Journal: Molecules

Article Title: Casticin Inhibits A375.S2 Human Melanoma Cell Migration/Invasion through Downregulating NF-κB and Matrix Metalloproteinase-2 and -1

doi: 10.3390/molecules21030384

Figure Lengend Snippet: Casticin affect the levels of associated proteins in migration and invasion of A375.S2 cells. Samples were obtained from nuclear extract preparation or A375.S2 cells (1 × 10 6 cells/dish) were kept in 10 cm dish and were treated with 0, 100, 125, 150, 175 and 200 nM of casticin for 24 h. Each total cell lysate was prepared for western blotting assay as described in the Materials and Methods Section and after blocking with 5% nonfat skim milk, the membrane was probed with primary antibodies for SOS1, Ras, p-ERK 1/2and p-MEK 1/2 ( A ), p-c-jun, c-jun, p-EGFR and MMP-1 ( B ).

Article Snippet: Primary antibodies against MMP-1 and -2, NF-κB p65, SOS1, Ras, p-ERK 1/2, p-MEK 1/2, p-c-jun, c-jun and p-EGFR and secondary antibodies were obtained from Cell Signaling Technology, Inc. (Beverly, MA, USA).

Techniques: Migration, Western Blot, Blocking Assay, Membrane

Journal: Cancers

Article Title: Lactate Oxidase Disrupts Lactate-Activated RAS and PI3K Oncogenic Signaling.

doi: 10.3390/cancers16162817

Figure Lengend Snippet: Figure 7. Model of lactate and LOX regulation of the HCAR1-RAS/PI3K signaling cascades. TME lactate activates HCAR1, which induces the assembly of a protein complex that includes the receptor itself, GRB2, SOS1, RAS, GAB1, p85, and p110β. Subsequently, the RAS and PI3K pathways are acti- vated and transduce the lactate-triggered signals, via phosphorylated ERK1/2 and AKT, respectively, to the downstream transcription factors that further mediate the transcription of the genes involved in promoting the signaling and cancer cell survival and growth (left panel). Introducing LOX to the TME depletes lactate and renders HCAR1 inactive for triggering the assembly of the RAS and PI3K signaling complex. The disassembled HCAR1-associated RAS and PI3K signaling proteins are targeted for degradation by proteasomes (right panel). TME, tumor microenvironment; PM, plasma membrane; TF, transcription factor; the criss cross lines in the right panel represent removal or loss of interaction or signaling.

Article Snippet: Antibodies for HCAR (Abcepta, CA, USA), GRB2 (Santa Cruz Biotechnology, TX, USA), SOS1 (Proteintech, IL, USA), KRAS (Santa Cruz Biotechnology, TX, USA), GAB1 (Novus Biologicals, CO, USA), p85 (Proteintech, IL, USA), p110β (Cell Signaling Technology, MA, USA), p110α (Boster Bio, CA, USA), pERK1/2 (Santa Cruz Biotechnology, TX, USA), ERK1/2 (Santa Cruz Biotechnology, TX, USA), AKT (Cell Signaling Technology, MA, USA), pAKTS473 (Cell Signaling Technology, MA, USA), pAKTT308 (Cell Signaling Technology, MA, USA), MCT1 (Santa Cruz Biotechnology, TX, USA), MCT4 (Santa Cruz Biotechnology, TX, USA), and Actin (Thermo Scientific, MA, USA) were used in this study.

Techniques: Clinical Proteomics, Membrane

Journal: Molecular cancer research : MCR

Article Title: Identification and characterization of oncogenic SOS1 mutations in lung adenocarcinoma

doi: 10.1158/1541-7786.MCR-18-0316

Figure Lengend Snippet: A: SOS1 mutations found in oncogene-negative lung adenocarcinomas and Noonan Syndrome.

Article Snippet: Antibodies: Antibodies against SOS1 (12409), vinculin (4650), MEK 1/2 (4694), p-MEK 1/2 (9121), ERK 1/2 (9107), p-ERK (4370), AKT1 (2967), and p-AKT (4058) were purchased from Cell Signaling Technology.

Techniques:

Journal: Molecular cancer research : MCR

Article Title: Identification and characterization of oncogenic SOS1 mutations in lung adenocarcinoma

doi: 10.1158/1541-7786.MCR-18-0316

Figure Lengend Snippet: A: Western blot showing RAS-GTP pulldown in serum-starved SOS1 wildtype and N233Y mutant cells, together with total RAS control. Levels of MEK, p-MEK, ERK, and p-ERK are also shown, together with SOS1 and beta-actin controls.

Article Snippet: Antibodies: Antibodies against SOS1 (12409), vinculin (4650), MEK 1/2 (4694), p-MEK 1/2 (9121), ERK 1/2 (9107), p-ERK (4370), AKT1 (2967), and p-AKT (4058) were purchased from Cell Signaling Technology.

Techniques: Western Blot, Mutagenesis, Control

Journal: Molecular cancer research : MCR

Article Title: Identification and characterization of oncogenic SOS1 mutations in lung adenocarcinoma

doi: 10.1158/1541-7786.MCR-18-0316

Figure Lengend Snippet: A: RNA sequencing was performed on three biological replicates of vector-control, SOS1 wildtype, SOS1 N233Y, and KRAS G12V NIH-3T3 cells. Normalized read counts were used to compute the distance between the samples based on Pearson correlation coefficient, and complete linkage was used to create clusters for the dendrogram.

Article Snippet: Antibodies: Antibodies against SOS1 (12409), vinculin (4650), MEK 1/2 (4694), p-MEK 1/2 (9121), ERK 1/2 (9107), p-ERK (4370), AKT1 (2967), and p-AKT (4058) were purchased from Cell Signaling Technology.

Techniques: RNA Sequencing, Plasmid Preparation, Control

Journal: Molecular cancer research : MCR

Article Title: Identification and characterization of oncogenic SOS1 mutations in lung adenocarcinoma

doi: 10.1158/1541-7786.MCR-18-0316

Figure Lengend Snippet: A: Model of SOS1 domain organization and interactions with partner signaling molecules. Second-site mutations were generated at the catalytic CDC25 (F929A), allosteric activating REM (W729E), and putative RacGEF (351LHYFELL357→351IIIRDII357) domains. Color scheme matches Figure 1A. Diagrams based on (22).

Article Snippet: Antibodies: Antibodies against SOS1 (12409), vinculin (4650), MEK 1/2 (4694), p-MEK 1/2 (9121), ERK 1/2 (9107), p-ERK (4370), AKT1 (2967), and p-AKT (4058) were purchased from Cell Signaling Technology.

Techniques: Generated

Journal: Molecular cancer research : MCR

Article Title: Identification and characterization of oncogenic SOS1 mutations in lung adenocarcinoma

doi: 10.1158/1541-7786.MCR-18-0316

Figure Lengend Snippet: A: SOS1 dependency scores from shRNA knockdown in AML cell lines were ranked (data from (33)). Cell lines with scores <=−2 are considered dependent on SOS1. OCI-AML5 has a SOS1 N233Y mutation and is labeled in red. OCI-AML3 has an NRAS Q61L mutation and is third from top.

Article Snippet: Antibodies: Antibodies against SOS1 (12409), vinculin (4650), MEK 1/2 (4694), p-MEK 1/2 (9121), ERK 1/2 (9107), p-ERK (4370), AKT1 (2967), and p-AKT (4058) were purchased from Cell Signaling Technology.

Techniques: shRNA, Knockdown, Mutagenesis, Labeling

Journal: PLoS ONE

Article Title: DOK3 Negatively Regulates LPS Responses and Endotoxin Tolerance

doi: 10.1371/journal.pone.0039967

Figure Lengend Snippet: BMM were stimulated with 1 µg/ml LPS for indicated time and cell lysates were immunoprecipitated with (A) anti-DOK3 antibody or (B) anti-Grb2 antibody followed by immunoblotting for DOK3, Grb2, and SOS1. As a control, isotype matched antibody was used for immunoprecipitation (Con Ig). Protein levels were quantified using Image J. (C) Wild type and DOK3 −/− BMM were stimulated with 1 µg/ml LPS for indicated time and immunoblotted for SOS1 and GAPDH. (D) BMM were treated with NH 4 CL or PBS (control) and were stimulated with 1 µg/ml LPS and immunoblotted for SOS1 and GAPDH. Data are representative of three independent experiments.

Article Snippet: The following antibodies were used: DOK1, DOK2, DOK3, Cbl-b, SHIP1 and SOS1 (Santa Cruz Biotechnology); GST, ERK, PERK, Grb2, IRAK-M, SOCS1, IκBα, and cleaved caspase3 (Cell Signaling Technology); GAPDH (Abcam); ubiquitin (Covance); 4G10-argarose conjugate (phosphotyrosine) and Flag M2 (Upstate Biotechnology); fluorescently-conjugated secondary antibodies (Invitrogen), PE-conjugated anti-TNFα (EBioscience).

Techniques: Immunoprecipitation, Western Blot, Control

Journal: PLoS ONE

Article Title: DOK3 Negatively Regulates LPS Responses and Endotoxin Tolerance

doi: 10.1371/journal.pone.0039967

Figure Lengend Snippet: (A) BMM were pretreated with 1 µg/ml LPS for 18 hours, rested in fresh media for 2 hours and re-stimulated along with naïve cells with 1 µg/ml LPS for indicated time and cell lysates were immunoblotted for DOK3, pERK, ERK, and GAPDH. (B) Tolerant BMM were stimulated with 1 µg/ml LPS and cell lysates were immunoprecipitated with anti-DOK3 antibody and immunoblotted for Grb2 and DOK3. (C) Tolerant wild type and DOK3-deficient BMM were re-stimulated with 1 µg/ml LPS and lysates immunoblotted for SOS1 and JNK (loading control). Data are representative of three independent experiments.

Article Snippet: The following antibodies were used: DOK1, DOK2, DOK3, Cbl-b, SHIP1 and SOS1 (Santa Cruz Biotechnology); GST, ERK, PERK, Grb2, IRAK-M, SOCS1, IκBα, and cleaved caspase3 (Cell Signaling Technology); GAPDH (Abcam); ubiquitin (Covance); 4G10-argarose conjugate (phosphotyrosine) and Flag M2 (Upstate Biotechnology); fluorescently-conjugated secondary antibodies (Invitrogen), PE-conjugated anti-TNFα (EBioscience).

Techniques: Immunoprecipitation, Control

Journal: PLoS ONE

Article Title: DOK3 Negatively Regulates LPS Responses and Endotoxin Tolerance

doi: 10.1371/journal.pone.0039967

Figure Lengend Snippet: In naïve cells, DOK3 associates with Grb2 and SOS1 constitutively. Upon LPS stimulation, DOK3 becomes ubiquitinated, possibly by Cbl-b, and degraded, thereby releasing Grb2. SOS1 is also degraded in a DOK3-dependent manner thus limiting ERK activation. In LPS-induced tolerant cells, DOK3 and SOS1 expression remains stable during repeated LPS challenge. DOK3 may limit ERK activation during tolerance by binding to and sequestering Grb2 and SOS1. Mediators of tolerance including IRAK-M, SHIP1, and SOCS1 are upregulated.

Article Snippet: The following antibodies were used: DOK1, DOK2, DOK3, Cbl-b, SHIP1 and SOS1 (Santa Cruz Biotechnology); GST, ERK, PERK, Grb2, IRAK-M, SOCS1, IκBα, and cleaved caspase3 (Cell Signaling Technology); GAPDH (Abcam); ubiquitin (Covance); 4G10-argarose conjugate (phosphotyrosine) and Flag M2 (Upstate Biotechnology); fluorescently-conjugated secondary antibodies (Invitrogen), PE-conjugated anti-TNFα (EBioscience).

Techniques: Activation Assay, Expressing, Binding Assay

Journal: EMBO Reports

Article Title: Phase‐separated foci of EML4‐ALK facilitate signalling and depend upon an active kinase conformation

doi: 10.15252/embr.202153693

Figure Lengend Snippet: A, B H3122 and H2228 cells were stained for anti‐ALK (green), anti‐GRB2 (red), anti‐SOS1 (red), anti‐pC‐KIT Y721 (red), anti‐PI3K p85β or anti‐pPLCγ2 Y759 and DAPI (blue). Scale bars, 10 μm; magnified views of a selected area are shown. C HEK293 cells transfected with YFP‐EM4L‐ALK V1 WT or V3 WT for 48 h and treated with 5% or 10% 1,6‐hexanediol. Representative still images after 5 min (taken from time‐lapse movies) are shown of cells treated with 10% 1,6‐hexanediol or DMSO. Scale bars, 10 μm; the arrowheads indicate cytoplasmic foci. D Box plot showing the number of cytoplasmic foci in DMSO and 5% or 10% Hexanediol (5 min). Data represent 5–10 counts from three independent experiments ( n = 3). Error bar represents SD of three biological replicates. **** P < 0.0001 in comparison with YFP‐EML4‐ALK V3 WT DMSO by one‐way ANOVA. E Representative Western blots of HEK293 transfected EML4‐ALK V1 WT and V3 WT for 48 h. Cells were treated with either 5% or 10% 1,6‐hexanediol for 5 min. DMSO was used as a control. Lysates were analysed for the phosphorylation and expression of the indicated antibodies. GAPDH was used as a loading control. Data representative of n = 2 experiments.

Article Snippet: Primary antibodies were against, α‐tubulin mouse (1:1,000; Sigma), α‐tubulin rabbit (1:800; 18251; Abcam), GFP (1:1,000; 6556; Abcam), GFP (1:1,000; sc‐9996; Santa Cruz Biotechnology), ALK rabbit (D5F3) (1:100; CST), ALK (31F12) mouse (1:100; CST), GRB2 rabbit (1:1,000; PA1‐10033; Invitrogen), GRB2 (1:100) mouse (C7; Santa Cruz Biotechnology), SOS1 mouse (1:100; MCA2887; Bio‐Rad;), PLCγ2 (Y759) rabbit (1:100; AP0785; ABclonal), PI3K p85β mouse (1:100; 1B180967; Abcam), c‐KIT (Y721) (1:100; 44‐494G; Thermo Fisher Scientific).

Techniques: Staining, Transfection, Comparison, Western Blot, Control, Phospho-proteomics, Expressing

Journal: EMBO Reports

Article Title: Phase‐separated foci of EML4‐ALK facilitate signalling and depend upon an active kinase conformation

doi: 10.15252/embr.202153693

Figure Lengend Snippet: A, B Inducible Beas2B V1 and V3 cells were stained with anti‐ALK (green), anti‐GRB2 (red), anti‐SOS1 (red), anti‐pC‐KIT Y721 (red), anti‐PI3K p85β or anti‐pPLCγ2 Y759 and DAPI (blue). Scale bars, 10 μm; magnified views of a selected area are shown. C–G Intensity profiles showing colocalisation between endogenous GRB2, SOS1, pC‐KIT Y721 , PI3K p85β or pPLCγ2 Y759 and ALK staining in different cell lines. R (Pearson’s correlation coefficient) measures the correlation between the indicated proteins and ALK signals. Pearson’s measurements are from 20 foci of 10–20 cells for each antibody combination. Data in all whisker plots represent counts from at least 10 cells, n = 3. The central dashed band indicates the minimum Pearson R value (0.5) required for colocalisation. All whisker boxplots indicate the minimum and maximum Pearson R values of each cell line. * P < 0.5, ** P < 0.01, *** P < 0.001, **** P < 0.0001 in comparison of each cell line by one‐way ANOVA.

Article Snippet: Primary antibodies were against, α‐tubulin mouse (1:1,000; Sigma), α‐tubulin rabbit (1:800; 18251; Abcam), GFP (1:1,000; 6556; Abcam), GFP (1:1,000; sc‐9996; Santa Cruz Biotechnology), ALK rabbit (D5F3) (1:100; CST), ALK (31F12) mouse (1:100; CST), GRB2 rabbit (1:1,000; PA1‐10033; Invitrogen), GRB2 (1:100) mouse (C7; Santa Cruz Biotechnology), SOS1 mouse (1:100; MCA2887; Bio‐Rad;), PLCγ2 (Y759) rabbit (1:100; AP0785; ABclonal), PI3K p85β mouse (1:100; 1B180967; Abcam), c‐KIT (Y721) (1:100; 44‐494G; Thermo Fisher Scientific).

Techniques: Staining, Whisker Assay, Comparison

Journal: EMBO Reports

Article Title: Phase‐separated foci of EML4‐ALK facilitate signalling and depend upon an active kinase conformation

doi: 10.15252/embr.202153693

Figure Lengend Snippet: Antibodies used for immunofluorescence (IF) and Western blotting (WB) and dilutions.

Article Snippet: Primary antibodies were against, α‐tubulin mouse (1:1,000; Sigma), α‐tubulin rabbit (1:800; 18251; Abcam), GFP (1:1,000; 6556; Abcam), GFP (1:1,000; sc‐9996; Santa Cruz Biotechnology), ALK rabbit (D5F3) (1:100; CST), ALK (31F12) mouse (1:100; CST), GRB2 rabbit (1:1,000; PA1‐10033; Invitrogen), GRB2 (1:100) mouse (C7; Santa Cruz Biotechnology), SOS1 mouse (1:100; MCA2887; Bio‐Rad;), PLCγ2 (Y759) rabbit (1:100; AP0785; ABclonal), PI3K p85β mouse (1:100; 1B180967; Abcam), c‐KIT (Y721) (1:100; 44‐494G; Thermo Fisher Scientific).

Techniques: Immunofluorescence, Western Blot

Journal: Developmental cell

Article Title: Twist1 Activation in Muscle Progenitor Cells Causes Muscle Loss Akin to Cancer Cachexia

doi: 10.1016/j.devcel.2018.05.026

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: REAGENTS or RESOURCES SOURCE IDENTIFIER Antibodies Atrogin1/Fbx32 Abcam ab168372 BrdU Cell Signaling 52925 Cleaved caspase 3 Cell Signaling 9661 Cytokeratin 19 Abcam ab52625 Dystrophin Abcam ab15277 elF3-f Abcam ab64177 MHC Abcam ab71808 Myostatin Abcam ab71808 Myostatin R&D Systems AF788 MuRF1 Cell Signaling 4305 Myc tag Cell Signaling 22765 Smad4 Santa Cruz sc-7966 Smad2 Cell Signaling 5339 pSmad2 Cell Signaling 3108 Twist1 Santa Cruz sc-81417 Pax7 Abcam Ab92317 Chemicals, Peptides, and Recombinant Proteins Activin A R&D Systems 338-AC-050 bFGF Sigma-Aldrich F3685 Collagenase D Sigma-Aldrich 11088858001 Collagenase type 1 Sigma-Aldrich SCR103 Dispase II Sigma-Aldrich 4942078001 D-luciferin Perkin Elmer 122799 JQ1 Sigma-Aldrich SML-1524 Myostatin R&D Systems 788-GB-010 puromycin Sigma-Aldrich P9620 Tamoxifen Sigma-Aldrich T5648 Critical Commercial Assays Activin A ELISA kit R&D Systems DAC00B SURVEYOR Mutation Detection Kit Integrated DNA Technologies 706025 Recombinant DNA Adenovirus-CMV.Cre (Ad.Cre) Baylor College Ad5-CMV-Cre Atrogin1-Luc This study N/A Mstn-Luc This study N/A MuRF1-Luc This study N/A pBABE-puro-mTwist1 Addgene 1783 pGIPZ-sh.Activin Dharmacon EG16323 pGIPZ-sh.control Dharmacon RHS4346 LentiCISPRv2 Addgene 52961 pSpCas9(BB)-2A-Puro (PX459) Addgene 62988 Experimental Models: Cell Lines B16 ATCC CRL-6322 Suit2 ATCC CRL-1687 MiaPaca2 ATCC CRL-1420 CT-26 ATCC CRL-2638 KP2 This study N/A KP1 This study N/A LLC ATCC CRL-1642 Experimental Models: Organisms/Strains CAG-Cre ERT2 Jackson Lab. 004682 CAG-Loxp-CAT-Loxp-Twist1 Jackson Lab. 018543 CAG-Loxp-Stop-Loxp-Luciferase NCI 01XAC Loxp-Stop-Loxp-Kras G12D NCI 01XJ6 NOD/SCID Jackson Lab. 001303 p16Ink4A-Luciferase Dr. Sharpless Burd et al., 2013 Pax7.Cre Jackson Lab. 010530 Pax7.Cre ERT2 Jackson Lab. 012476 Pdx1.Cre NCI 01XL5 Prrx1.Cre Jackson Lab. 005584 MCK.Cre Jackson Lab. 006475 Trp53.Loxp/Loxp Jackson Lab. 008462 Twist1.Loxp/Loxp Dr. Xu Baylor College of Medicine Open in a separate window KEY RESOURCES TABLE CONTACT FOR REAGENT AND RESOURCE SHARING Further information and requests for reagents may be directed to and will be fulfilled by the Lead Contact Azeddine Atfi, rf.mresni@ifta.eniiddeza ; ude.cmu@iftaa EXPERIMENTAL MODEL AND SUBJECT DETAILS Mice All animal experiments were approved by the Institutional Animal Care and Use Committee (IACUC) of the University of Mississippi Medical Center.

Techniques: Recombinant, Enzyme-linked Immunosorbent Assay, Mutagenesis

Journal: Cancer Research

Article Title: Suppression of MicroRNA-9 by Mutant EGFR Signaling Upregulates FOXP1 to Enhance Glioblastoma Tumorigenicity

doi: 10.1158/0008-5472.can-13-2117

Figure Lengend Snippet: Figure 3. The Ras/PI3K/AKT axis is required for miR-9 repression. A, miR-9 expression was determined in cells with mutant DEGFR alleles bearing tyrosine to phenylalanine substitutions. The U87DY5 mutant with impaired binding to Ras shows upregulation of miR-9 (, P 0.005). B, SOS1 silencing upregulates miR-9 in U87DEGFR cells (, P 0.004). C, H-Ras G12V suppresses miR-9 (, P 0.006). D, treatment of U87DEGFR cells with a Raf inhibitor does not modulate miR-9 (left). D, introduction of wild-type PTEN (, P 0.004) or dead kinase AKT (DKAKT; , P 0.04) relieved the suppression of miR-9 in U87DEGFR cells (middle and right).

Article Snippet: SOS1 siRNAs and control siRNAswere obtained fromSigmaAldrich and Santa Cruz Biotechnology.

Techniques: Expressing, Mutagenesis, Binding Assay