Journal: iScience

Article Title: Jchain -diphtheria toxin receptor mice allow for depletion of antibody-secreting cells and analysis of differentiation kinetics

doi: 10.1016/j.isci.2025.113946

Figure Lengend Snippet: Validation of DTR gene expression by ASCs from J-DTR mice (A) Representative flow cytometry pseudocolor plots showing gating of CD19 + CD138 -/LO B cells and CD138 HI CD267(TACI) + ASCs in total spleen or cells purified with STEMCELL Technologies Pan-B and CD138 (ASC) isolation kits. Numbers in plots indicate percentages of gated populations within total live singlets. (B) Quantification of ASC percentages in total spleen or purified cells using Pan-B and ASC isolation kits. (C–E) Relative gene expression of (C) Prdm1 , (D) Jchain , and (E) DTR ( HBEGF ) in cells purified using Pan-B and ASC isolation kits. All values are relative to the expression of Actb . (F) DTR expression normalized to Prdm1 expression in WT and J-DTR ASCs. (G) Jchain expression normalized to Prdm1 expression in WT and J-DTR ASCs. (B–G) Symbols represent individual 3–7 months old female (orange) and male (blue) mice. Data pooled from 3 independent experiments. Horizontal lines represent mean ± standard error of the mean (SEM). WT spleen, Pan-B and ASC: female n = 2, male n = 3; J-DTR spleen, Pan-B and ASC: female n = 3, male n = 3. (B) Statistics: One-way ANOVA with Dunnett’s multiple comparisons test with ASCs for each genotype set as the control column. (C–G) Statistics: Unpaired Student’s t test between genotypes or cell types (related to and ).

Article Snippet: TaqMan Human HBEGF (DTR) (Hs00961129_m1) , Thermo Fisher Scientific , Cat# 4331182.

Techniques: Biomarker Discovery, Gene Expression, Flow Cytometry, Purification, Isolation, Expressing, Control

Journal: Oncotarget

Article Title: Novel functional anti-HER3 monoclonal antibodies with potent anti-cancer effects on various human epithelial cancers

doi: 10.18632/oncotarget.27414

Figure Lengend Snippet: ( A ) Scheme for the sandwich ELISA (sELISA) with soluble HER3-GFP proteins for the selection of anti-HER3 mAbs. ( B ) An example of first screening of anti-HER3 mAbs using sELISA. ( C ) Second and third FCM screenings of anti-HER3 mAbs using HEK293 transfectants expressing GFP-fused HER family proteins. ( D ) Representative FCM histograms showing the reactivity of anti-HER3 mAb (Ab4) with human cancer cell lines.

Article Snippet: Subconfluent LS-174T cell were seeded in each well of 6-well plates (Corning Japan, Tokyo) at a destiny of 3 × 10 5 cells/well, and transfected with HER3 siRNA mixture (ERBB3HSS140802, 140813 and 176604 Stealth RNAi, each 10 pmol, Invitrogen) in 2 mL medium using Lipofectamine RNAiMAX (Invitrogen).

Techniques: Sandwich ELISA, Selection, Expressing

Journal: Oncotarget

Article Title: Novel functional anti-HER3 monoclonal antibodies with potent anti-cancer effects on various human epithelial cancers

doi: 10.18632/oncotarget.27414

Figure Lengend Snippet: ( A ) Specificity of anti-HER3 mAbs was determined by siRNA-mediated knockdown in LS-LM4 cells. Histograms were shown by black (without anti-HER3 mAbs) and red (with anti-HER3 mAbs) lines. ( B ) Specificity of anti-HER3 mAbs was determined by and CRIPR/Cas9-based knockout in SW1116 cells. Histograms were shown by red (without anti-HER3 mAbs) and blue (with anti-HER3 mAbs) lines. ( C ) Nearest germline variable region (VH and VL) segments of mAbs are shown. Usage of different germlines in CDR segments is highlighted with different colors. ( D ) Heat map from sequence homology (%) of CDR amino acids (identity) was determined using Pairwise Sequence Alignment ( https://www.ebi.ac.uk/Tools/psa/ ).

Article Snippet: Subconfluent LS-174T cell were seeded in each well of 6-well plates (Corning Japan, Tokyo) at a destiny of 3 × 10 5 cells/well, and transfected with HER3 siRNA mixture (ERBB3HSS140802, 140813 and 176604 Stealth RNAi, each 10 pmol, Invitrogen) in 2 mL medium using Lipofectamine RNAiMAX (Invitrogen).

Techniques: Knock-Out, Sequencing

Journal: Oncotarget

Article Title: Novel functional anti-HER3 monoclonal antibodies with potent anti-cancer effects on various human epithelial cancers

doi: 10.18632/oncotarget.27414

Figure Lengend Snippet: Reactivity of anti-HER3 mAb (Ab4) with human various normal, non-cancer and cancer cell lines was analyzed by FCM, and mAb binding was shown by ΔMFI.

Article Snippet: Subconfluent LS-174T cell were seeded in each well of 6-well plates (Corning Japan, Tokyo) at a destiny of 3 × 10 5 cells/well, and transfected with HER3 siRNA mixture (ERBB3HSS140802, 140813 and 176604 Stealth RNAi, each 10 pmol, Invitrogen) in 2 mL medium using Lipofectamine RNAiMAX (Invitrogen).

Techniques: Binding Assay

Journal: Oncotarget

Article Title: Novel functional anti-HER3 monoclonal antibodies with potent anti-cancer effects on various human epithelial cancers

doi: 10.18632/oncotarget.27414

Figure Lengend Snippet: ( A ) Scheme for the epitope analysis of anti-HER3 rat mAbs with HER3 transfectants. ( B ) 3D bar graph for the binding inhibition (%) by all combinations of anti-HER3 rat mAbs. ( C ) Scheme for the Patritumab binding in the presence of excess anti-HER3 rat mAbs. ( D ) Binding of Patritumab to transfectants in the presence of control rat IgG or anti-HER3 rat mAbs.

Article Snippet: Subconfluent LS-174T cell were seeded in each well of 6-well plates (Corning Japan, Tokyo) at a destiny of 3 × 10 5 cells/well, and transfected with HER3 siRNA mixture (ERBB3HSS140802, 140813 and 176604 Stealth RNAi, each 10 pmol, Invitrogen) in 2 mL medium using Lipofectamine RNAiMAX (Invitrogen).

Techniques: Binding Assay, Inhibition

Journal: Oncotarget

Article Title: Novel functional anti-HER3 monoclonal antibodies with potent anti-cancer effects on various human epithelial cancers

doi: 10.18632/oncotarget.27414

Figure Lengend Snippet: ( A ) Scheme for the internalization of HER3 by mAbs. Human cancer cells were treated with anti-HER3 mAbs at 37° C or 4° C for 1~1.5 h, and cell-surface HER3 proteins were analyzed by FCM. ( B ) Internalization of cell-surface HER3 proteins of LS-174T and T47D by anti-HER3 mAbs was shown as the internalization (%). ( C ) Scheme for the binding inhibition of mAbs to HEK293 cells expressing HER3-GFP in the presence of NRG1 (upper). Representative binding of anti-HER3 mAb in the absence (-) or presence (+) of NRG1(lower). ( D ) Histogram (left) for the binding of mAb to HEK293 cells expressing HER3-GFP in NRG1(+) or NRG1(-). Bar graph (right) shows binding inhibition of anti-HER3 mAbs with HEK293 cells expressing HER3-GFP in NRG (+).

Article Snippet: Subconfluent LS-174T cell were seeded in each well of 6-well plates (Corning Japan, Tokyo) at a destiny of 3 × 10 5 cells/well, and transfected with HER3 siRNA mixture (ERBB3HSS140802, 140813 and 176604 Stealth RNAi, each 10 pmol, Invitrogen) in 2 mL medium using Lipofectamine RNAiMAX (Invitrogen).

Techniques: Binding Assay, Inhibition, Expressing

Journal: Oncotarget

Article Title: Novel functional anti-HER3 monoclonal antibodies with potent anti-cancer effects on various human epithelial cancers

doi: 10.18632/oncotarget.27414

Figure Lengend Snippet: ( A ) Inhibition of NRG1-induced tyrosine phosphorylation of HER3 proteins by anti-HER3 mAbs in human cancer cells. ( B ) Effects of anti-HER3 mAbs on the cell growth of NRG1-treated BT474 cells. ( C ) Immunohistochemical staining of human colon carcinoma tissues with anti-HER3 mAb (Ab1). ( D ) Immunofluorescent staining of human colon cancer-derived CTOS with anti-HER3 mAbs (Ab1 and Ab3).

Article Snippet: Subconfluent LS-174T cell were seeded in each well of 6-well plates (Corning Japan, Tokyo) at a destiny of 3 × 10 5 cells/well, and transfected with HER3 siRNA mixture (ERBB3HSS140802, 140813 and 176604 Stealth RNAi, each 10 pmol, Invitrogen) in 2 mL medium using Lipofectamine RNAiMAX (Invitrogen).

Techniques: Inhibition, Immunohistochemical staining, Staining, Derivative Assay

Journal: Oncotarget

Article Title: Novel functional anti-HER3 monoclonal antibodies with potent anti-cancer effects on various human epithelial cancers

doi: 10.18632/oncotarget.27414

Figure Lengend Snippet: ( A ) PCA by the binding inhibition analyses of anti-HER3 mAbs. ( B ) PCA by the amino acid identity of CDR of anti-HER3 mAbs. ( C ) Correlation diagram about seven anti-HER3 mAbs between %CDR homology and binding inhibition (%). ( D ) Summary table showing various features of seven anti-HER3 mAbs. ( E ) In vitro ffects of anti-HER3 mAb (Ab4) or patritumab on the cell growth of MCF7 cells with or without Erlotinib. ( F ) Anti-tumor effects of anti-HER3 mAb (Ab4) or patritumab on BT474 human breast cancer cells were evaluated ( n = 4). Anti-tumor effects of anti-HER3 mAb (Ab4) on LS-174T ( G , n = 3) and LS-LM4 ( H , n = 8) human colon cancer cells were evaluated.

Article Snippet: Subconfluent LS-174T cell were seeded in each well of 6-well plates (Corning Japan, Tokyo) at a destiny of 3 × 10 5 cells/well, and transfected with HER3 siRNA mixture (ERBB3HSS140802, 140813 and 176604 Stealth RNAi, each 10 pmol, Invitrogen) in 2 mL medium using Lipofectamine RNAiMAX (Invitrogen).

Techniques: Binding Assay, Inhibition, In Vitro

Journal: iScience

Article Title: SARS-CoV-2 antigen-carrying extracellular vesicles activate T cell responses in a human immunogenicity model

doi: 10.1016/j.isci.2023.108708

Figure Lengend Snippet: Characterization of 293F cell-derived EVs carrying SARS-CoV-2 spike protein (A) Schematic of EV engineering strategy using 293F cells including SARS-CoV-2 spike expression construct (see Figure S1 ) with spike protein depicted in blue. Schematic created with Biorender.com . (B) Histogram plot of particle size distribution comparing 293F EVs and Spike EVs as determined by NTA. The number of particles falling within each size bin was normalized to total particle counts and are presented as the proportion of total particles. Data represent the mean ± standard error of the mean (SEM). N = 3 independent experiments. Statistical comparisons were carried out by unpaired, two-tailed Student’s t test where p < 0.05 would be considered significant. (C) Mean and mode particle size of 293F EVs and Spike EVs as determined by NTA. Data represent the mean ± standard error of the mean (SEM). N = 3 independent experiments. Statistical comparisons were carried out by unpaired, two-tailed Student's t test where p < 0.05 would be considered significant. n.s. indicates not significant. (D) Multiplexed bead-based profiling assay of 37 surface antigens on 293F EVs and Spike EVs confirming the presence of expected EV surface markers. Data represent the median fluorescent intensity (MFI) of APC signal (reflecting CD63-APC, CD81-APC and CD9-APC counterstaining) following the subtraction of isotype controls. N = 1 independent experiment. (E) Representative western blots characterizing the presence of SARS-CoV-2 spike protein in Spike EVs and markers known to be present in EV preparations (CD63, CD9, Flotillin, TSG101, GAPDH) as well as the absence of cell contaminant markers (GM130, Calnexin). (F) Confirmation of SARS-CoV-2 spike protein presence in Spike EVs through competitive Spike-ACE2 binding assay. Spike EVs inhibit the binding of fluorescently labeled, recombinant SARS-CoV-2 spike protein competitively, in a dose-responsive manner, while 293F EVs demonstrate little inhibition. Data are mean ± SEM. N = 2 independent experiments and 2 technical replicates per sample per run. (G) Transmission electron micrographs of unstained Spike EVs confirming expected EV morphology. SARS-CoV-2 spike protein presence on 293F Spike EVs was also visually confirmed using immunogold labeling where 10 nm gold particles are seen associating with 293F Spike EVs.

Article Snippet: Mouse Anti-Flotillin-1, Clone 18 , BD Transduction Laboratories , Cat#610820; RRID: AB_398139.

Techniques: Derivative Assay, Expressing, Construct, Two Tailed Test, Western Blot, Binding Assay, Labeling, Recombinant, Inhibition, Transmission Assay