Journal: Communications Biology

Article Title: Activation of TMEM16E scramblase induces ligand independent growth factor receptor signaling and macropinocytosis for membrane repair

doi: 10.1038/s42003-025-07465-6

Figure Lengend Snippet: a Schematic of phospholipid rearrangement by TMEM16E-dependent scramblase and conformational changes of GFRs (EGFR and PDGFR). GFRs consist of an extracellular domain (gray), transmembrane domain (blue), juxtamembrane domain (magenta), and tyrosine kinase domain (purple). Positively charged amino acids of the juxtamembrane domain interact with negatively charged phosphatidylserine (PS) in inactive GFRs. Treatment with A23187 induces TMEM16E-dependent scramblase. The interaction between the juxtamembrane domain and PS is broken, and the juxtamembrane region dimerizes and is converted to an active form by clustering with negatively charged PI(4,5)P 2 . Activation of GFRs induces macropinocytosis. The images and every material element by the authors using Microsoft Office PowerPoint 365 (Microsoft). b Representative images of 1 μM A23187-induced scrambling in cells expressing RF-Dead, RF-Sac1, or RF-PJ with TMEM16E. Cells were treated with 100 nM rapamycin for 10 min in DMEM before imaging. The white dashed square box is magnified in the images in the right panel. Scale bars, 10 μm, and magnified images scale bar, 2 μm. c Analysis of AV fluorescence intensity at 15 min (RF-Dead, n = 11; RF-Sac1, n = 13; RF-PJ, n = 12). ROIs selected plasma membrane (PM), and cytoplasmic regions (Cyto). The ratio of the cytoplasmic region to the plasma membrane (F Cyto /F PM ratio) was calculated. Analysis was performed by one-way ANOVA followed by Dunnett’s post-hoc test (Compared to Dead, PM Sac1, ns = 0.9218; PM PJ, ns = 0.3227; Cyto Sac1, ns = 0.844; Cyto PJ, * P = 0.0345; F Cyto /F PM ratio Sac1, ns = 0.9936; F Cyto /F PM ratio PJ, **** P < 0.0001). Data are mean ± SEM.

Article Snippet: Human cDNAs of EGFR (Plasmid #133749) and PDGFR (Plasmid #136455) were purchased from Addgene, and coding regions were subcloned into a pcDNA 3.1(+) vector.

Techniques: Activation Assay, Expressing, Imaging, Fluorescence, Clinical Proteomics, Membrane

Journal: Communications Biology

Article Title: Activation of TMEM16E scramblase induces ligand independent growth factor receptor signaling and macropinocytosis for membrane repair

doi: 10.1038/s42003-025-07465-6

Figure Lengend Snippet: a Schematic diagram of the action of inhibitors on EGFR and PDGFR. Afatinib inhibits phosphorylation by covalent binding to C797 located in the tyrosine kinase (TK) domain of EGFR. Imatinib binds to the ATP allosteric pocket and blocks the binding of ATP. The images and every material element by the authors using Microsoft Office PowerPoint 365 (Microsoft). b Representative images of TMEM16E-dependent scramblase calculated as AV fluorescence in the plasma membrane (PM) over that in cytoplasm (Cyto) for control, after application of 1 μM afatinib for 1 h, and after 20 μM imatinib for 1 h in DMEM. The yellow dashed line indicates the plasma membrane region and the white dashed square box is magnified in the images in the right panel. Scale bars, 10 μm, and magnified images scale bar, 2 μm. c AV fluorescence intensity 15 min after induction of scramblase in cells treated with GFR inhibitors (Control, n = 16; Afatinib (afat), n = 7; Imatinib (Imat), n = 17). The ratio of the cytoplasmic region (Cyto) to the plasma membrane (PM) was calculated. Analysis was performed by one-way ANOVA followed by Dunnett’s post-hoc test (Compared to Control, PM Afat, ns = 0.9189; PM Imat, ns = 0.5111; Cyto Afat, *** P = 0.0002; Cyto Imat, ** P = 0.0063; F Cyto /F PM ratio Afat, **** P < 0.0001; F Cyto /F PM ratio Imat, * P = 0.0392). d Protein gels of co-immunoprecipitates performed with anti-HA or IgG in cells expressing TMEM16E-GFP, PDGFR-Hax3, and EGFR-Hax3. The input represents 5% of the lysate used for immunoprecipitation (IP) ( n = 3). The result of co-immunoprecipitation with anti-HA, TMEM16E- GFP was detected with PDGFR-HAx3 and EGFR-HAx3. The summary histogram indicates an interaction of PDGFRxHA3 and EGFR-HAx3 with TMEM16E-GFP. Analysis was performed by one-way ANOVA followed by Dunnett’s post-hoc test (Compared to only cells expressing TMEM16E, with PDGFR, ** P = 0.0026; with EGFR, ** P = 0.0021). e RT-PCR determined the endogenous expression levels of EGFR and PDGFR in HEK293T cells ( n = 6). *** P < 0.001, compared with control, analyzed by Student’s t-test. f Protein gels of co-immunoprecipitates performed with anti-HA or IgG in cells expressing EGFR-HAx3, TMEM16E-GFP, E584Q-GFP, and TMEM16F-GFP. g Analysis of co-immunoprecipitation with anti-HA and TMEM16E-GFP, E584Q-GFP, and TMEM16F-GFP. The summary shows that EGFR-HAx3 interacts with TMEM16E and E584Q, but not TMEM16F ( n = 4). Analysis was performed by one-way ANOVA followed by Dunnett’s post-hoc test (Compared to cells expressing EGFR-HAx3 with TMEM16E WT, with E584Q, ns = 0.8639; with TMEM16F, **** P < 0.0001). h Protein gels of co- immunoprecipitates performed with anti-HA or IgG in cells expressing TMEM16E-GFP and HAx3-M 1 R. Co-immunoprecipitation with anti-HA was detected only with HAx3-M 1 R, but not with TMEM16E-GFP ( n = 3). Analysis of the interaction between TMEM16E-GFP with HAx3-M 1 R. ns = 0.2040, when compared with IgG, analyzed by Student’s t-test. Data are mean ± SEM.

Article Snippet: Human cDNAs of EGFR (Plasmid #133749) and PDGFR (Plasmid #136455) were purchased from Addgene, and coding regions were subcloned into a pcDNA 3.1(+) vector.

Techniques: Phospho-proteomics, Binding Assay, Fluorescence, Clinical Proteomics, Membrane, Control, Expressing, Immunoprecipitation, Reverse Transcription Polymerase Chain Reaction

Journal: Cell reports

Article Title: Time-resolved proximity labeling of protein networks associated with ligand-activated EGFR

doi: 10.1016/j.celrep.2022.110950

Figure Lengend Snippet:

Article Snippet: Gateway entry clones pDONR223-EGFR-WT (#81926) were purchased from Addgene. pLEX-305 (#41390) lentiviral backbone vector was engineered to substitute the hPKG (phosphoglycerate kinase) promoter with the CMV (human cytomegalovirus) promoter plus APEX2 tag in the C-terminus of the expression gene. pDONR223_EGFR_WT (#81926) were transferred to pLEX305_Cterm_APEX2 destination vector using Gateway LR Clonase II enzyme mix (Thermo Fisher) based on the provided protocol.

Techniques: Transduction, Recombinant, Mass Spectrometry, Sequencing, Software

Journal: Scientific Reports

Article Title: Design of immunogens to present a tumor-specific cryptic epitope

doi: 10.1038/s41598-025-94295-5

Figure Lengend Snippet: Rabbit polyclonal antibodies elicited by the epitope-peptide keyhole limpet hemocyanin (KLH) conjugate and the designed immunogens bound weakly, and in some cases not at all, to tumor cells. ( A ) FACS analysis of the final antisera from the indicated rabbits (with eliciting immunogens in parentheses) binding to tumor cell lines. None of the antisera exhibited as high a binding affinity to any of the cell lines as the monoclonal ch806 binding to the three cell lines U87MG wt EGFR , U87MG EGFRvIII , and A431. Nevertheless, small shifts in the fluorescence distributions—indicative of some binding—were observed for some pairs of cell lines and antisera, as indicated by the asterisks. In particular, the antisera from rabbit 2759 and 2842 exhibited binding to the same three cell lines as ch806. The red lines are the binding data for the monoclonal antibody ch806 or the antisera, the green lines are those for human IgG1 control (Biolegend, Catalog No. 403102), and the black lines are for the pre-immunization bleed of the corresponding animals. ( B ) For the indicated rabbits (eliciting immunogens in parentheses), characterization of the binding of purified pAbs to the tumor cell lines by an On-Cell Western assay. All pAbs demonstrated some binding to the cell line expressing EGFRvIII mutant, suggesting that they targeted an epitope that was more exposed in this mutant. All pAbs elicited by the designed immunogens, however, showed binding only at high concentrations, consistent with their low ELISA titers against the epitope peptide. Insets (in which the y-axis is also normalized fluorescence): Ch806 was able to compete against the binding of the polyclonal antibodies from rabbits 2759, 2749, and 2842 to U87MG wt EGFR cells, suggesting that these polyclonal antibodies contain antibody molecules that bind to the same EGFR epitope on the tumor cells as the ch806 antibody. The rabbit pAb concentrations in these competition experiments were 67 nM (blue) and 0 nM (gray). The inhibition of binding by the ch806 antibody was only partial even at high ch806 concentrations, suggesting that the pAbs may have recognized a partial segment of the ch806-epitope that is inaccessible in normal EGFR conformations.

Article Snippet: Stable cell lines of U87MG wt EGFR and U87MG EGFRvIII were produced by Genscript under a research contract.

Techniques: Binding Assay, Fluorescence, Control, Purification, Western Blot, Expressing, Mutagenesis, Enzyme-linked Immunosorbent Assay, Inhibition