Journal: Stem cells (Dayton, Ohio)

Article Title: Neural stem cells secreting anti-HER2 antibody improve survival in a preclinical model of HER2 overexpressing breast cancer brain metastases

doi: 10.1002/stem.2109

Figure Lengend Snippet: Inhibition of growth and HER2 signaling by HER2Ab-NSCs cells. (A) Inhibition in growth of BT474 cells using supernatant of HER2Ab-NSCs. (B) Co-Culture assay using BT474 cells with either vector control or HER2Ab-NSCs cells. (C-D) Inhibition of PI3K-AKT signaling using purified anti-HER2Ab released by NSC. Right panel in C and D demonstrates significant decrease in relative densitometric units. Trastuzumab was used as positive control in A, C and D. The experiments were repeated three times. * indicates p<0.05 and ** indicates p<0.01.

Article Snippet: ELISA plates (Corning, Pittsburg, PA) were coated overnight with 2μg/mL of recombinant human HER2 Fc chimera (R&D Systems, Minneapolis, MN).

Techniques: Inhibition, Co-culture Assay, Plasmid Preparation, Control, Purification, Positive Control

Journal: Stem cells (Dayton, Ohio)

Article Title: Neural stem cells secreting anti-HER2 antibody improve survival in a preclinical model of HER2 overexpressing breast cancer brain metastases

doi: 10.1002/stem.2109

Figure Lengend Snippet: Binding of anti-HER2Ab to HER2 overexpressing breast cancer cells. (A) BT474Br were stained with trastuzumab as a positive control (B-F) MCF 7, MDA-MB-361, BT474Br, ZR-75-30 and SKBR3 cells stained with anti-HER2Ab secreted by NSC. The experiments were repeated two times.

Article Snippet: ELISA plates (Corning, Pittsburg, PA) were coated overnight with 2μg/mL of recombinant human HER2 Fc chimera (R&D Systems, Minneapolis, MN).

Techniques: Binding Assay, Staining, Positive Control

Journal: International journal of molecular sciences

Article Title: Tumor Site-Specific Cleavage Improves the Antitumor Efficacy of Antibody-Drug Conjugates.

doi: 10.3390/ijms241311011

Figure Lengend Snippet: Figure 2. Characterization of the conjugates. (A) SDS—PAGE of the purified ZHER2-MMAE, Herceptin-MMAE, and Fc-U-ZHER2-MMAE. Lane 1, uncoupled raw proteins; Lane 2, conjugates. (B) Western blot analysis of ZHER2-MMAE, Herceptin-MMAE, and Fc-U-ZHER2-MMAE using HRP- labeled mouse anti-IgG Fc antibody, HRP-labeled mouse anti-His antibody, or HRP-labeled mouse anti-MMAE antibody. (C) ELISA assays of the binding ability of the conjugates with HER2 receptor. The human HER2 protein was coated on the plates and incubated with different concentrations of Herceptin-MMAE, ZHER2-MMAE or Fc-U-ZHER2-MMAE at 37 ◦C for 2 h, and then analyzed with mouse anti-MMAE monoclonal antibody by ELISA. Data are expressed as the mean ± SEM (n = 3). (D) EC50 data calculated with GraphPad Prism 5.0 software.

Article Snippet: Briefly, microtiter plates were coated with human HER2 protein (ACRO biosystems, Cat # HE2-H521y, Beijing, China) in PBS overnight at 4 ◦C.

Techniques: SDS Page, Western Blot, Labeling, Enzyme-linked Immunosorbent Assay, Binding Assay, Incubation, Software

Journal: Nature immunology

Article Title: St3gal1 and βII-spectrin pathways control CAR-T cell migration to target tumor sites

doi: 10.1038/s41590-023-01498-x

Figure Lengend Snippet: A. Gating strategy for detection of hHER2-CAR-T cells. A median of > 90% of mouse CD8 T cells transduced with the hHER2-CAR retrovirus were positive for the transgene as judged by flow cytometry analysis. B. B16F10 cells were transfected with mammalian human HER2 construct with lipofectamine. Cells were grown in the presence of G418 and sorted twice to generate single cell clones. C. CAR-T-mediated B16-HER2 cell killing assay. Apoptotic cells were stained for Annexin V and analyzed by flow cytometry. D. Whole mouse lung before (left) and after (right) CUBIC clearing.

Article Snippet: Biotinylated goat anti-rat IgG (H+L) was purchased from Vector Lab. Recombinant human ErbB2/Her2 Fc chimera protein and human ErbB2/Her2-Alexa Fluor-594 were obtained from R&D systems.

Techniques: In Vivo, Transduction, Flow Cytometry, Transfection, Construct, Clone Assay, Staining

Journal: Nature immunology

Article Title: St3gal1 and βII-spectrin pathways control CAR-T cell migration to target tumor sites

doi: 10.1038/s41590-023-01498-x

Figure Lengend Snippet: A. Generation of GFP-ROSAβII-spectrin mouse model (Rosa26tm(CAG-LSL-Sptbn1-IRES-GFP)). Mouse Sptbn1 cDNA was inserted into the CAG-STOP-GFP-Rosa targeting vector, CTV, between a floxed Stop cassette and the internal ribosome entry site (IRES) followed by the enhanced Green Fluorescent Protein gene (eGFP). Transcription is under control of the CAG promoter. The targeting vector contained Rosa26 homology arms (1 kb 5′ and 3.8 kb 3′), so that the entire loxP-stop-loxP-Tmc2-IRES-GFP transcriptional cassette was inserted into the first intron of Rosa26 gene on chromosome 6. B & C. Flow cytometry analysis of IFNγ and TNFα expression in hHER2-CAR transfected T cells from GFP-ROSAβII-spectrin mouse (TβII-spectrin) after treated with PBS or Tat-Cre Recombinase. Cells were co-cultured with B16-HER2 cells. Data represent mean ± SEM. n = 9. D. CAR-mediated B16-HER2 cell killing assay with TβII-spectrin cells after treated with PBS or Tat-Cre Recombinase. Cell death was stained for NucSpot. Data represent mean ± SEM. n = 9. E. Activated CD4+ T cell (OT-II) migration on ICAM-1 coated plates ± CXCL12. Cells were treated with PTx (6 hr) or Gallein (30 min) where indicated. Data were collected from 2 independent experiments (n = 2, 17–34 individual cells per mouse). Data represent mean ± SEM Statistical analyses were performed using one-way ANOVA with Bonferroni post-test. *P = 0.007. F. The pie charts depict the proportion of CD4 T cells distributed in the tumour, blood, LN/spleen, or lung/liver 72 h post-injection. G. Expression levels of βII-spectrin and St3gal1 in human CD4 and CD8 T cells (before and after activation). Loading control: β-actin. Representative western blot images from three independent experiments are shown. H. Expression levels of βII-spectrin in human CD8 memory T cells (CD8+CD45RO+CD45RA–CD56–CD57–). Loading control: β-actin. Representative western blot images from three independent experiments are shown.

Article Snippet: Biotinylated goat anti-rat IgG (H+L) was purchased from Vector Lab. Recombinant human ErbB2/Her2 Fc chimera protein and human ErbB2/Her2-Alexa Fluor-594 were obtained from R&D systems.

Techniques: Expressing, Plasmid Preparation, Control, Flow Cytometry, Transfection, Cell Culture, Staining, Migration, Injection, Activation Assay, Western Blot

Journal: bioRxiv

Article Title: LegoBody: facile generation of bispecific and multi-specific antibodies

doi: 10.1101/2019.12.25.888586

Figure Lengend Snippet: ( A ) The scheme to produce bispecific antibodies. ( B ) Two bispecific antibodies, Ipili-Dara-KIH and Ipili-Her-KIH, were generated, both of which show a predominant component with a molecular weight of 150 kDa. A small fraction of contamination, likely caused by incorrectly paired homodimers, can be observed at the half size of an antibody (labeled with ‘*’). Under reducing condition, separated heavy and light chains, ∼50 kDa and ∼25 kDa respectively, were observed. ( C, D ) Bispecific Ipili-Dara-KIH binding to CTLA4-Fc ( C ) and CD38 ( D ), in comparison to the original monoclonal antibodies. ( E, F ) Bispecific Ipili-Her-KIH binding to CTLA4-Fc ( E ) and ErbB2-Fc ( F ), in comparison to the original monoclonal antibodies.

Article Snippet: Extracellular domain Fc fusions for human CTLA4 (CTLA4-Fc) and ErbB2 (ErbB2-Fc) were purchased from Abcam (ab180054 and ab168896).

Techniques: Generated, Molecular Weight, Labeling, Binding Assay, Comparison, Bioprocessing

Journal: Cancer medicine

Article Title: Protein lysine methyltransferase SMYD3 is involved in tumorigenesis through regulation of HER2 homodimerization.

doi: 10.1002/cam4.1099

Figure Lengend Snippet: Figure 2. Knockdown of SMYD3 attenuates HER2 activity. (A and B) Effects of SMYD3 knockdown on HER2 phosphorylation levels in ZR-75-1 cells (A) and MCF7 cells (B). Cells were transfected with one control siRNA (siNC) or either of two SMYD3 siRNAs (#1 and #2). After incubation for 96 h, cell extracts were fractionated into cytoplasmic and nuclear proteins, then samples were immunoblotted with anti-phospho HER2 (Tyr 1248) (#2247), anti-HER2 (29D8), anti-SMYD3 (D2Q4V), anti-ACTB (#4967), and anti-Histone H3 (ab1791). (C) The known three-dimensional structure of the ECD of HER2. Only part of domain I (blue) and domain II (orange) is shown. The side-chain amino group of Lys 175 in domain I makes a hydrogen bond with the backbone carbonyl group of Gly 223 in domain II. The drawing was prepared from the Protein Data Bank (entry code, 3WLW)[33] using Molecular Operating Environment (MOE), 2015.10 (Chemical Computing Group Inc.).

Article Snippet: Recombinant GST- HER2 (H00002064- P01, Novus biologicals, Littleton, CO) was incubated with SMYD3 enzyme and 2 μCi S- adenosyl- l- [methyl- [3]H]- methionine (SAM; PerkinElmer, Branchburg, NJ) in a mixture of methylase activity buffer (50 mmol/L Tris- HCl at pH 8.8, 10 mmol/L dithiothreitol (DTT), and 10 mmol/L MgCl2), for 3 h at 30°C.

Techniques: Knockdown, Activity Assay, Phospho-proteomics, Transfection, Control, Incubation

Journal: Cancer medicine

Article Title: Protein lysine methyltransferase SMYD3 is involved in tumorigenesis through regulation of HER2 homodimerization.

doi: 10.1002/cam4.1099

Figure Lengend Snippet: Figure 3. SMYD3-mediated methylation enhances the formation of HER2 homodimer. (A) HeLa cells were transfected with FLAG-HER2 and HA- HER2, with Mock vector or SMYD3-expressing vector. After 24 h of incubation, cells were treated with 0 or 100 ng/mL of EGF. Cell extracts were immunoprecipitated with anti-FLAG® M2 affinity gel, and immunoblotted with anti-HA (Y-11), anti-FLAG (F7425), anti-HER2 (29D8), anti-EGFR (D38B1), anti-SMYD3 (D2Q4V), and anti-ACTB (#4967). (B and C) 293T cells were transfected with HA-HER2-WT, and FLAG-HER2-WT or FLAG-HER2- K175A in the presence of SMYD3 expression vector and incubated for 48 h. Cell lysates were immunoprecipitated with anti-HA-agarose. (B) or anti- FLAG® M2 affinity gel. (C), then immunoblotted with anti-FLAG (F7425), anti-HA (Y-11), anti-SMYD3 (D2Q4V), and anti-ACTB (#4967). (D) 293T cells were cotransfected with FLAG-HER2-WT or FLAG-HER2-K175A, and Mock vector or SMYD3-expressing vector. After 48 h of incubation, cell lysates were immunoprecipitated with anti-FLAG® M2 affinity gel and immunoblotted with anti-FLAG (F7425), anti-phospho HER2 (Tyr 1248) (#2247), anti- SMYD3 (D2Q4V), and anti-ACTB (#4967). The signal intensities of phosphorylated HER2 were quantified, and normalized by each FLAG level.

Article Snippet: Recombinant GST- HER2 (H00002064- P01, Novus biologicals, Littleton, CO) was incubated with SMYD3 enzyme and 2 μCi S- adenosyl- l- [methyl- [3]H]- methionine (SAM; PerkinElmer, Branchburg, NJ) in a mixture of methylase activity buffer (50 mmol/L Tris- HCl at pH 8.8, 10 mmol/L dithiothreitol (DTT), and 10 mmol/L MgCl2), for 3 h at 30°C.

Techniques: Methylation, Transfection, Plasmid Preparation, Expressing, Incubation, Immunoprecipitation

Journal: Cancer medicine

Article Title: Protein lysine methyltransferase SMYD3 is involved in tumorigenesis through regulation of HER2 homodimerization.

doi: 10.1002/cam4.1099

Figure Lengend Snippet: Figure 4. Effect of SMYD3-mediated HER2 methylation on downstream pathways. (A) HeLa cells were transfected with FLAG-HER2-WT or FLAG- HER2-K175A, and incubated for 48 h. Cell lysates were immunoblotted with anti-AKT (C67E7), anti-phospho AKT (Ser 473) (587F11), anti-PLCγ1 (D9H10, anti-phospho PLCγ1 (Tyr 783) (#2821), anti-ERK1/2 (#9102), anti-phospho ERK1/2 (Thr202/Tyr204) (D13.14.4E), anti-FLAG (F7425), anti- SMYD3 (D2Q4V), and anti-ACTB (#4967). (B) The schema of the effect of SMYD3-mediated methylation on HER2 function.

Article Snippet: Recombinant GST- HER2 (H00002064- P01, Novus biologicals, Littleton, CO) was incubated with SMYD3 enzyme and 2 μCi S- adenosyl- l- [methyl- [3]H]- methionine (SAM; PerkinElmer, Branchburg, NJ) in a mixture of methylase activity buffer (50 mmol/L Tris- HCl at pH 8.8, 10 mmol/L dithiothreitol (DTT), and 10 mmol/L MgCl2), for 3 h at 30°C.

Techniques: Methylation, Transfection, Incubation