation response to EGF. The reactivation (catalytic cysteine reduction: SH) of the phosphatase activity of inactivated RPTPγ (oxidated catalytic cysteine: SOH) by vesicular recycling through the cytoplasm via the RE (curved orange arrows), together with vesicular recycling and dephosphorylation of ligandless EGFRp, reverts ligandless EGFRp to basal levels at the PM when growth factor levels decline. In dependence on EGF concentration (green arrow), accumulation of liganded EGF‐EGFR in clathrin coated pits generate stable ubiquitinated (Ub) EGF‐EGFR complexes that unidirectionally traffic to the LE via the EE (green arrow), from which they couple to proliferative effectors. High, receptor saturating, levels of EGF (right diagram) thereby lead to a faster accumulation of EGF‐EGFR in endosomes, depletion of promigratory EGFRp monomers at the PM, and predominantly proliferative EGF‐EGFR signaling from endosomes. In this branch, EGF‐EGFRp signal duration is determined by the dephosphorylating activities of ER‐associated TCPTP (green) and PTP1B (cyan) while the receptor complexes traffic to the LE via the EE.

Article Snippet: WT, RPTPγ‐KO, and p22 phox ‐KO MCF7 cells were seeded on Lab‐Tek™ chambered cover glass slides (ThermoFisher), transfected with EGFR‐mCitrine or BFP‐tkRas and incubated in cell culture medium with 0.5% FBS for at least 6 h before the experiment.

Techniques: Activity Assay, Inhibition, De-Phosphorylation Assay, Concentration Assay

Journal: Antioxidants

Article Title: Targeting M2 Macrophages with a Novel NADPH Oxidase Inhibitor

doi: 10.3390/antiox12020440

Figure Lengend Snippet: Confocal imaging of living cells (Left A – F) ( A , B ): monocytes isolated from blood of healthy donors; the red signal is the NOX2 and mitotracker in ( A , B ), respectively, the grey signal corresponds to the mitotracker and CellRox deep red reagent in ( A , B ), respectively; ( C , D ) Monocyte-derived macrophages (with CSF1-without NS1 treatment) using the same markers used for the living monocytes in ( A , B ); ( E , F ) the macrophages were treated with NS1 after their CSF1-induced differentiation from monocytes using the same markers as in ( A , B ); the green signal in ( F ) is the fluorescence of NS1; the ROS levels are quantified by the cell-permeable CellROX deep red reagent shown in light grey, the mitochondria are monitored by the red mitotracker reagent as quantified in ( G ); Co-localization experiments in fixed cells (Right H – L) The images ( H – L ) present co-localization experiments in fixed monocytes or CSF1-treated monocytes differentiated into macrophages . Each antibody is mentioned above each image, the merge, representing the colocalization is shown in yellow. The intensity of the colocalization is quantified in ( M ). We tested colocalization between partners of the activated NOX2 complex in “M2” macrophages formed by membrane bound NOX2 and p22 phox with p67 phox and p47 phox (p40 phox and rac1 are not labeled). It is known that p67 phox and p47 phox are located in the cytoplasm of the resting cells in agreement with the images shown in ( H ); ( I , J ) are p22 phox -p47 phox and p22 phox -p67 phox co-localization signals in macrophages without NS1, respectively; ( K , L ) are p22 phox -p47 phox and p22 phox -p67 phox co-localizations with NS1, respectively. Scale bars are 3 µm. Quantification of the 3D images is shown in ( M ) and was performed with Imaris 7.3 software ( http://www.bitplane.com/imaris , accessed on 6 February 2023).

Article Snippet: Mitotracker (red or far-red) and CellRox deep red were purchased from Thermo-Fischer and Invitrogen (Les Ulys, France), respectively; antibodies against NOX2 (ab80897) and p22 phox (ab75941) were acquired from Abcam; p47 phox (sc7660) and p67 phox (sc7663) were acquired from Santa Cruz; and NS1 was synthesized by Innoverda according to the published synthesis [ ].

Techniques: Imaging, Isolation, Derivative Assay, Fluorescence, Labeling, Software

Journal: Molecular Systems Biology

Article Title: Glucose deprivation activates a metabolic and signaling amplification loop leading to cell death

doi: 10.1038/msb.2012.20

Figure Lengend Snippet: NADPH oxidase- and mitochondria-derived ROS contribute to glucose withdrawal-induced phospho-tyrosine signaling. ( A ) Inhibition of NOX inhibits glucose withdrawal-induced signaling. LN18, T98, and U87 cells were starved of glucose and pyruvate in the presence of DMSO or DPI (1 μM). Western blotting demonstrated that NOX activity is required for the induction of phospho-tyrosine signaling. ( B ) Knockdown of the NOX subunit p22 phox attenuates phospho-tyrosine signaling following glucose withdrawal. U87 cells were reverse transfected with control, non-targeting siRNA or siRNA against p22 phox , DUOX1 or DUOX2. Forty-eight hours later, cells were starved of glucose and pyruvate for 5 h. Western blotting demonstrated that knockdown of p22 phox but not DUOX1/2 attenuated glucose withdrawal-induced phospho-tyrosine signaling. p22 phox knockdown efficiency was >90% . ( C ) LN18, T98, and U87 cells were starved of glucose and pyruvate in the presence of either DMSO or BAPTA-AM (25 μM). Western blotting with an anti-phospho-tyrosine antibody demonstrated that chelation of intracellular Ca 2+ by BAPTA-AM completely abrogated glucose withdrawal-induced phospho-tyrosine signaling. Treatment with extracellular EDTA (25 μM) had no effect. Actin and GRB2 served as equal loading controls. ( D ) ρ 0 derivatives of T98 and U87 cells do not exhibit upregulation of phospho-tyrosine signaling or activation of Src in response to glucose withdrawal. ( E ) Catalase rescues parental but not ρ 0 cells from glucose withdrawal-induced cell death. Cells were starved of glucose and pyruvate with or without catalase (1 kU/ml), and viability was measured by Trypan blue exclusion 24 h later.

Article Snippet: siRNA constructs targeting p22 phox (M-011020) and a non-targeting control siRNA (D-001206) were purchased from Thermo Scientific.

Techniques: Derivative Assay, Inhibition, Western Blot, Activity Assay, Transfection, Activation Assay

Journal: International Journal of Nanomedicine

Article Title: Therapeutic effects of proteoliposomes on X-linked chronic granulomatous disease: proof of concept using macrophages differentiated from patient-specific induced pluripotent stem cells

doi: 10.2147/IJN.S128611

Figure Lengend Snippet: Optimized production of NOX2/p22 phox liposomes using the cell-free expression system. Notes: ( A ) Effect of magnesium and potassium concentrations on cell-free production of p22 phox protein. In vitro expression was performed in 96-well plates with a volume of 50 µL. ( B ) Effect of lipid composition on the expression of p22 phox subunit. Total protein fraction was obtained after the cell-free expression reaction, while the proteoliposome fraction was separated after a discontinuous sucrose gradient to separate them from liposomes and aggregated proteins. ( C ) Effect of the reaction time variation on NOX2 and p22 phox expression. Reactions were carried out at 30°C for 2 h, 4 h, 6 h or 16 h, in batch format (100 µL) and separated by SDS-PAGE on a 15% gel. P22 phox and NOX2 detection bands are indicated with stars. ( D ) Effect of the variation of iron and hemin concentration on protein expression. In vitro expression was performed in 96-well plates with a volume of 50 µL. In all experiments, monoclonal anti-His HRP-conjugated antibody and anti-NOX2 antibodies (clone 44.1) were used for the detection of p22 phox and NOX2, respectively. Optimal concentrations were indicated. * Indicates the location of NOX2 and p22 phox . Abbreviations: NTPs, nucleotide triphosphates; PLs, proteoliposomes; SDS-PAGE, sodium dodecyl sulfate polyacrylamide gel electrophoresis.

Article Snippet: Paraformaldehyde (PFA), fetal bovine serum (FBS), goat serum, Iscove’s Modified Dulbecco’s Medium (IMDM), Alexa Fluor ® 488-conjugated goat-F(ab′) 2 fragment anti-mouse IgG1 (H+L), anti-NOX2 antibody (clone 54.1) and anti-p22 phox antibody (clone 44.1) were from Thermo Fisher Scientific (Courtaboeuf, France).

Techniques: Expressing, In Vitro, SDS Page, Concentration Assay, Polyacrylamide Gel Electrophoresis

Journal: International Journal of Nanomedicine

Article Title: Therapeutic effects of proteoliposomes on X-linked chronic granulomatous disease: proof of concept using macrophages differentiated from patient-specific induced pluripotent stem cells

doi: 10.2147/IJN.S128611

Figure Lengend Snippet: In vitro physicochemical characterization of NOX2/p22 phox and negative liposomes. Notes: ( A ) Particle size distribution of NOX2/p22 phox and negative liposomes by DLS. ( B ) Coomassie blue staining of NOX2/p22 phox and negative liposomes (The * and ▲ indicate the location of NOX2 and p22 phox respectively) and Western blot analysis of NOX2/p22 phox and negative liposomes using monoclonal antibodies against NOX2 and p22 phox . ( C ) Dithionite-reduced minus-oxidized spectra of NOX2/p22 phox and negative liposomes. Abbreviations: DLS, dynamic light scattering; OD, optical density; PLs, proteoliposomes.

Article Snippet: Paraformaldehyde (PFA), fetal bovine serum (FBS), goat serum, Iscove’s Modified Dulbecco’s Medium (IMDM), Alexa Fluor ® 488-conjugated goat-F(ab′) 2 fragment anti-mouse IgG1 (H+L), anti-NOX2 antibody (clone 54.1) and anti-p22 phox antibody (clone 44.1) were from Thermo Fisher Scientific (Courtaboeuf, France).

Techniques: In Vitro, Staining, Western Blot

Journal: International Journal of Nanomedicine

Article Title: Therapeutic effects of proteoliposomes on X-linked chronic granulomatous disease: proof of concept using macrophages differentiated from patient-specific induced pluripotent stem cells

doi: 10.2147/IJN.S128611

Figure Lengend Snippet: In vitro NADPH oxidase activity of NOX2/p22 phox and negative liposomes. Notes: ( A ) Representative results of the NADPH oxidase activity of NOX2/p22 phox (8 pmol cytochrome b 558 ) and negative liposomes in a cell-free system assay in the presence of the recombinant p47 phox , p67 phox and Rac (1 µM), arachidonic acid (400 µM) and NBT (100 µM), stimulated or not with NADPH (200 µM). ( B ) NADPH oxidase activity was expressed in moles of superoxide O 2 •− produced/s/mol of heme and measured before (total activity) or after SOD addition (SOD non-inhibitable activity). Cell-free system assay was performed in the same experimental conditions as ( A ). Abbreviations: DPI, diphenyleneiodonium; NADPH, nicotinamide adenine dinucleotide phosphate; NBT, nitroblue tetrazolium; OD, optical density; O 2 •− , superoxide anion; SOD, superoxide dismutase; PLs, proteoliposomes.

Article Snippet: Paraformaldehyde (PFA), fetal bovine serum (FBS), goat serum, Iscove’s Modified Dulbecco’s Medium (IMDM), Alexa Fluor ® 488-conjugated goat-F(ab′) 2 fragment anti-mouse IgG1 (H+L), anti-NOX2 antibody (clone 54.1) and anti-p22 phox antibody (clone 44.1) were from Thermo Fisher Scientific (Courtaboeuf, France).

Techniques: In Vitro, Activity Assay, Recombinant, Produced

Journal: International Journal of Nanomedicine

Article Title: Therapeutic effects of proteoliposomes on X-linked chronic granulomatous disease: proof of concept using macrophages differentiated from patient-specific induced pluripotent stem cells

doi: 10.2147/IJN.S128611

Figure Lengend Snippet: Analysis of the membrane delivery of NOX2 and p22 phox subunits in X 0 -CGD iPSC-derived macrophages after NOX2/p22 phox liposome treatment. Notes: ( A ) Flow cytometry analysis of NOX2 and p22 phox expression using monoclonal antibodies in WT and untreated X 0 -CGD macrophages (black curve), and X 0 -CGD macrophages treated for 8 h with NOX2/p22 phox (red curve) or negative (green curve) liposomes. Isotype controls are represented by gray-filled curves. MFIs were indicated for each condition, and the shift of fluorescence was calculated as the percentage of increased fluorescence compared to untreated macrophages. ( B ) Confocal microscopy images showing the staining of NOX2 subunit with 7D5 antibody and AF488-conjugated secondary antibody (green) in WT and X 0 -CGD macrophages treated for 8 h with NOX2/p22 phox or negative liposomes. Nuclei were counterstained with Hoechst 33258 (blue); scale bars =20 µm. The same observations were obtained in at least two experiments. Abbreviations: CGD, chronic granulomatous disease; iPSC, induced pluripotent stem cell; MFIs, mean fluorescence intensities; MΦ, macrophages; WT, wild type; X 0 -CGD, X 0 -linked CGD; XCGD, X-linked CGD.

Article Snippet: Paraformaldehyde (PFA), fetal bovine serum (FBS), goat serum, Iscove’s Modified Dulbecco’s Medium (IMDM), Alexa Fluor ® 488-conjugated goat-F(ab′) 2 fragment anti-mouse IgG1 (H+L), anti-NOX2 antibody (clone 54.1) and anti-p22 phox antibody (clone 44.1) were from Thermo Fisher Scientific (Courtaboeuf, France).

Techniques: Derivative Assay, Flow Cytometry, Expressing, Fluorescence, Confocal Microscopy, Staining

Journal: International Journal of Nanomedicine

Article Title: Therapeutic effects of proteoliposomes on X-linked chronic granulomatous disease: proof of concept using macrophages differentiated from patient-specific induced pluripotent stem cells

doi: 10.2147/IJN.S128611

Figure Lengend Snippet: Location of NOX2 in liposome-treated X 0 -CGD iPSC-derived macrophages after C . albicans phagocytosis. Notes: ( A ) Confocal microscopy images showing the staining of NOX2 subunit with 7D5 antibody and AF488-conjugated secondary antibody (green) in X 0 -CGD macrophages treated for 8 h with NOX2/p22 phox or negative liposomes and then for 4 h with C . albicans strain at an MOI of 3:1. ( B ) Z-stack images (top to bottom of cell) of an NOX2/p22 phox liposome-treated X 0 -CGD macrophage incubated for 4 h with C . albicans strain at an MOI of 3:1. Nuclei were counterstained with Hoechst 33258 (blue); scale bars =20 µm in ( A ) and 10 µm in ( B ). The same observations were obtained in at least two experiments. Abbreviations: C. albicans; Candida albicans , CGD, chronic granulomatous disease; iPSC, induced pluripotent stem cell; MOI, multiplicity of infection; X 0 -CGD, X 0 -linked CGD; XCGD, X-linked CGD.

Article Snippet: Paraformaldehyde (PFA), fetal bovine serum (FBS), goat serum, Iscove’s Modified Dulbecco’s Medium (IMDM), Alexa Fluor ® 488-conjugated goat-F(ab′) 2 fragment anti-mouse IgG1 (H+L), anti-NOX2 antibody (clone 54.1) and anti-p22 phox antibody (clone 44.1) were from Thermo Fisher Scientific (Courtaboeuf, France).

Techniques: Derivative Assay, Confocal Microscopy, Staining, Incubation, Infection

Journal: International Journal of Nanomedicine

Article Title: Therapeutic effects of proteoliposomes on X-linked chronic granulomatous disease: proof of concept using macrophages differentiated from patient-specific induced pluripotent stem cells

doi: 10.2147/IJN.S128611

Figure Lengend Snippet: Analysis of in cellulo toxicity and NADPH oxidase activity restoration in X 0 -CGD iPSC-derived macrophages. Notes: ( A ) Viability of CGDX 0 iPSC-derived macrophages after 8, 12 and 24 h of treatment. Untreated cells are used as positive control (100%) and DMSO-treated cells as negative control. Data are expressed as mean ± SEM ( n =3). The differences of viability between the untreated cells and the liposome-treated cells were analyzed using the nonparametric Mann–Whitney test. ( B ) Morphological images of X 0 -CGD iPSC-derived macrophages after 8, 12 and 24 h of treatment with NOX2/p22 phox liposomes or incubated with IMDM only (magnification ×10). ( C ) X 0 -CGD macrophages were treated for 8 h with NOX2/p22 phox or negative liposomes. Then, WT and X 0 -CGD macrophages were stimulated with PMA (resting; scale bars =50 µm). Abbreviations: CGD, chronic granulomatous disease; DMSO, dimethylsulfoxide; IMDM, Iscove’s Modified Dulbecco’s Medium; iPSC, induced pluripotent stem cell; NADPH, nicotinamide adenine dinucleotide phosphate; ns, nonsignificant; PMA, phorbolmyristate-acetate; SEM, standard error of the mean; WT, wild type; X 0 -CGD, X 0 -linked CGD.

Article Snippet: Paraformaldehyde (PFA), fetal bovine serum (FBS), goat serum, Iscove’s Modified Dulbecco’s Medium (IMDM), Alexa Fluor ® 488-conjugated goat-F(ab′) 2 fragment anti-mouse IgG1 (H+L), anti-NOX2 antibody (clone 54.1) and anti-p22 phox antibody (clone 44.1) were from Thermo Fisher Scientific (Courtaboeuf, France).

Techniques: Activity Assay, Derivative Assay, Positive Control, Negative Control, MANN-WHITNEY, Incubation, Modification

Journal: eLife

Article Title: EROS is a selective chaperone regulating the phagocyte NADPH oxidase and purinergic signalling

doi: 10.7554/eLife.76387

Figure Lengend Snippet: ( A–C ) Mouse constructs encoding EROS and gp91 phox were co-transfected into NIH3T3 ( A ), COS-7 ( B ), and HEK293T ( C ) cell lines. gp91 phox expression was analysed by immunoblotting; arrow indicates gp91 phox band; ns: non-specific band. ( D–F ) gp91 phox and p22 phox expression in HEK293T cells following transfection with the indicated human constructs. ( G ) Left panel: analysis of the stability of the different forms of gp91 phox (indicated by the arrows) following transfection in HEK293T cells in the presence or absence of EROS and treatment with 10 μg/mL cycloheximide. Right panel: quantitation of the cycloheximide assay (mean of four independent experiments; error bars indicate SD) represented as a fold change of gp91 phox in cells expressing gp91 phox and EROS vectors relative to gp91 phox vector alone at 0 hr and normalised to actin expression. Actin and vinculin were used as loading control. ( H ) Stability of endogenous gp91 phox in PLB985 neutrophil-like cells overexpressing lentivirus (LV) EROS-GFP vector (MW ≈ 41 kDa) and treated with 10 μg/mL cycloheximide. ( I–J ) gp91 phox expression following lentiviral transduction of EROS-GFP, gp91 phox, or both in differentiated PL985 knockout (KO) for p22 phox ( I ) or EROS ( J ). Data are representative of three independent experiments. See also and . Figure 1—source data 1. Raw unedited blots for . Figure 1—source data 2. Raw unedited blots for . Figure 1—source data 3. Raw unedited blots for . Figure 1—source data 4. Uncropped gels used for .

Article Snippet: Cells were transfected at 60–80% confluency with 1.6–2 µg of the following constructs: mouse gp91 phox , mouse GFP-tagged gp91 phox , mouse EROS, human EROS, human gp91 phox , human mRFP-tagged gp91 phox , human p22 phox , human NOX4, mouse GFP-tagged P2X7, human P2X1, and human P2X4; using Lipofectamine RNAiMAX (Thermo Fisher) reagent for HEK293T, HEK293, and COS-7 cells and Lipofectamine 2000 (Thermo Fisher) reagent for NIH3T3 cells, following the manufacturer’s recommendation.

Techniques: Construct, Transfection, Expressing, Western Blot, Quantitation Assay, Plasmid Preparation, Transduction, Knock-Out

Journal: eLife

Article Title: EROS is a selective chaperone regulating the phagocyte NADPH oxidase and purinergic signalling

doi: 10.7554/eLife.76387

Figure Lengend Snippet: ( A–D ) Immunoprecipitation (IP) and size-exclusion chromatography (SEC) analysis of protein complexes associated with EROS. ( A ) IP of EROS in HEK293-F cells expressing StrepII-FLAG-tagged EROS, gp91 phox -GFP, and p22 phox with Western blot for gp91 phox . Lysates treated with peptide N-glycosidase F (PNGaseF) or endoglycosidase H (EndoH) served as reference; FG: fully glycosylated; PG: partially glycosylated; NG: non-glycosylated; Tot: total lysate; RT: run through; Elu: eluate. ( B ) SEC profile of EROS-IP eluate indicating protein (280 nm) and heme (414 nm) content. ( C ) Immunoblot analysis of gp91 phox -GFP, EROS-FLAG, and endogenous p22 phox in SEC fractions 9–14 and 15–18. ( D ) SEC profile of EROS eluate from HEK293-F cells expressing EROS-FLAG, gp91 phox, and p22 phox constructs and treated with heme biosynthesis inhibitor succinyl acetone (10 µg/ml). ( E ) IP of StrepII-FLAG-tagged EROS in HEK293-F treated with succinyl acetone. ( F ) Interaction between gp91 phox and EROS assessed through luminescence production in live HEK293T cells expressing the indicated plasmids fused with the large (LgBIT) or small (SmBIT) fragment of the NanoLuc luciferase (see ‘Methods’). Halo Tag (HT)-SmBIT is the negative control; RLU: relative luminescence unit. ( G ) Yeast growth phenotypes obtained with the specified selective media using gp91 phox bait plasmid and EROS prey plasmid. L: leucine; W: tryptophan; H: histidine; DBD: DNA binding domain of Gal4; AD: activation domain of Gal4 (see ‘Methods’). ( H ) EROS localisation in HEK293 cells transfected with EROS construct (top panel; 3D stack) or EROS and Lap2-GFP constructs (bottom panel; single plane), fixed, permeabilised, and labelled with anti-EROS and anti-calnexin antibodies. Scale bar = 5 μm. Data are representative of at least three independent experiments; error bars indicate SEM of triplicates. See also and . Figure 2—source data 1. Raw unedited blots for . Figure 2—source data 2. Uncropped gels used for .

Article Snippet: Cells were transfected at 60–80% confluency with 1.6–2 µg of the following constructs: mouse gp91 phox , mouse GFP-tagged gp91 phox , mouse EROS, human EROS, human gp91 phox , human mRFP-tagged gp91 phox , human p22 phox , human NOX4, mouse GFP-tagged P2X7, human P2X1, and human P2X4; using Lipofectamine RNAiMAX (Thermo Fisher) reagent for HEK293T, HEK293, and COS-7 cells and Lipofectamine 2000 (Thermo Fisher) reagent for NIH3T3 cells, following the manufacturer’s recommendation.

Techniques: Immunoprecipitation, Size-exclusion Chromatography, Expressing, Western Blot, Construct, Luciferase, Negative Control, Plasmid Preparation, Binding Assay, Activation Assay, Transfection

Journal: eLife

Article Title: EROS is a selective chaperone regulating the phagocyte NADPH oxidase and purinergic signalling

doi: 10.7554/eLife.76387

Figure Lengend Snippet: Diagram depicting the role of EROS in gp91 phox biosynthesis and formation of the heterodimer with p22 phox .

Article Snippet: Cells were transfected at 60–80% confluency with 1.6–2 µg of the following constructs: mouse gp91 phox , mouse GFP-tagged gp91 phox , mouse EROS, human EROS, human gp91 phox , human mRFP-tagged gp91 phox , human p22 phox , human NOX4, mouse GFP-tagged P2X7, human P2X1, and human P2X4; using Lipofectamine RNAiMAX (Thermo Fisher) reagent for HEK293T, HEK293, and COS-7 cells and Lipofectamine 2000 (Thermo Fisher) reagent for NIH3T3 cells, following the manufacturer’s recommendation.

Techniques:

Journal: eLife

Article Title: EROS is a selective chaperone regulating the phagocyte NADPH oxidase and purinergic signalling

doi: 10.7554/eLife.76387

Figure Lengend Snippet:

Article Snippet: Cells were transfected at 60–80% confluency with 1.6–2 µg of the following constructs: mouse gp91 phox , mouse GFP-tagged gp91 phox , mouse EROS, human EROS, human gp91 phox , human mRFP-tagged gp91 phox , human p22 phox , human NOX4, mouse GFP-tagged P2X7, human P2X1, and human P2X4; using Lipofectamine RNAiMAX (Thermo Fisher) reagent for HEK293T, HEK293, and COS-7 cells and Lipofectamine 2000 (Thermo Fisher) reagent for NIH3T3 cells, following the manufacturer’s recommendation.

Techniques: Knock-Out, Generated, Over Expression