Journal: Cancers

Article Title: Drug-Induced Resistance and Phenotypic Switch in Triple-Negative Breast Cancer Can Be Controlled via Resolution and Targeting of Individualized Signaling Signatures

doi: 10.3390/cancers13195009

Figure Lengend Snippet: TNBC tissues are represented by different patient-specific signaling signatures, majority of which do not include EGFR. ( A ) Fold changes in expression levels of EGFR and pEGFR in TNBC and non-TNBC tumors are shown. Y1068 and Y1173 are EGFR phosphorylation sites; ( B ) Examples for patient-specific sets of active unbalanced processes are shown. Each sample harbors a set of 1–3 active unbalanced processes (PaSSS), represented schematically by a barcode. In each barcode active unbalanced processes are represented by black or gray squares, inactive white. Negative/positive amplitude denotes how the patients are correlated with respect to a particular process. Suggested PaSSS-based therapies appear below each barcode; ( C ) Heterogeneity index of 3 subgroups, represented by a ratio between the number of distinct PaSSSs and the number of samples in each subset, is shown for the TNBC subset of tissues, the entire set (3467 samples from 11 cancer types) and the subset of non-TNBC samples. (Abbreviations: TNBC—Triple Negative Breast Cancer, PaSSS—Patient-specific signaling signature, EGFR—Epidermal Growth Factor Receptor, VEGFR2—Vascular Endothelial Growth Factor Receptor 2, Her2—Human Epidermal growth factor Receptor 2, Src—Proto-oncogene tyrosine-protein kinase Src).

Article Snippet: The following conjugated antibodies were used: anti-p-EGFR (Y1068) (R&D Systems, Minneapolis, MN, USA, cat. no. IC3570G), anti-p-ERK2 (Thr202/Tyr204) (BioLegend, San Diego, CA, USA, cat. No. 675503), anti-p-S6 (Ser235/236) (BioLegend, cat. no. 608605), and anti-GAPDH (Santa Cruz Biotechnology, Dallas, Texas, USA, cat. no. sc-47724AF594).

Techniques: Expressing, Phospho-proteomics

Journal: Oncogene

Article Title: Akt kinase-interacting protein1, a novel therapeutic target for lung cancer with EGFR-activating and gatekeeper mutations.

doi: 10.1038/onc.2012.446

Figure Lengend Snippet: Figure 1. Aki1 expression and association to EGFR in EGFR mutant human lung cancer cell lines. (a) EGFR mutant human lung cancer cell lines (PC-9, HCC827 and H1975), EGFR wild-type human lung cancer cell lines (A549 and PC14PE6) and human lung fibroblast cell lines (MRC-5 and IMR-90) were lysed and the indicated proteins were detected by western blotting. (b) EGFR mutant lung cancer cell lines were treated with or without EGF (50 ng/ml) or IGF-1 (50 ng/ml) for 10 min. Then, cells were lysed and the indicated proteins were detected by western blotting with or without immunoprecipitation of Aki1.

Article Snippet: Oncogene (2013) 4427 – 4435 & 2013 Macmillan Publishers Limited (40D4), anti-phospho-Akt (Thr308), anti-phospho-Akt (Ser473), anti-Par-4, anti-cleaved PARP (Asp214), anti-phospho-S6 ribosomal protein (Ser235/ 236), anti-S6 ribosomal protein (5G10), anti-phospho-IGF-1 R (Tyr1131), DYKDDDDK (FLAG) tag antibody or anti-b-actin (13E5) antibodies (1:1000 dilution; Cell Signaling Technology, Danvers, MA, USA), anti-Aki1 (1:1000 dilution; Bethyl Laboratories, Montgomery, TX, USA), anti-PDK1 (1:1000 dilution; Santa Cruz Biotechnology, Santa Cruz, CA, USA), and anti-human EGFR (1 mg/ml) or anti-human IGF-1 R (0.1mg/ml) antibody (R&D Systems, Minneapolis, MN, USA).

Techniques: Expressing, Mutagenesis, Western Blot, Immunoprecipitation

Journal: Oncogene

Article Title: Akt kinase-interacting protein1, a novel therapeutic target for lung cancer with EGFR-activating and gatekeeper mutations.

doi: 10.1038/onc.2012.446

Figure Lengend Snippet: Figure 3. Comparison of efficacy between Aki1 knockdown and EGFR inhibition on cell viability. Cells were treated with Aki1-1 siRNA, EGFR siRNA, control scramble siRNA or erlotinib (1 mM). (a) After 72-h incubation, cell viability was determined by MTT assay. (b) After 24-h incubation, cells were lysed and the indicated proteins were detected by western blotting.

Article Snippet: Oncogene (2013) 4427 – 4435 & 2013 Macmillan Publishers Limited (40D4), anti-phospho-Akt (Thr308), anti-phospho-Akt (Ser473), anti-Par-4, anti-cleaved PARP (Asp214), anti-phospho-S6 ribosomal protein (Ser235/ 236), anti-S6 ribosomal protein (5G10), anti-phospho-IGF-1 R (Tyr1131), DYKDDDDK (FLAG) tag antibody or anti-b-actin (13E5) antibodies (1:1000 dilution; Cell Signaling Technology, Danvers, MA, USA), anti-Aki1 (1:1000 dilution; Bethyl Laboratories, Montgomery, TX, USA), anti-PDK1 (1:1000 dilution; Santa Cruz Biotechnology, Santa Cruz, CA, USA), and anti-human EGFR (1 mg/ml) or anti-human IGF-1 R (0.1mg/ml) antibody (R&D Systems, Minneapolis, MN, USA).

Techniques: Comparison, Knockdown, Inhibition, Control, Incubation, MTT Assay, Western Blot

Journal: Oncogene

Article Title: Akt kinase-interacting protein1, a novel therapeutic target for lung cancer with EGFR-activating and gatekeeper mutations.

doi: 10.1038/onc.2012.446

Figure Lengend Snippet: Figure 4. Therapeutic effects of Aki1 knockdown against lung cancer cells with EGFR T790M secondary mutation in vivo. H1975 cells (5 106 cells per 100 ml of PBS) were injected subcutaneously into the flanks of 5-week-old male SCID mice. After cell inoculation, 50 mg of either scramble or Aki1 siRNA complexed with invivofectamine was injected intratumorally on days 5 and 8. (a) Tumor size was measured twice a week and tumor volume was calculated as described in Materials and methods. (b) Macroscopic appearance of the tumors harvested on day 26. (c) The harvested tumors were examined for Aki1, and the inhibition of downstream signaling molecule, S6, in tumors by western blotting.

Article Snippet: Oncogene (2013) 4427 – 4435 & 2013 Macmillan Publishers Limited (40D4), anti-phospho-Akt (Thr308), anti-phospho-Akt (Ser473), anti-Par-4, anti-cleaved PARP (Asp214), anti-phospho-S6 ribosomal protein (Ser235/ 236), anti-S6 ribosomal protein (5G10), anti-phospho-IGF-1 R (Tyr1131), DYKDDDDK (FLAG) tag antibody or anti-b-actin (13E5) antibodies (1:1000 dilution; Cell Signaling Technology, Danvers, MA, USA), anti-Aki1 (1:1000 dilution; Bethyl Laboratories, Montgomery, TX, USA), anti-PDK1 (1:1000 dilution; Santa Cruz Biotechnology, Santa Cruz, CA, USA), and anti-human EGFR (1 mg/ml) or anti-human IGF-1 R (0.1mg/ml) antibody (R&D Systems, Minneapolis, MN, USA).

Techniques: Knockdown, Mutagenesis, In Vivo, Injection, Inhibition, Western Blot

Journal: Oncogene

Article Title: Akt kinase-interacting protein1, a novel therapeutic target for lung cancer with EGFR-activating and gatekeeper mutations.

doi: 10.1038/onc.2012.446

Figure Lengend Snippet: Figure 2. Effects of Aki1 siRNA on cell viability and apoptosis in EGFR mutant human lung cancer cell lines. Cells were treated with Aki1-1 or control scramble siRNA. (a) After 72-h incubation, cell viability was determined by MTT assay. (b) After 24-h incubation with control scramble siRNA (lanes 1, 3 and 5) or Aki1-1 siRNA (lanes 2, 4 and 6), cells were lysed and the indicated proteins were detected by western blotting. (c) After 48-h incubation, cell apoptosis was determined with an Annexin V-FITC Apoptosis Detection Kit I. The numbers show percentages of early apoptotic cells.

Article Snippet: Oncogene (2013) 4427 – 4435 & 2013 Macmillan Publishers Limited (40D4), anti-phospho-Akt (Thr308), anti-phospho-Akt (Ser473), anti-Par-4, anti-cleaved PARP (Asp214), anti-phospho-S6 ribosomal protein (Ser235/ 236), anti-S6 ribosomal protein (5G10), anti-phospho-IGF-1 R (Tyr1131), DYKDDDDK (FLAG) tag antibody or anti-b-actin (13E5) antibodies (1:1000 dilution; Cell Signaling Technology, Danvers, MA, USA), anti-Aki1 (1:1000 dilution; Bethyl Laboratories, Montgomery, TX, USA), anti-PDK1 (1:1000 dilution; Santa Cruz Biotechnology, Santa Cruz, CA, USA), and anti-human EGFR (1 mg/ml) or anti-human IGF-1 R (0.1mg/ml) antibody (R&D Systems, Minneapolis, MN, USA).

Techniques: Mutagenesis, Control, Incubation, MTT Assay, Western Blot

Journal: Oncogene

Article Title: Akt kinase-interacting protein1, a novel therapeutic target for lung cancer with EGFR-activating and gatekeeper mutations.

doi: 10.1038/onc.2012.446

Figure Lengend Snippet: Figure 5. Effects of Aki1 knockdown combined with new generation EGFR TKI in lung cancer with EGFR T790M secondary mutation. H1975 cells were treated with Aki1-1 or control scramble siRNA in the presence or absence of erlotinib (1 mM), CL-387, 785 (0.3 mM), BIBW2992 (0.1 mM) or WZ4002 (0.1 mM). (a) After 72-h incubation, cell viability was determined by MTT assay. *Po0.01, one-way ANOVA. (b) After 24-h incubation, cells were lysed and the indicated proteins were detected by western blotting. (c) Schema showing the role of Aki1 in cells with wild-type EGFR and mutant EGFR.

Article Snippet: Oncogene (2013) 4427 – 4435 & 2013 Macmillan Publishers Limited (40D4), anti-phospho-Akt (Thr308), anti-phospho-Akt (Ser473), anti-Par-4, anti-cleaved PARP (Asp214), anti-phospho-S6 ribosomal protein (Ser235/ 236), anti-S6 ribosomal protein (5G10), anti-phospho-IGF-1 R (Tyr1131), DYKDDDDK (FLAG) tag antibody or anti-b-actin (13E5) antibodies (1:1000 dilution; Cell Signaling Technology, Danvers, MA, USA), anti-Aki1 (1:1000 dilution; Bethyl Laboratories, Montgomery, TX, USA), anti-PDK1 (1:1000 dilution; Santa Cruz Biotechnology, Santa Cruz, CA, USA), and anti-human EGFR (1 mg/ml) or anti-human IGF-1 R (0.1mg/ml) antibody (R&D Systems, Minneapolis, MN, USA).

Techniques: Knockdown, Mutagenesis, Control, Incubation, MTT Assay, Western Blot

Journal: Oncogene

Article Title: Akt kinase-interacting protein1, a novel therapeutic target for lung cancer with EGFR-activating and gatekeeper mutations.

doi: 10.1038/onc.2012.446

Figure Lengend Snippet: Figure 6. Aki1 is frequently expressed in EGFR mutant lung cancer. Clinical specimens from EGFR mutant lung cancer patients were stained for Aki1 by immunohistochemistry. (a) A total of 56 tumor specimens with EGFR-activating mutations were obtained from 56 lung adenocarcinoma patients. Of the 56 patients, 42 were EGFR-TKI naive, 7 tumors were from patients who showed intrinsic resistance to the EGFR-TKIs, gefitinib or erlotinib. Another seven tumors were from patients who showed acquired resistance to EGFR-TKIs. Of 42 EGFR-TKI naive tumors, the presence of Aki1 protein was scored as 2 þ in 31 tumors (74%), 1 þ in 8 tumors (19%) and – in 3 tumors (7%). Aki1 protein was detected diffusely in all of seven tumors with intrinsic resistance: 2 þ in 4 (57%), 1 þ in 3 (43%). Aki1 protein was detected diffusely in all of seven tumors with acquired resistance: 2 þ in 6 (86%), 1 þ in 1 (14%). (b) Representative staining results are shown.

Article Snippet: Oncogene (2013) 4427 – 4435 & 2013 Macmillan Publishers Limited (40D4), anti-phospho-Akt (Thr308), anti-phospho-Akt (Ser473), anti-Par-4, anti-cleaved PARP (Asp214), anti-phospho-S6 ribosomal protein (Ser235/ 236), anti-S6 ribosomal protein (5G10), anti-phospho-IGF-1 R (Tyr1131), DYKDDDDK (FLAG) tag antibody or anti-b-actin (13E5) antibodies (1:1000 dilution; Cell Signaling Technology, Danvers, MA, USA), anti-Aki1 (1:1000 dilution; Bethyl Laboratories, Montgomery, TX, USA), anti-PDK1 (1:1000 dilution; Santa Cruz Biotechnology, Santa Cruz, CA, USA), and anti-human EGFR (1 mg/ml) or anti-human IGF-1 R (0.1mg/ml) antibody (R&D Systems, Minneapolis, MN, USA).

Techniques: Mutagenesis, Staining, Immunohistochemistry

Journal: Science Advances

Article Title: TET2 regulates early and late transitions in exhausted CD8 + T cell differentiation and limits CAR T cell function

doi: 10.1126/sciadv.adp9371

Figure Lengend Snippet: ( A ) Left: Schematic of TRAC (top) and TET2 (bottom) loci alongside rAAV6 KI vectors. Right: Sanger sequencing electropherogram confirming integration of TRAC and TET2 KI constructs, underlined with dashed line. ( B ) Example plots of TET2 and TRAC-CAR19 single KI or dual TET2-TRAC-CAR19 KI T cells. ( C ) Example plots of CD3 loss detected by flow in TRAC-CAR19-KI T cells. ( D and E ) Schematic of in vitro ADCC assay (D) to deplete CRISPR-edited TET2-KI T cells. Example plots and data (E) of EGFR expression on TET2-KI T cells alone or in an NK cell coculture ± cetuximab incubation, gated on CD56 − populations, n = 4. ( F ) Cumulative fold expansion of TRAC-CAR19 and TET2-TRAC-CAR19 T cells during restimulation and at day 25, arrows represent addition of irradiated K562-CD19 + target cells, n = 5. ( G ) Proportions of CD4 + versus CD8 + T cells in TRAC-CAR19 and TET2-TRAC-CAR19 T cells after stimulation, n = 7. ( H ) Example plots showing distribution of central (CCR7 + CD45RO + ) and effector (CCR7 − CD45RO + ) memory-associated markers in CD8 + CAR T cell populations after restimulation, with summary after five stimulations, n = 5. ( I ) SPICE plot showing distribution of IR coexpression in CD8 + TRAC-CAR19 and TET2-TRAC-CAR19 T cells after 1 (acute) and 5 (chronic) stimulations, n = 6. ( J ) Data shown as means ± SEM [(F) and (G)] or individual values [(E) and (H)] from independent donors. ns P > 0.05; * P < 0.05; ** P < 0.01; *** P < 0.001 by paired t test. Schematics [(A and (D)] created with BioRender.com .

Article Snippet: The following day, TET2 KI T cells were incubated with a cetuximab biosimilar (R&D Systems, no. MAB9577) at a concentration of 2000 ng/ml for 20 min. T cells were then cocultured at a 1:10 ratio with NK cells, with T cell numbers normalized to EGFR + expression.

Techniques: Sequencing, Construct, In Vitro, ADCC Assay, CRISPR, Expressing, Incubation, Irradiation

Journal: bioRxiv

Article Title: Unveiling DNA Origami Interaction Dynamics on Living Cell Surfaces by Single Particle Tracking

doi: 10.1101/2024.12.23.628980

Figure Lengend Snippet: a) Schematic on the DNA origami self-assembly approach, combining the p7560 viral scaffold, the staple strands (core staples, 18 staples with extended handles for antibody/aptamer functionalization and 6 staples with extended handles for fluorescent dye functionalization). Folding and purification of the NR via poly-ethylene glycol precipitation was confirmed by agarose gel electrophoresis. b) Schematic representation of anti-EGFR conjugation approach to a ssDNA oligonucleotide, via click chemistry, and to Cy3, via NHS-ester. A native PAGE shows further migration of the oligo conjugated antibody towards the positive pole (lane 2). c) Conjugation of the anti-EGFR antibodies or aptamers to the NRs. The agarose gel shows differences the migration height of the different NRs, confirming their correct functionalization. Legend: Sc=Scaffold, NR=Folded DNA origami nanorod, NR_18Ab= NRs functionalized with anti-EGFR antibody, and NR_18Apt= NR functionalized with anti-EGFR aptamers. d) Atomic force microscopy images of the non-functionalized NRs (upper panel) and the NRs coated with 18 antibodies (lower panel).

Article Snippet: P7560 DNA scaffold (Tilibit nanosystems, Germany), Staples and modified staples (Integrated DNA technologies Europe), PEG8000 (Sigma-Aldrich, USA), InVivoMab anti-human EGFR antibody (Bio X cell, USA), NHS ester-(PEG) 4 -DBCO (Vector Labs, USA), Cyanine3 NHS ester (Lumiprobe, USA), Amicon 10 and 100 kDa weight cut-off (Sigma-Aldrich, USA), mini-PROTEAN TGX precast gels (BIO-RAD Laboratories Inc, USA)

Techniques: Purification, Agarose Gel Electrophoresis, Conjugation Assay, Clear Native PAGE, Migration, Microscopy

Journal: bioRxiv

Article Title: Unveiling DNA Origami Interaction Dynamics on Living Cell Surfaces by Single Particle Tracking

doi: 10.1101/2024.12.23.628980

Figure Lengend Snippet: non-denaturing SDS page gel of the EGFR antibody (first lane) and the oligo conjugated EGFR antibody (lane 2-5, increasing concentration of antibody)

Article Snippet: P7560 DNA scaffold (Tilibit nanosystems, Germany), Staples and modified staples (Integrated DNA technologies Europe), PEG8000 (Sigma-Aldrich, USA), InVivoMab anti-human EGFR antibody (Bio X cell, USA), NHS ester-(PEG) 4 -DBCO (Vector Labs, USA), Cyanine3 NHS ester (Lumiprobe, USA), Amicon 10 and 100 kDa weight cut-off (Sigma-Aldrich, USA), mini-PROTEAN TGX precast gels (BIO-RAD Laboratories Inc, USA)

Techniques: SDS Page, Concentration Assay

Journal: bioRxiv

Article Title: Unveiling DNA Origami Interaction Dynamics on Living Cell Surfaces by Single Particle Tracking

doi: 10.1101/2024.12.23.628980

Figure Lengend Snippet: Immunostaining of the EGFR on Hek 293T cells (left) and the MDA MD 468 cells (right). Images were taken via confocal laser scanning microscopy.

Article Snippet: P7560 DNA scaffold (Tilibit nanosystems, Germany), Staples and modified staples (Integrated DNA technologies Europe), PEG8000 (Sigma-Aldrich, USA), InVivoMab anti-human EGFR antibody (Bio X cell, USA), NHS ester-(PEG) 4 -DBCO (Vector Labs, USA), Cyanine3 NHS ester (Lumiprobe, USA), Amicon 10 and 100 kDa weight cut-off (Sigma-Aldrich, USA), mini-PROTEAN TGX precast gels (BIO-RAD Laboratories Inc, USA)

Techniques: Immunostaining, Confocal Laser Scanning Microscopy

Journal: bioRxiv

Article Title: Unveiling DNA Origami Interaction Dynamics on Living Cell Surfaces by Single Particle Tracking

doi: 10.1101/2024.12.23.628980

Figure Lengend Snippet: Agarose gel of nanorods conjugated with 8 or 18 EGFR antibodies and 8 or 18 aptamers.

Article Snippet: P7560 DNA scaffold (Tilibit nanosystems, Germany), Staples and modified staples (Integrated DNA technologies Europe), PEG8000 (Sigma-Aldrich, USA), InVivoMab anti-human EGFR antibody (Bio X cell, USA), NHS ester-(PEG) 4 -DBCO (Vector Labs, USA), Cyanine3 NHS ester (Lumiprobe, USA), Amicon 10 and 100 kDa weight cut-off (Sigma-Aldrich, USA), mini-PROTEAN TGX precast gels (BIO-RAD Laboratories Inc, USA)

Techniques: Agarose Gel Electrophoresis