Journal: Research Square

Article Title: Post-fast refeeding enhances intestinal stem cell-mediated regeneration and tumourigenesis through mTORC1-dependent polyamine synthesis

doi: 10.21203/rs.3.rs-2320717/v1

Figure Lengend Snippet:

Article Snippet: Linsitinib (OSI-906) , Med Chem Express , HY-10191.

Techniques: Recombinant, Plasmid Preparation, Western Blot, SYBR Green Assay, RNA Sequencing, Software, RNAscope

Journal: OncoTargets and Therapy

Article Title:

Mitofusin1 Is a Major Mediator in Glucose-Induced Epithelial-to-Mesenchymal Transition in Lung Adenocarcinoma Cells

doi: 10.2147/ott.s238714

Figure Lengend Snippet: Figure 5 High glucose led to the induction of autophagy in the LAD cell line through MFN1. (A and B) Expression of LC3B-II, BECN-1 and SQSTM1 in A549 cells from the NG+NC, HG+NC, and HG+siMFN1 groups. (C) Cells were transfected with the eGFP-mRFP-LC3 plasmid and exposed to different concentrations of glucose for 24 h. Yellow and red dots refer to autolysosomes and autophagosomes respectively. Scale bar = 50 µm. Data shown are mean ± SEM. *P < 0.05, **P < 0.01 vs NG+NC group. #P < 0.05, ##P < 0.01 vs HG+NC group (n = 6). Abbreviations: LAD, lung adenocarcinoma; NC, non-targeted control; NG, normal glucose; HG, high glucose. MFN1, mitofusin1; siMFN1, small interfering RNA of MFN1; LC3B, microtubule-associated proteins 1A/1B light chain 3B; BECN, beclin-1; SQSTM, sequestosome 1; eGFP, enhanced green fluorescent protein; mRFP, monomer red fluorescent protein.

Article Snippet: Materials Antibodies against SQSTM1 (PB0458, 1:400) was obtained from Boster Biological Technology Co. Ltd. Antibody against MFN1 (ab107129), LC3B (ab48394), Pink (ab23707), Parkin (ab77924) and Snail (ab53519) were purchased from Abcam.

Techniques: Expressing, Transfection, Plasmid Preparation, Control, Small Interfering RNA

Journal: Molecular Oncology

Article Title: Activation of the EGFR / PI3K / AKT pathway limits the efficacy of trametinib treatment in head and neck cancer

doi: 10.1002/1878-0261.13500

Figure Lengend Snippet: Epidermal growth factor receptor (EGFR) limits trametinib efficacy by hyperactivating the phosphatidylinositol‐4,5‐bisphosphate 3‐kinase/protein kinase B (PI3K/AKT) pathway. (A) 4‐day proliferation assay testing trametinib (50 n m ) efficacy in shControl and shEGFR CAL33 and HSC3 cell lines. Error bars indicate SEM. Data represent a representative experiment from three independent experiments. (B) (top) Tumor volume of the shControl or shEGFR CAL33 and HSC3 cell‐derived xenografts injected to NSG mice subsequently treated daily with trametinib (0.5 mg·kg −1 i.p.) or vehicle ( n = 5). Error bars indicate SEM. (bottom) change in tumor volume from first to last day of treatment. (C) Western blot for the indicated proteins following 24 h of 20 n m trametinib treatment in shControl and shEGFR‐3 CAL33 and HSC3 cell lines. Numbers indicate the fold change in protein level normalized to Actin. Data represent a representative experiment from three independent experiments. (D) Western blot for the indicated proteins following 24 h of 20 n m trametinib treatment with or without 5 μ m EGFR inhibitor erlotinib, in CAL33 and HSC3 head and neck squamous cell carcinoma cell lines. Numbers indicate the fold change in protein level normalized to Actin. Data represent a representative experiment from three independent experiments. Statistical significance was calculated using the unpaired t ‐test (** P < 0.01, *** P < 0.001, **** P < 0.0001).

Article Snippet: Erlotinib (HY‐50896) was purchased from MedChemExpress, Monmouth Junction, NJ, USA.

Techniques: Proliferation Assay, Derivative Assay, Injection, Western Blot

Journal: Cell research

Article Title: Lysosomal EGFR acts as a Rheb-GEF independent of its kinase activity to activate mTORC1.

doi: 10.1038/s41422-025-01110-x

Figure Lengend Snippet: Fig. 1 Lysosomal EGFR is crucial for mTORC1 activation. a, b Quantification of the p-T389-S6K1/S6K1 (a) and p-S473-AKT/AKT (b) ratios in NSCLC patients tested by IHC. Data are presented as means ± SD. Two-tailed unpaired t-test. c Afatinib causes much stronger inhibition of mTORC1 activation than erlotinib, although both are capable of suppressing EGFR tyrosine kinase activity. PC9 and HCC827 cells were treated with erlotinib or afatinib at the indicated dose for 12 h and analyzed by western blotting. d LY3000328 or Dyngo-4a impairs the activation of mTORC1 in cells harboring mutant EGFR. PC9, HCC827, or NCI-H1975 cells were treated with DMSO, 50 μM LY3000328 for 24 h, or 50 μM Dyngo-4a for 2 h, and analyzed by western blotting.

Article Snippet: Reagents were obtained from the following sources: antibodies against phospho-Y1068 EGFR (2234S), EGFR (4267 L), TSC2 (4308S), Rheb (13879S), mTOR (2983S), phospho-T389 S6K1 (9234S), S6K1 (9202S), phospho-S473 AKT (4060S), phospho-Thr202/Tyr204-p44/42 MAPK (Erk1/2) (4370S), 4EBP1 (9644S), Phospho-4E-BP1 (Thr37/46) (2855S), β-actin (4970S), GFP (2956S), FLAG (14793S), HA (3724S and 2367S), V5 (13202S), and LAMP1 (9091S) from Cell Signaling Technology; an antibody against EGFR (AF231SP) from Bio-Techne includes R&D Systems; an antibody against GAPDH (FD0063) from Fudebio-tech; an antibody against EGFR (GTX628887) from GeneTex; an antibody against AKT (10176-2-AP) and normal rabbit IgG (30000-0-AP) from Proteintech; an antibody against Rheb (H00006009M01) from Abnova; antibodies against ERK1/2 (AF1051) and Alexa Fluor647 (A0468) from Beyotime; antibodies conjugated with Alexa Fluor-594 (A-11005 and A-11012), Alexa Fluor-488 (A-21202 and A-21206), Alexa Fluor-647 (A32849) and Hoechst 33342 (62249) from Invitrogen; horseradish-peroxidase (HRP)-conjugated (W401B and W402B) antibodies from Promega; anti-FLAG affinity gel (B23012) from Bimake; anti-V5 agarose affinity gel antibody produced in mouse (A7345), monoclonal antiHA-agarose antibody produced in mouse (A2095), protein A agarose (P3476), GTPγS (G8634), and GDP (G7127) from Sigma‒Aldrich; animal-free recombinant human EGF (96-AF-100-15) from PeproTech; Dyngo-4a (S7163), erlotinib HCl (S1023), afatinib (S1011), and MK-2206 2HCl (S1078) from Selleck; LY3000328 (HY-15533) from MCE; RhoGEF Exchange Assay Kit (BK100) from Cytoskeleton; an active Rheb-GTP kit (26910) from NewEastBio; antifade mounting medium (P0128M) from Beyotime.

Techniques: Activation Assay, Two Tailed Test, Inhibition, Activity Assay, Western Blot, Mutagenesis

Journal: Cell research

Article Title: Lysosomal EGFR acts as a Rheb-GEF independent of its kinase activity to activate mTORC1.

doi: 10.1038/s41422-025-01110-x

Figure Lengend Snippet: Fig. 2 EGFR-TKD directly binds Rheb. a Co-localization of endogenous mutant EGFR with Rheb and LAMP1 (top) or mTOR (bottom). Immunofluorescence analysis of endogenous Rheb (green), EGFR (pink) and LAMP1 (top) or mTOR (bottom) (red) in EGFR-mutated cells using super-resolution SIM. Scale bar, 1 μm (enlarged view, 0.1 μm). b Afatinib inhibits the interaction between EGFR and Rheb in cells. PC9 cells treated with or without 25 nM erlotinib or afatinib for 12 h were subjected to immunoprecipitation and analyzed by western blotting. c The interaction between EGFR and Rheb is independent of EGFR kinase activity. HEK-293T cells stably expressing empty vector, HA-tagged EGFR WT or KD were subjected to immunoprecipitation and analyzed by western blotting. d Afatinib disrupts the EGFR–Rheb interaction in vitro. Purified EGFR-TKD was incubated with GST or GST-tagged Rheb1–169 (Rheb, unless specified) with or without 100 μM erlotinib or 100 μM afatinib, precipitated with GST beads, and subjected to SDS-PAGE analysis. Coomassie blue staining is shown.

Article Snippet: Reagents were obtained from the following sources: antibodies against phospho-Y1068 EGFR (2234S), EGFR (4267 L), TSC2 (4308S), Rheb (13879S), mTOR (2983S), phospho-T389 S6K1 (9234S), S6K1 (9202S), phospho-S473 AKT (4060S), phospho-Thr202/Tyr204-p44/42 MAPK (Erk1/2) (4370S), 4EBP1 (9644S), Phospho-4E-BP1 (Thr37/46) (2855S), β-actin (4970S), GFP (2956S), FLAG (14793S), HA (3724S and 2367S), V5 (13202S), and LAMP1 (9091S) from Cell Signaling Technology; an antibody against EGFR (AF231SP) from Bio-Techne includes R&D Systems; an antibody against GAPDH (FD0063) from Fudebio-tech; an antibody against EGFR (GTX628887) from GeneTex; an antibody against AKT (10176-2-AP) and normal rabbit IgG (30000-0-AP) from Proteintech; an antibody against Rheb (H00006009M01) from Abnova; antibodies against ERK1/2 (AF1051) and Alexa Fluor647 (A0468) from Beyotime; antibodies conjugated with Alexa Fluor-594 (A-11005 and A-11012), Alexa Fluor-488 (A-21202 and A-21206), Alexa Fluor-647 (A32849) and Hoechst 33342 (62249) from Invitrogen; horseradish-peroxidase (HRP)-conjugated (W401B and W402B) antibodies from Promega; anti-FLAG affinity gel (B23012) from Bimake; anti-V5 agarose affinity gel antibody produced in mouse (A7345), monoclonal antiHA-agarose antibody produced in mouse (A2095), protein A agarose (P3476), GTPγS (G8634), and GDP (G7127) from Sigma‒Aldrich; animal-free recombinant human EGF (96-AF-100-15) from PeproTech; Dyngo-4a (S7163), erlotinib HCl (S1023), afatinib (S1011), and MK-2206 2HCl (S1078) from Selleck; LY3000328 (HY-15533) from MCE; RhoGEF Exchange Assay Kit (BK100) from Cytoskeleton; an active Rheb-GTP kit (26910) from NewEastBio; antifade mounting medium (P0128M) from Beyotime.

Techniques: Mutagenesis, Immunoprecipitation, Western Blot, Activity Assay, Stable Transfection, Expressing, Plasmid Preparation, In Vitro, Incubation, SDS Page, Staining

Journal: Cell research

Article Title: Lysosomal EGFR acts as a Rheb-GEF independent of its kinase activity to activate mTORC1.

doi: 10.1038/s41422-025-01110-x

Figure Lengend Snippet: Fig. 3 EGFR is a GEF for Rheb. a Afatinib decreases the level of GTP-bound Rheb in PC9 and HCC827 cells. PC9 and HCC827 cells treated with or without 25 nM erlotinib or 25 nM afatinib for 12 h, were subjected to immunoprecipitation using Rheb-GTP agarose and analyzed by western blotting. b The Rheb-D60V mutant preferentially interacts with endogenous WT EGFR. HEK-293T cells were transfected with empty vector, 3× FLAG-Rheb-WT, -D60V, or -Q64L as indicated, subjected to immunoprecipitation, and analyzed by western blotting. c EDTA increases the interaction between EGFR and Rheb at endogenous levels. HeLa cells were lysed in the absence or presence of EDTA, subjected to immunoprecipitation, and analyzed by western blotting. d EGFR preferentially interacts with nucleotide-free or GDP-bound Rheb in vitro. Purified EGFR-TKD was incubated with GST, nucleotide-free GST-Rheb, GST-Rheb with GDP, or GST-Rheb with GTP as indicated, and then precipitated with GST beads and subjected to SDS-PAGE analysis. Coomassie blue staining is shown. e Top, the reconstitution peaks in the size-exclusion chromatography analysis of the purified EGFR-TKD-WT and Rheb-D60V complex. The reconstitution peak for the complex is shown by a dotted line and observed at 10.22 mL. Bottom, SDS-PAGE analysis of each reconstitution sample. The peak for the complex of EGFR-TKD and Rheb is indicated. f EGFR induces Rheb nucleotide exchange from the GDP-bound to the GTP-bound state. A guanine nucleotide exchange assay was performed in vitro using purified EGFR-TKD and Rheb. The relative fluorescence reflects the guanine nucleotide exchange activity. The initial fluorescence intensity was set to 1. Human RhoGEF Dbs (hDbs) and RhoA were used as positive controls. Curves are representative of three independent experiments. Data are presented as means ± SEM. g EGFR induces Rheb nucleotide exchange in a dose-dependent manner. An in vitro guanine nucleotide exchange assay was performed using purified Rheb and a concentration gradient of EGFR-TKD-WT as indicated. h LAMP2-V5-HER2-ICD, LAMP2-V5-IGF1R-ICD, or LAMP2-V5-c-MET-ICD failed to activate mTORC1. HEK-293T cells stably expressing LAMP2-V5-EGFR-TKD-WT, LAMP2-V5-HER2-ICD, LAMP2-V5-IGF1R-ICD, or LAMP2-V5-c-MET-ICD were serum-starved for 24 h and analyzed by western blotting.

Article Snippet: Reagents were obtained from the following sources: antibodies against phospho-Y1068 EGFR (2234S), EGFR (4267 L), TSC2 (4308S), Rheb (13879S), mTOR (2983S), phospho-T389 S6K1 (9234S), S6K1 (9202S), phospho-S473 AKT (4060S), phospho-Thr202/Tyr204-p44/42 MAPK (Erk1/2) (4370S), 4EBP1 (9644S), Phospho-4E-BP1 (Thr37/46) (2855S), β-actin (4970S), GFP (2956S), FLAG (14793S), HA (3724S and 2367S), V5 (13202S), and LAMP1 (9091S) from Cell Signaling Technology; an antibody against EGFR (AF231SP) from Bio-Techne includes R&D Systems; an antibody against GAPDH (FD0063) from Fudebio-tech; an antibody against EGFR (GTX628887) from GeneTex; an antibody against AKT (10176-2-AP) and normal rabbit IgG (30000-0-AP) from Proteintech; an antibody against Rheb (H00006009M01) from Abnova; antibodies against ERK1/2 (AF1051) and Alexa Fluor647 (A0468) from Beyotime; antibodies conjugated with Alexa Fluor-594 (A-11005 and A-11012), Alexa Fluor-488 (A-21202 and A-21206), Alexa Fluor-647 (A32849) and Hoechst 33342 (62249) from Invitrogen; horseradish-peroxidase (HRP)-conjugated (W401B and W402B) antibodies from Promega; anti-FLAG affinity gel (B23012) from Bimake; anti-V5 agarose affinity gel antibody produced in mouse (A7345), monoclonal antiHA-agarose antibody produced in mouse (A2095), protein A agarose (P3476), GTPγS (G8634), and GDP (G7127) from Sigma‒Aldrich; animal-free recombinant human EGF (96-AF-100-15) from PeproTech; Dyngo-4a (S7163), erlotinib HCl (S1023), afatinib (S1011), and MK-2206 2HCl (S1078) from Selleck; LY3000328 (HY-15533) from MCE; RhoGEF Exchange Assay Kit (BK100) from Cytoskeleton; an active Rheb-GTP kit (26910) from NewEastBio; antifade mounting medium (P0128M) from Beyotime.

Techniques: Immunoprecipitation, Western Blot, Mutagenesis, Transfection, Plasmid Preparation, In Vitro, Incubation, SDS Page, Staining, Size-exclusion Chromatography, Activity Assay, Concentration Assay, Stable Transfection, Expressing

Journal: Cell research

Article Title: Lysosomal EGFR acts as a Rheb-GEF independent of its kinase activity to activate mTORC1.

doi: 10.1038/s41422-025-01110-x

Figure Lengend Snippet: Fig. 4 EGFR-Glu804 is a potential glutamic finger indispensable for GEF activity. a AlphaFold2-Multimer prediction of EGFR-TKD complexed with Rheb (amino acids 1–184). Glu804 of EGFR is shown as a stick-and-ball model and is in close proximity to the nucleotide- binding pocket of Rheb (left), which is similar to the cytohesin-2–Arf1 complex structure (PDB: 1R8Q). GDP Guanosine diphosphate, G3P Guanosine-3’-monophosphate-5’-diphosphate. b EGFR-E804K does not bind Rheb in cells. HEK-293T cells stably expressing SFB-Rheb were transfected with HA-tagged EGFR-WT, -KD, or -E804K, then subjected to immunoprecipitation using anti-HA antibody, and analyzed by western blotting. c EGFR-E804K does not directly bind Rheb in vitro. Purified EGFR-TKD-WT or -E804K was incubated with GST or GST-tagged Rheb as indicated, precipitated with GST beads, and subjected to SDS-PAGE. Coomassie blue staining is shown. d EGFR-E804K failed to induce Rheb nucleotide exchange. An in vitro guanine nucleotide exchange assay was performed using purified EGFR-TKD-WT or -E804K and Rheb as indicated and analyzed as described in Fig. 3f. e LAMP2-V5-EGFR-TKD-WT or -KD, but not LAMP2-V5-EGFR-TKD-E804K, triggers the activation of mTORC1. HEK-293T cells stably expressing the indicated plasmids were serum-starved for 24 h and analyzed by western blotting. f Afatinib, but not erlotinib, impairs the EGFR-mediated Rheb nucleotide exchange. An in vitro guanine nucleotide exchange assay was performed using purified Rheb and EGFR-TKD-WT with the addition of erlotinib or afatinib as indicated and analyzed as described in Fig. 3f. g Afatinib inhibits mTORC1 activation in TSC2-deficient MEFs. WT and TSC2-deficient MEFs were serum-starved, treated with MK-2206 (10 μM), erlotinib (10 μM), or afatinib (10 μM) for 24 h, stimulated with 100 ng/mL EGF for 30 min and analyzed by western blotting. h Afatinib decreases the level of GTP- bound Rheb. TSC2-deficient MEFs treated with or without 10 μM erlotinib or 10 μM afatinib as indicated for 24 h were subjected to immunoprecipitation using Rheb-GTP agarose and analyzed by western blotting. i MK-2206 does not inhibit mTORC1 activation in PC9 and HCC827 cells. PC9 and HCC827 cells were treated with MK-2206 (1 μM), erlotinib (25 nM), or afatinib (25 nM) for 12 h as indicated, and analyzed by western blotting.

Article Snippet: Reagents were obtained from the following sources: antibodies against phospho-Y1068 EGFR (2234S), EGFR (4267 L), TSC2 (4308S), Rheb (13879S), mTOR (2983S), phospho-T389 S6K1 (9234S), S6K1 (9202S), phospho-S473 AKT (4060S), phospho-Thr202/Tyr204-p44/42 MAPK (Erk1/2) (4370S), 4EBP1 (9644S), Phospho-4E-BP1 (Thr37/46) (2855S), β-actin (4970S), GFP (2956S), FLAG (14793S), HA (3724S and 2367S), V5 (13202S), and LAMP1 (9091S) from Cell Signaling Technology; an antibody against EGFR (AF231SP) from Bio-Techne includes R&D Systems; an antibody against GAPDH (FD0063) from Fudebio-tech; an antibody against EGFR (GTX628887) from GeneTex; an antibody against AKT (10176-2-AP) and normal rabbit IgG (30000-0-AP) from Proteintech; an antibody against Rheb (H00006009M01) from Abnova; antibodies against ERK1/2 (AF1051) and Alexa Fluor647 (A0468) from Beyotime; antibodies conjugated with Alexa Fluor-594 (A-11005 and A-11012), Alexa Fluor-488 (A-21202 and A-21206), Alexa Fluor-647 (A32849) and Hoechst 33342 (62249) from Invitrogen; horseradish-peroxidase (HRP)-conjugated (W401B and W402B) antibodies from Promega; anti-FLAG affinity gel (B23012) from Bimake; anti-V5 agarose affinity gel antibody produced in mouse (A7345), monoclonal antiHA-agarose antibody produced in mouse (A2095), protein A agarose (P3476), GTPγS (G8634), and GDP (G7127) from Sigma‒Aldrich; animal-free recombinant human EGF (96-AF-100-15) from PeproTech; Dyngo-4a (S7163), erlotinib HCl (S1023), afatinib (S1011), and MK-2206 2HCl (S1078) from Selleck; LY3000328 (HY-15533) from MCE; RhoGEF Exchange Assay Kit (BK100) from Cytoskeleton; an active Rheb-GTP kit (26910) from NewEastBio; antifade mounting medium (P0128M) from Beyotime.

Techniques: Activity Assay, Binding Assay, Stable Transfection, Expressing, Transfection, Immunoprecipitation, Western Blot, In Vitro, Incubation, SDS Page, Staining, Activation Assay

Journal: Cell research

Article Title: Lysosomal EGFR acts as a Rheb-GEF independent of its kinase activity to activate mTORC1.

doi: 10.1038/s41422-025-01110-x

Figure Lengend Snippet: Fig. 5 Aberrant Rheb-GEF activity of EGFR impairs mTORC1 activation and cell growth. a mTORC1 activation was inhibited in EGFR-E804K knock-in PC9 cells. EGFR-E804K knock-in PC9 cells were analyzed by western blotting. b The EGFR–Rheb interaction was disrupted in EGFR- E804K knock-in cells. EGFR-E804K knock-in PC9 cell lysates were subjected to immunoprecipitation using IgG or anti-EGFR antibody and analyzed by western blotting. c The level of GTP-bound Rheb was decreased in EGFR-E804K knock-in cells. EGFR-E804K knock-in PC9 cell lysates were subjected to immunoprecipitation using Rheb-GTP agarose and analyzed by western blotting. d Cell vitality was decreased in EGFR-E804K knock-in cells. CCK-8 assays were performed to examine the proliferation in parental and EGFR-E804K knock-in PC9 cells (n = 4). Two-way ANOVA was used. e, f Cell proliferation was diminished in EGFR-E804K knock-in cells. Colony formation (e) and EdU (f) assays were used to evaluate the proliferation ability of parental and EGFR-E804K knock-in PC9 cells (n = 3). Two-tailed unpaired t-test. g EGFR-E804K knock-in PC9 cells exhibit reduced sensitivity to afatinib compared to parental cells. Parental and EGFR-E804K knock-in PC9 cells were treated with indicated concentrations of erlotinib or afatinib for 72 h (n = 3). The IC50 values were as follows: 20.59 nM and 0.97 nM for parental cells treated with erlotinib and afatinib, respectively; 25.89 nM and 17.44 nM for EGFR-E804K knock-in cells treated with erlotinib and afatinib, respectively. h Representative parental and EGFR-E804K knock-in PC9 tumors surgically removed. i EGFR-E804K knock-in effectively inhibited tumor growth in vivo. Tumor volumes in mice were measured and calculated at specified time intervals following implantation. Two-tailed unpaired t-test. j EGFR-E804K knock-in tumors weighed less than those derived from parental cells. Five weeks after implantation, the mice were euthanized, and the tumors were excised and weighed. Two-tailed unpaired t-test was used.

Article Snippet: Reagents were obtained from the following sources: antibodies against phospho-Y1068 EGFR (2234S), EGFR (4267 L), TSC2 (4308S), Rheb (13879S), mTOR (2983S), phospho-T389 S6K1 (9234S), S6K1 (9202S), phospho-S473 AKT (4060S), phospho-Thr202/Tyr204-p44/42 MAPK (Erk1/2) (4370S), 4EBP1 (9644S), Phospho-4E-BP1 (Thr37/46) (2855S), β-actin (4970S), GFP (2956S), FLAG (14793S), HA (3724S and 2367S), V5 (13202S), and LAMP1 (9091S) from Cell Signaling Technology; an antibody against EGFR (AF231SP) from Bio-Techne includes R&D Systems; an antibody against GAPDH (FD0063) from Fudebio-tech; an antibody against EGFR (GTX628887) from GeneTex; an antibody against AKT (10176-2-AP) and normal rabbit IgG (30000-0-AP) from Proteintech; an antibody against Rheb (H00006009M01) from Abnova; antibodies against ERK1/2 (AF1051) and Alexa Fluor647 (A0468) from Beyotime; antibodies conjugated with Alexa Fluor-594 (A-11005 and A-11012), Alexa Fluor-488 (A-21202 and A-21206), Alexa Fluor-647 (A32849) and Hoechst 33342 (62249) from Invitrogen; horseradish-peroxidase (HRP)-conjugated (W401B and W402B) antibodies from Promega; anti-FLAG affinity gel (B23012) from Bimake; anti-V5 agarose affinity gel antibody produced in mouse (A7345), monoclonal antiHA-agarose antibody produced in mouse (A2095), protein A agarose (P3476), GTPγS (G8634), and GDP (G7127) from Sigma‒Aldrich; animal-free recombinant human EGF (96-AF-100-15) from PeproTech; Dyngo-4a (S7163), erlotinib HCl (S1023), afatinib (S1011), and MK-2206 2HCl (S1078) from Selleck; LY3000328 (HY-15533) from MCE; RhoGEF Exchange Assay Kit (BK100) from Cytoskeleton; an active Rheb-GTP kit (26910) from NewEastBio; antifade mounting medium (P0128M) from Beyotime.

Techniques: Activity Assay, Activation Assay, Knock-In, Western Blot, Immunoprecipitation, CCK-8 Assay, Two Tailed Test, In Vivo, Derivative Assay

Journal: Cell research

Article Title: Lysosomal EGFR acts as a Rheb-GEF independent of its kinase activity to activate mTORC1.

doi: 10.1038/s41422-025-01110-x

Figure Lengend Snippet: Fig. 6 BIEGi-1 suppresses cancer cell growth. a Chemical structure of BIEGi-1. b Structural model of EGFR-TKD complexed with BIEGi-1. EGFR- TKD is shown in surface representation and BIEGi-1 in ball-and-stick. The ATP-binding site (orange) and predicted Rheb-GEF interface (blue) of EGFR are indicated. c BIEGi-1 impairs the EGFR-mediated Rheb nucleotide exchange. An in vitro guanine nucleotide exchange assay was performed using purified Rheb and EGFR-TKD-WT with the addition of erlotinib, afatinib or BIEGi-1 and analyzed as described in Fig. 3f. d BIEGi-1 abolishes the interaction between EGFR and Rheb in cells. PC9 cells treated with or without 25 nM BIEGi-1 for 12 h were subjected to immunoprecipitation and analyzed by western blotting. e BIEGi-1 inhibits mTORC1 activation in EGFR-mutated cells. PC9 and HCC827 cells were treated with 25 nM erlotinib, afatinib, or BIEGi-1 for 12 h as indicated, and analyzed by western blotting. f CCK8 assays of PC-9 and HCC827 cells exposed to BIEGi-1. The IC50 values were 17 nM and 20 nM for PC9 and HCC827 cells, respectively. Data points represent the means ± SD (n = 3). g BIEGi-1 and afatinib, but not erlotinib, inhibit the activation of mTORC1 induced by LAMP2-V5-EGFR-TKD. HEK-293T cells stably expressing the indicated plasmids were serum-starved, treated with 200 nM BIEGi-1, erlotinib or afatinib for 24 h, and analyzed by western blotting. WT and KD refer to LAMP2-V5-EGFR-TKD-WT and LAMP2-V5-EGFR-TKD-KD, respectively. h Model illustrating that lysosomal EGFR acts as a GEF for Rheb, with Glu804 serving as a glutamic finger to activate mTORC1. Inhibition of both GEF and kinase activities of EGFR by BIEGi-1 restricts mTORC1 activity, leading to impaired cell growth.

Article Snippet: Reagents were obtained from the following sources: antibodies against phospho-Y1068 EGFR (2234S), EGFR (4267 L), TSC2 (4308S), Rheb (13879S), mTOR (2983S), phospho-T389 S6K1 (9234S), S6K1 (9202S), phospho-S473 AKT (4060S), phospho-Thr202/Tyr204-p44/42 MAPK (Erk1/2) (4370S), 4EBP1 (9644S), Phospho-4E-BP1 (Thr37/46) (2855S), β-actin (4970S), GFP (2956S), FLAG (14793S), HA (3724S and 2367S), V5 (13202S), and LAMP1 (9091S) from Cell Signaling Technology; an antibody against EGFR (AF231SP) from Bio-Techne includes R&D Systems; an antibody against GAPDH (FD0063) from Fudebio-tech; an antibody against EGFR (GTX628887) from GeneTex; an antibody against AKT (10176-2-AP) and normal rabbit IgG (30000-0-AP) from Proteintech; an antibody against Rheb (H00006009M01) from Abnova; antibodies against ERK1/2 (AF1051) and Alexa Fluor647 (A0468) from Beyotime; antibodies conjugated with Alexa Fluor-594 (A-11005 and A-11012), Alexa Fluor-488 (A-21202 and A-21206), Alexa Fluor-647 (A32849) and Hoechst 33342 (62249) from Invitrogen; horseradish-peroxidase (HRP)-conjugated (W401B and W402B) antibodies from Promega; anti-FLAG affinity gel (B23012) from Bimake; anti-V5 agarose affinity gel antibody produced in mouse (A7345), monoclonal antiHA-agarose antibody produced in mouse (A2095), protein A agarose (P3476), GTPγS (G8634), and GDP (G7127) from Sigma‒Aldrich; animal-free recombinant human EGF (96-AF-100-15) from PeproTech; Dyngo-4a (S7163), erlotinib HCl (S1023), afatinib (S1011), and MK-2206 2HCl (S1078) from Selleck; LY3000328 (HY-15533) from MCE; RhoGEF Exchange Assay Kit (BK100) from Cytoskeleton; an active Rheb-GTP kit (26910) from NewEastBio; antifade mounting medium (P0128M) from Beyotime.

Techniques: Binding Assay, In Vitro, Immunoprecipitation, Western Blot, Activation Assay, Stable Transfection, Expressing, Inhibition, Activity Assay

Journal: Journal of Translational Medicine

Article Title: Transcriptomic insights into UTUC: role of inflammatory fibrosis and potential for personalized treatment

doi: 10.1186/s12967-023-04815-y

Figure Lengend Snippet: Tyrosine kinase inhibitors demonstrate significant potential in UTUC treatment. A Both CTRPv2 and Prism share 204 common drugs. B Risk scores for all cell lines in the CCL database, urothelial cell lines, and metastatic urothelial cell lines. C Drug screening methodology for CTRPv2 and Prism. D Results of shared expression analysis (r < -0.2, Spearman correlation) between selected drugs from CTRPv2 and risk scores. E Normalized AUC values for drugs selected by CTRPv2 in high-risk and low-risk groups. F Results of shared expression analysis (Spearman correlation, r < -0.2) between selected drugs from Prism and risk scores. G Normalized AUC values for drugs selected by Prism in high-risk and low-risk groups. H Validation of drug selection results from CTRPv2 and Prism in the CMap database, to identify more potential drugs for targeted UTUC treatment. I , J Identification of kidney fibrosis in the Aristolochic acid induced kidney disease mouse model using Masson's trichrome staining in the Erlotinib and saline groups. K Potential mode of action for tyrosine kinase inhibitors. ( p ≤ 0.05 as *, p ≤ 0.01 as **, p ≤ 0.001 as ***, p ≤ 0.0001 as ****)

Article Snippet: The mice were divided into two groups, one group was gavaged with Erlotinib (T0373, TargetMol, USA), 50 mg/kg/d, and the other group was gavaged with saline, for a duration of 2 weeks.

Techniques: Expressing, Selection, Staining, Saline