Journal: iScience

Article Title: FAM65A, as a potential predictor of prognosis, promotes colorectal cancer progression via activating Ras/ERK/RSK signaling

doi: 10.1016/j.isci.2026.114662

Figure Lengend Snippet: FAM65A binds to Ras and activates the Ras/ERK signaling to mediate RSK activation (A) The volcano plot analysis results for the FAM65A high-expression and low-expression groups from the TCGA database were shown. (B) The KEGG and GO results were shown. (C) The GSEA results were shown. (D) GSEA on DEGs between the FAM65A high-expression group and low-expression group in the Reactome database were shown. (E) IP was performed to detect the binding of FAM65A and Ras/p-RSK. (F) IP was performed to detect the binding of Ras and FAM65A/p-RSK. (G) Immunofluorescence was performed to detect the co-localization of FAM65A and Ras. Scale bars, 20 μm. (H) Western blot analysis the Ras and p -ERK expression in FAM65A knockdown or overexpression cells. Data are presented as mean ± SEM of biologically independent experiments.

Article Snippet: p -ERK , Proteintech , Cat# 28733-1-AP; RRID: AB_2881202.

Techniques: Activation Assay, Expressing, Binding Assay, Immunofluorescence, Western Blot, Knockdown, Over Expression

Journal: iScience

Article Title: FAM65A, as a potential predictor of prognosis, promotes colorectal cancer progression via activating Ras/ERK/RSK signaling

doi: 10.1016/j.isci.2026.114662

Figure Lengend Snippet: Ras/ERK signaling activation was indispensable for FAM65A-mediated RSK activation and CRC progression (A) Western blot analysis of Ras and p -ERK expression in HCT116-FAM65A cells treated with 10 μM Abd-7, or without treatment. (B) Results from the CCK8 cell proliferation assay conducted on HCT116-FAM65A cells with and without the application of Abd-7, n = 3, ∗∗∗ p < 0.001. (C) Colony formation assay performed on HCT116-FAM65A cells treated with Abd-7 or not. (D) Quantitative analysis of the colony formation assay results, n = 3, ∗∗∗ p < 0.001. (E) Results from the EdU assay conducted on HCT116-FAM65A cells with and without the application of Abd-7. Scale bars, 100 μm. (F) Quantitative analysis of the EdU assay results, n = 3, ∗∗∗ p < 0.001. (G) Western blot analysis of Ki-67, cleaved Caspase 3, Bcl-2, and Bax expression in HCT116-FAM65A cells treated with Abd-7 or not. (H) Results from the apoptosis assay conducted on HCT116-FAM65A cells treated with Abd-7 or not. Scale bars, 50 μm. (I) Quantitative analysis of the apoptosis experiments, n = 3, ∗∗∗ p < 0.001. (J) Results from the Transwell migration assay conducted on HCT116-FAM65A cells with and without the application of Abd-7. Scale bars, 50 μm. (K) Quantitative analysis of the Transwell migration assay results, n = 3, ∗∗∗ p < 0.001. (L) Results from the wound healing assay performed on HCT116-FAM65A cells treated with Abd-7 or not. Scale bars, 50 μm. (M) Quantitative analysis of the wound healing assay results, n = 3, ∗∗∗ p < 0.001. (N) Western blot analysis the expression of EMT markers in HCT116-FAM65A cells treated with Abd-7 or not. (O) Proposed model of FAM65A in CRC progression. Data are presented as mean ± SEM of biologically independent experiments.

Article Snippet: p -ERK , Proteintech , Cat# 28733-1-AP; RRID: AB_2881202.

Techniques: Activation Assay, Western Blot, Expressing, Proliferation Assay, Colony Assay, EdU Assay, Apoptosis Assay, Transwell Migration Assay, Wound Healing Assay

Journal: iScience

Article Title: FAM65A, as a potential predictor of prognosis, promotes colorectal cancer progression via activating Ras/ERK/RSK signaling

doi: 10.1016/j.isci.2026.114662

Figure Lengend Snippet: Knockdown of FAM65A inhibits tumor progression in vivo (A) LOVO-shCtrl and LOVO-shFAM65A cells were administered into the fourth fat pad of nude mice, and the resulting tumor growth curves were subsequently generated, n = 5, ∗ p < 0.05. (B) The tumors excised from mice across various experimental groups are presented. (C) Hematoxylin and Eosin (HE) staining results of lung tissue from the different groups is displayed. (D) A quantitative analysis of metastatic lung nodules is provided, n = 5, ∗∗ p < 0.01. (E) IHC results for FAM65A, Ki-67, p -RSK, p -ERK, Ras, N-cadherin, vimentin, cleaved Caspase 3, ZO-1, and E-cadherin in tumor tissues are illustrated. (F) A quantitative analysis of the IHC results is included. Data are presented as mean ± SEM of biologically independent experiments, n = 5, ∗∗∗ p < 0.001.

Article Snippet: p -ERK , Proteintech , Cat# 28733-1-AP; RRID: AB_2881202.

Techniques: Knockdown, In Vivo, Generated, Staining

Journal: bioRxiv

Article Title: Comparative analysis of wavelength-specific UV stress granule formation

doi: 10.64898/2026.03.15.711948

Figure Lengend Snippet: Representative images of SG formation by G3BP1 and TIA1 signals in wt U2OS cells treated as indicated. Cells were either untreated, treated with 250μM As III for 1h, or exposed to 15mJ/cm 2 of UV. UV samples were harvested and processed for imaging 4 hours after exposure. Yellow arrows indicate SGs. Scale bar = 20μm.

Article Snippet: The following antibodies were used in this study; G3BP1 (Proteintech® 13057 (rabbit) and 66486 (mouse)), S6 Ribosomal protein (RPS6) (ABclonal A6058), TIA1 (Proteintech® 12133), RACK1 (Proteintech® 66940), TRAF2 (Proteintech® 26846 (rabbit) 67315 (mouse)), Geminin (Proteintech® 10802), DHX9 (Proteintech®17721), cytokeratin 8 (Invitrogen, 06318), and eIF3n (Santa Cruz, 137214).

Techniques: Imaging

Journal: bioRxiv

Article Title: Comparative analysis of wavelength-specific UV stress granule formation

doi: 10.64898/2026.03.15.711948

Figure Lengend Snippet: (A) Representative images of SG formation by G3BP1 and TIA1 signals in HaCaT cells treated as indicated. Cells were either untreated, treated with 250μM As III for 1h, or exposed to 15mJ/cm 2 of UV. UV samples were harvested and processed for imaging 4 hours after exposure. (B) Quantification of SG formation by G3BP1 and TIA1 signals in wt U2OS, HaCaT, and wt MEF cells treated as indicated. Cells were either untreated, treated with 250μM As III for 1h, or exposed to 15mJ/cm 2 of UV. UV samples were harvested and processed for imaging 4 hours after exposure. At least 250 cells and 3 fields of view were scored to calculate percent SG formation. n =3; error bars are ±SD; ns P > 0.05, ** P ≤ 0.01, **** P ≤ 0.0001 by one-way ANOVA and Dunnett’s post-hoc test. (C) Quantification of SG formation by G3BP1 and TIA1 signals in wt U2OS cells treated as indicated. Cells were either untreated, treated with 250μM As III for 1h, or exposed to 150mJ/cm 2 of UV. UV samples were harvested and processed for imaging 4 hours after exposure. At least 250 cells and 3 fields of view were scored to calculate percent SG formation. n =3; error bars are ±SD; ns P > 0.05, **** P ≤ 0.0001 by one-way ANOVA and Dunnett’s post-hoc test. Scale bar = 20μm.

Article Snippet: The following antibodies were used in this study; G3BP1 (Proteintech® 13057 (rabbit) and 66486 (mouse)), S6 Ribosomal protein (RPS6) (ABclonal A6058), TIA1 (Proteintech® 12133), RACK1 (Proteintech® 66940), TRAF2 (Proteintech® 26846 (rabbit) 67315 (mouse)), Geminin (Proteintech® 10802), DHX9 (Proteintech®17721), cytokeratin 8 (Invitrogen, 06318), and eIF3n (Santa Cruz, 137214).

Techniques: Imaging

Journal: Cell Death & Disease

Article Title: Arginine dependency in omental metastasis of epithelial ovarian cancer reveals a therapeutic vulnerability

doi: 10.1038/s41419-026-08606-3

Figure Lengend Snippet: A Scheme of L -arginine-immobilized NHS magnetic beads and the enrichment strategy for arginine-binding proteins. B Volcano plot of arginine- and leucine-binding proteins. C , D GO analysis of arginine-binding proteins in A2780 cells. E Venn diagram showing arginine-binding proteins that were upregulated in both 500 μM arginine-treated A2780 ovarian cancer cells and omentum metastases. F Relative abundance of DDX3X protein captured by arginine or leucine-coupled magnetic beads. G WB detection of DDX3X in A2780 cells with arginine treatment, or lysate from omentum metastasis, and elution after purification with L -leucine- or L -arginine-immobilized NHS magnetic beads. H Molecular docking between DDX3X (HMDB ID: HMDB0000517) and L -arginine (PubChem CID: 6322) performed using Discovery Studio. I Molecular dynamics (MD) simulation was performed using Gromacs. The backbone root mean square deviation (RMSD) of the DDX3X- L -arginine complex over the 100 ns MD simulation was shown. J Surface plasmon resonance analysis for the binding of L -arginine to DDX3X. Data were shown as the mean ± SD. The p value was calculated using an unpaired two-tailed Student’s t -test ( F ). ** p < 0.01.

Article Snippet: Permeabilized cells were then sequentially incubated with a primary rabbit anti-DDX3X antibody (Cat. No. 11115-1-AP, Proteintech, Wuhan, China) and a secondary Goat Anti-Rabbit IgG antibody (Cat. No. AS070, ABclonal, Wuhan, China).

Techniques: Magnetic Beads, Binding Assay, Purification, SPR Assay, Two Tailed Test

Journal: Cell Death & Disease

Article Title: Arginine dependency in omental metastasis of epithelial ovarian cancer reveals a therapeutic vulnerability

doi: 10.1038/s41419-026-08606-3

Figure Lengend Snippet: A Representative confocal microscopy images and immunofluorescence analysis of DDX3X in A2780 cells treated with arginine at the indicated times. Scale bars, 10 μm. B WB analyses of DDX3X, histone H3, and tubulin protein levels in cytoplasmic and nuclear fractions of A2780 cells under the indicated treatments. C Representative confocal microscopy images of DDX3X immunofluorescence in A2780 cells treated as indicated. Scale bars, 10 μm. D WB analyses of DDX3X, CRM1, histone H3, and tubulin protein levels in the cytoplasmic and nuclear fractions of A2780 cells under the indicated treatments. E Representative confocal microscopy images of Flag immunofluorescence in A2780 shDDX3X cells treated as indicated. Scale bars, 10 μm. F WB analyses of Flag, histone H3, and tubulin protein levels in the cytoplasmic and nuclear fractions of A2780 shDDX3X cells under the indicated treatments. G Quantification of the nuclear-to-cytoplasmic ratio of DDX3X fluorescence intensity in A2780 cells treated with arginine as indicated. H Quantification of the nuclear-to-cytoplasmic ratio of DDX3X fluorescence intensity in A2780 shDDX3X cells treated with arginine as indicated. I Representative images and quantification of immunohistochemical staining of DDX3X in primary tumors and omentum metastases ( n = 20). Scale bars, 50 μm. J Immunohistochemical quantification of nuclear DDX3X in primary tumors and omentum metastases ( n = 20). Scale bars,50 μm. Data were shown as the mean ± SEM. The p value was calculated using one-way ANOVA ( A , G , H ), and unpaired two-tailed Student’s t -test ( I , J ). * p < 0.05, ** p < 0.01.

Article Snippet: Permeabilized cells were then sequentially incubated with a primary rabbit anti-DDX3X antibody (Cat. No. 11115-1-AP, Proteintech, Wuhan, China) and a secondary Goat Anti-Rabbit IgG antibody (Cat. No. AS070, ABclonal, Wuhan, China).

Techniques: Confocal Microscopy, Immunofluorescence, Fluorescence, Immunohistochemical staining, Staining, Two Tailed Test

Journal: Cell Death & Disease

Article Title: Arginine dependency in omental metastasis of epithelial ovarian cancer reveals a therapeutic vulnerability

doi: 10.1038/s41419-026-08606-3

Figure Lengend Snippet: A GO analysis of differentially expressed genes between DDX3X knockdown and control A2780 cells. B GO analysis of differentially expressed genes between A2780 cells with 100 and 500 μM arginine. C The relative levels of reactive oxygen species (ROS) were measured using the dihydroethidium (DHE) fluorescence method in fresh primary tumor and omental tissues ( n = 5). D Representative image and quantification of immunohistochemical staining of γH2AX and 8-OHdG in primary tumors and omentum metastases ( n = 20). Scale bars, 50 μm. E WB analyses of the protein levels of H2AX, γH2AX, and GAPDH in metastases from mice fed with the indicated diets. F Representative images and quantification of γH2AX immunofluorescence staining in A2780 cells treated with cisplatin and under the indicated treatments. Scale bars, 10 μm. G Representative images and quantification of the comet assay in A2780 cells treated with cisplatin and under the indicated treatments. Scale bars, 10 μm. H Representative images and quantification of immunofluorescence staining of γH2AX in A2780 cells treated with H 2 O 2 (1 mM final concentration) and under the indicated treatments. Scale bars, 10 μm. I Representative images and quantification of comet assay in A2780 cells treated with H 2 O 2 (1 mM final concentration) and under the indicated treatments. Scale bars, 10 μm. Data were shown as the mean ± SEM. The p value was calculated using one-way ANOVA ( F - I ), and unpaired two-tailed Student’s t -test ( C , D ). * p < 0.05, ** p < 0.01.

Article Snippet: Permeabilized cells were then sequentially incubated with a primary rabbit anti-DDX3X antibody (Cat. No. 11115-1-AP, Proteintech, Wuhan, China) and a secondary Goat Anti-Rabbit IgG antibody (Cat. No. AS070, ABclonal, Wuhan, China).

Techniques: Knockdown, Control, Fluorescence, Immunohistochemical staining, Staining, Immunofluorescence, Single Cell Gel Electrophoresis, Concentration Assay, Two Tailed Test

Journal: Cell Death & Disease

Article Title: Arginine dependency in omental metastasis of epithelial ovarian cancer reveals a therapeutic vulnerability

doi: 10.1038/s41419-026-08606-3

Figure Lengend Snippet: A The bar graph shows the fold changes in DDR gene expression following treatment with 500 μM arginine or DDX3X knockdown. B , C WB analyses for the protein level of ATM, Phospho-ATM (Ser 1981), ATR, Phospho-ATR, and GAPDH in metastasis of mice treated with the indicated diets. D , E WB showing the protein level of DDX3X, ATM, Phospho-ATM (Ser 1981), CHK2, Phospho-CHK2 (Thr68), P53, Phospho-P53 (Ser15), H2AX, γH2AX, and GAPDH in A2780 or HEY A8 cells with indicated treatments. F WB analyses for the protein level of ATM, Phospho-ATM (Ser 1981), H2AX, γH2AX, and GAPDH in A2780 cells with indicated treatments. G – I Representative images and quantification of immunofluorescence staining of γH2AX, comet assay in A2780 cells treated with cisplatin and ATM inhibitors (AZD0156[10 μM] and AZ31[5 μM]). Scale bars, 10 μm. J WB analyses for the protein level of CHK2, Phospho-CHK2 (Thr68), H2AX, γH2AX, and GAPDH in A2780 cells with the indicated treatments. K – M Representative images and quantification of immunofluorescence staining of γH2AX, comet assay in A2780 cells treated with cisplatin and CHK2 inhibitors (CCT241533 [5 μM] and PHI-101[1 μM]). Scale bars, 10 μm. Data were shown as the mean ± SEM from three independent experiments. The p value was calculated using one-way ANOVA ( H , I , L , M ). * p < 0.05, ** p < 0.01.

Article Snippet: Permeabilized cells were then sequentially incubated with a primary rabbit anti-DDX3X antibody (Cat. No. 11115-1-AP, Proteintech, Wuhan, China) and a secondary Goat Anti-Rabbit IgG antibody (Cat. No. AS070, ABclonal, Wuhan, China).

Techniques: Gene Expression, Knockdown, Immunofluorescence, Staining, Single Cell Gel Electrophoresis

Journal: Cell Death & Disease

Article Title: Arginine dependency in omental metastasis of epithelial ovarian cancer reveals a therapeutic vulnerability

doi: 10.1038/s41419-026-08606-3

Figure Lengend Snippet: A Experimental scheme illustrating orthotopic ovarian cancer and intraperitoneal ovarian cancer mice with arginase (10 μg/kg, intraperitoneal injection, three times a week) or DDX3X inhibitor (RK-33, 50 mg/kg, intraperitoneal injection, three times a week) administration. B Plasma arginine levels in arginase-injected mice were quantified using a biochemical assay kit at 24 h post-injection. C , D Representative IVIS bioluminescence images and quantitative luminescence analysis of mice with indicated treatments at 30 days after i.o. injection of ID8-Luc cells ( n = 5/group). E Representative images and weight of ovaries from orthotopic ovarian tumor mice with indicated treatments at 60 days after cell injection ( n = 5/group). Scale bars, 1 cm. F Representative IVIS bioluminescence images and quantitative luminescence analysis of mice with indicated treatments at 30 days after i.p. injection of ID8-Luc cells ( n = 5/group). G Kaplan–Meier analysis of mice with i.p. injection of ID8-Luc cells treated as indicated. ( n = 10/group). H – J Representative images of peritoneal metastasis ( H ), total number of metastatic deposits ( I ), and ascites volume ( J ) in mice with indicated treatments at 35 days after i.p. injection ( n = 5/group). K – M Representative images, tumor weight, and size of subcutaneous xenograft tumors in nude mice 30 days after injection of A2780 cisplatin-resistant cells, with the indicated treatments ( n = 5/group). Data were shown as the mean ± SD. The p value was calculated using unpaired two-tailed Student’s t -test ( A ), two-way ANOVA ( D – F , I , J , L ), and one-way ANOVA ( M ). Survival curves were generated with the Kaplan–Meier method and analyzed using the log-rank test ( G ). * p < 0.05, ** p < 0.01.

Article Snippet: Permeabilized cells were then sequentially incubated with a primary rabbit anti-DDX3X antibody (Cat. No. 11115-1-AP, Proteintech, Wuhan, China) and a secondary Goat Anti-Rabbit IgG antibody (Cat. No. AS070, ABclonal, Wuhan, China).

Techniques: Injection, Clinical Proteomics, Quantitative Luminescence, Two Tailed Test, Generated

Journal: Journal of Hematology & Oncology

Article Title: Heterodimerization of T cell engaging bispecific antibodies to enhance specificity against pancreatic ductal adenocarcinoma

doi: 10.1186/s13045-024-01538-5

Figure Lengend Snippet: Tumor-associated antigen expression (normalized MFI, mean fluorescence intensity) in PDAC cell lines

Article Snippet: Either recombinant human EGFR (R&D system: Cat# 9565-ER), HER2 (Sino Biological: Cat# 10,004-H08H), or both were immobilized on CM5 chips.

Techniques: Expressing, Fluorescence

Journal: Journal of Hematology & Oncology

Article Title: Heterodimerization of T cell engaging bispecific antibodies to enhance specificity against pancreatic ductal adenocarcinoma

doi: 10.1186/s13045-024-01538-5

Figure Lengend Snippet: Structure and in vitro activity of PDAC-directed T cell-engaging bispecific antibodies ( A ) IgG-[L]-scFv structure of BsAbs. CH, constant heavy chain; CL, constant light chain; scFv, single chain variable fragment; VH, variable heavy chain; VL, variabl light chain. ( B ) Flow cytometric analysis displaying fluorescent intensities of IgG-[L]-scFv BsAbs bound to human PDAC cells. IgG-[L]-scFv BsAbs evaluated were specific for human EGFR, HER2, MSLN, c-MET, and B7-H3. ( C ) Antibody-dependent T cell-mediated cytotoxicity (ADTC) as a function of increasing doses of BsAbs against PDAC cell lines. Ratio of effector T cells to target PDAC cells (E: T ratio) was set to 10:1

Article Snippet: Either recombinant human EGFR (R&D system: Cat# 9565-ER), HER2 (Sino Biological: Cat# 10,004-H08H), or both were immobilized on CM5 chips.

Techniques: In Vitro, Activity Assay

Journal: Journal of Hematology & Oncology

Article Title: Heterodimerization of T cell engaging bispecific antibodies to enhance specificity against pancreatic ductal adenocarcinoma

doi: 10.1186/s13045-024-01538-5

Figure Lengend Snippet: In vitro sensitivities (EC50, pM) to target antigen-specific bispecific antibodies in PDAC cell lines

Article Snippet: Either recombinant human EGFR (R&D system: Cat# 9565-ER), HER2 (Sino Biological: Cat# 10,004-H08H), or both were immobilized on CM5 chips.

Techniques: In Vitro

Journal: Journal of Hematology & Oncology

Article Title: Heterodimerization of T cell engaging bispecific antibodies to enhance specificity against pancreatic ductal adenocarcinoma

doi: 10.1186/s13045-024-01538-5

Figure Lengend Snippet: EGFR and HER2 T-BsAb heterodimerization and in vitro kinetics ( A ) Schematic of BsAb heterodimerization by controlled Fab Arm Exchange. Parental BsAbs bore 2 Fabs specific to EGFR or HER2 and 2 scFvs specific for CD3 (‘2 + 2’). Reduction of inter-H-chain disulfide bonds and subsequent heterodimerization (driven by F405L and K409R amino acid substitutions) yielded EGFRxHER2 BsAbs bearing 1 EGFR Fab, 1 HER2 Fab, and 2 CD3 scFv (‘1 + 1 + 2’). ( B ) SPR analysis of T-BsAbs binding to EGFR (left panel), HER2 (middle panel), and EGFR x HER2 (right panel) proteins. Representative normalized sensorgrams at 20nM were shown. ( C ) IHC staining of two cell-derived PDAC xenograft tumors SW1990 (top) and BxPC-3 (bottom). OCT-embedded tumor sections were stained with EGFRxEGFR, HER2xHER2, or EGFRxHER2 BsAbs. Control slides were either unstained (SW1990) or incubated with a control antibody (BxPC-3). ( D ) Antibody-dependent T cell-mediated cytotoxicity assays (ADTC) against SW1990 (left) and BxPC-3 (right). Cytotoxicity measured by Chromium-51 release. Ratio of effector T cells to target PDAC cells (E: T ratio) was set to 10:1. EC50s (SW1990; BxPC-3): EGFRxEGFR: 165.8fM; 31.5fM, HER2xHER2: 18.5pM; 7.14pM, EGFRxHER2: 699.6fM; 128.8fM. CD33xCD33 BsAb was included as a negative control

Article Snippet: Either recombinant human EGFR (R&D system: Cat# 9565-ER), HER2 (Sino Biological: Cat# 10,004-H08H), or both were immobilized on CM5 chips.

Techniques: In Vitro, Binding Assay, Immunohistochemistry, Derivative Assay, Staining, Control, Incubation, Negative Control

Journal: Journal of Hematology & Oncology

Article Title: Heterodimerization of T cell engaging bispecific antibodies to enhance specificity against pancreatic ductal adenocarcinoma

doi: 10.1186/s13045-024-01538-5

Figure Lengend Snippet: Avidities of EGFR and HER2 T-BsAbs

Article Snippet: Either recombinant human EGFR (R&D system: Cat# 9565-ER), HER2 (Sino Biological: Cat# 10,004-H08H), or both were immobilized on CM5 chips.

Techniques: Protein Binding

Journal: Journal of Hematology & Oncology

Article Title: Heterodimerization of T cell engaging bispecific antibodies to enhance specificity against pancreatic ductal adenocarcinoma

doi: 10.1186/s13045-024-01538-5

Figure Lengend Snippet: Heterodimeric EGFR and HER2 T-BsAbs impede PDAC growth ( A ) In vivo antitumor effect of BsAbs in the presence of human T cells against SW1990 cell line xenografts; 3 × 10 6 cells of SW1990 were subcutaneously implanted into mice. T-BsAbs were administered twice per week and 2 × 10 7 of luciferase-transduced T cells (Luc(+) T cells) were administered once per week for 2 weeks to treat the tumors. ( B ) In vivo anti-tumor effects of 10 µg EGFR and HER2 T-BsAbs. ( C ) Relative body weight of mice during treatment and ( D ) overall survival were plotted. ( E ) Bioluminescence imaging (BLI) of Luc(+) T cells. Quantitation of bioluminescence intensity in the lesions of tumors. Representative images (right) were taken on day 7

Article Snippet: Either recombinant human EGFR (R&D system: Cat# 9565-ER), HER2 (Sino Biological: Cat# 10,004-H08H), or both were immobilized on CM5 chips.

Techniques: In Vivo, Luciferase, Imaging, Quantitation Assay

Journal: Journal of Hematology & Oncology

Article Title: Heterodimerization of T cell engaging bispecific antibodies to enhance specificity against pancreatic ductal adenocarcinoma

doi: 10.1186/s13045-024-01538-5

Figure Lengend Snippet: T-BsAbs drive T cell infiltration, activation, and function in PDAC ( A ) Treatment of SW1990 tumor-bearing mice with EGFR and HER2 T-BsAbs; 3 × 10 6 cells of SW1990 were subcutaneously implanted into mice. Once tumor reached ∼500mm 3 , mice received a single infusion of 2 × 10 7 T cells and were treated with 10µg T-BsAb, administered twice per week. Tumors were harvested for analysis on day 10 after initiation of treatment. Mice that exhibited 50% or more reduction in tumor volume by day 10 were excluded from this and subsequent analyses. ( B ) Flow cytometric analysis of the in vivo effect of T-BsAb treatments on T cell infiltration into harvested SW1990 PDAC tumors. Infiltrating T cells were identified by detection of human CD45 + and human CD4 + or CD8 + . ( C ) Frequency of T cell activation indicated by detection of IFN-γ + TNF-α + T cells and expression of CD69 among CD4 + or CD8 + T cells. ( D ) Frequency of CD11b + intratumoral myeloid cells and prevalence of PDL1 expression

Article Snippet: Either recombinant human EGFR (R&D system: Cat# 9565-ER), HER2 (Sino Biological: Cat# 10,004-H08H), or both were immobilized on CM5 chips.

Techniques: Activation Assay, In Vivo, Expressing

Journal: Journal of Hematology & Oncology

Article Title: Heterodimerization of T cell engaging bispecific antibodies to enhance specificity against pancreatic ductal adenocarcinoma

doi: 10.1186/s13045-024-01538-5

Figure Lengend Snippet: ADTC sensitivities (EC50, pM) of SW1990 lines to EGFR and HER2 T-BsAbs

Article Snippet: Either recombinant human EGFR (R&D system: Cat# 9565-ER), HER2 (Sino Biological: Cat# 10,004-H08H), or both were immobilized on CM5 chips.

Techniques:

Journal: Journal of Hematology & Oncology

Article Title: Heterodimerization of T cell engaging bispecific antibodies to enhance specificity against pancreatic ductal adenocarcinoma

doi: 10.1186/s13045-024-01538-5

Figure Lengend Snippet: FACS binding (EC50, pM) of EGFR and HER2 T-BsAbs to SW1990 lines

Article Snippet: Either recombinant human EGFR (R&D system: Cat# 9565-ER), HER2 (Sino Biological: Cat# 10,004-H08H), or both were immobilized on CM5 chips.

Techniques: Binding Assay

Journal: Journal of Hematology & Oncology

Article Title: Heterodimerization of T cell engaging bispecific antibodies to enhance specificity against pancreatic ductal adenocarcinoma

doi: 10.1186/s13045-024-01538-5

Figure Lengend Snippet: EGFRxHER2 T-BsAbs efficacy is lost in EGFR and HER2-single-positive PDAC ( A ) Mean fluorescent intensities determined by flow cytometry and ( B ) antibody-dependent T cell-mediated cytotoxicity (ADTC) against SW1990-EGFR-KO (left) and SW1990-HER2-KO (right) cell lines. Ratio of effector T cells to target PDAC cells (E: T ratio) was set to 10:1. ( C ) Tumor growth of subcutaneous SW1990 EGFR-KO (left) and HER2-KO (right) tumors. One infusion of 2 × 10 7 T cells was administered. Two doses of T-BsAbs (10 µg each) were given by i.v. injection

Article Snippet: Either recombinant human EGFR (R&D system: Cat# 9565-ER), HER2 (Sino Biological: Cat# 10,004-H08H), or both were immobilized on CM5 chips.

Techniques: Flow Cytometry, Injection

Journal: The Journal of Neuroscience

Article Title: The Autism Protein Ube3A/E6AP Remodels Neuronal Dendritic Arborization via Caspase-Dependent Microtubule Destabilization

doi: 10.1523/JNEUROSCI.1511-17.2017

Figure Lengend Snippet: Increased E6AP overexpression in 2X Tg mice leads to decreased XIAP expression. A, Hippocampal brain lysates were collected from WT or 2X Tg mice from P5 to P40 and E6AP levels were measured by Western blot. Tubulin was probed as a loading control. B, Quantification of E6AP Western blot intensity; n = 3 independent experiments. C, Immunostaining of E6AP in somatosensory cortex slices obtained from P15 WT or 2X Tg mice. Scale bar, 100 μm. D, Quantification showed an increase in E6AP signal intensity in Tg mice; n = 20 slices. E, GFP AAV2 virus was injected into the brain ventricles of WT and 2X Tg mice at P0. Brain slices were prepared at P40 and imaged. Scale bar, 100 μm. A portion of the image of layer-V neurons was enlarged for clarity. Scale bar, 50 μm. Cortical-layer development and neuron density were also analyzed in 2X Tg mice (Fig. 6-1, ). F, XIAP staining in somatosensory cortex slices from P15 WT or 2X Tg mice. Scale bar, 100 μm. G, Quantification showed a decrease in XIAP signal intensity in 2X Tg mice; n = 20 slices. H, MG132 (10 mm, 1.5 μl in each ventricle) was injected into the brain of both WT and 2X Tg mice at P3 for 12 h. Brain lysates were probed for ubiquitin signals. An elevated ubiquitination amount was detected in 2X Tg mice under MG132 treatment. Error bars represent SEM, *p < 0.05, ****p < 0.0001.

Article Snippet: The following cDNA plasmids were obtained from Addgene: p4054-E6AP (#8658), E6AP C820A (#37602), pEBB-XIAP (#11558), and pCDNA3-caspase-3 C163A (#11814). mCherry-tubulin wild-type (WT) and mCherry-tubulin K40A were kind gifts from Dr. Saudou Frederic (Institut Curie). pHR-pTRE-iCre-mCherry and pHR-rtTA (Tet-ON) were generously provided by Wilson Wong.

Techniques: Over Expression, Expressing, Western Blot, Immunostaining, Injection, Staining

Journal: The Journal of Neuroscience

Article Title: The Autism Protein Ube3A/E6AP Remodels Neuronal Dendritic Arborization via Caspase-Dependent Microtubule Destabilization

doi: 10.1523/JNEUROSCI.1511-17.2017

Figure Lengend Snippet: E6AP autism mouse model neurons show impairment in spine maturation and reduction in dendritic branching. A, Brain lysates collected from WT or 2X Tg mice at P15 were probed for E6AP, XIAP, caspase-3, cleaved caspase-3, cleaved tubulin, and total tubulin. GAPDH was also probed as a loading control. B–D, Quantification analysis of Western blots for XIAP, cleaved caspase-3, and cleaved tubulin; n = 3 for each. E, At P15, brains of WT and 2X Tg mice were subjected to Golgi staining. Representative images of spine morphology of layer-V somatosensory cortical neurons are shown. F, Mean spine density decreased in 2X Tg mice; n = 10 neurons. G, Mean spine length increased in 2X Tg mice; n = 10 neurons. H, I, The percentage and number of filopodia increased in 2X Tg mice; n = 10 neurons. J, Representative layer-V pyramidal neuron tracing images of Golgi staining from P15 WT and 2X Tg mouse brain slices. K, L, Measurement of average dendrite number and total dendritic length in pyramidal neurons; n = 12 neurons. Error bars represent SEM, *p < 0.05, **p < 0.01 (Fig. 7-1, ). Summary of the E6AP-dependent dendritic remodeling pathway.

Article Snippet: The following cDNA plasmids were obtained from Addgene: p4054-E6AP (#8658), E6AP C820A (#37602), pEBB-XIAP (#11558), and pCDNA3-caspase-3 C163A (#11814). mCherry-tubulin wild-type (WT) and mCherry-tubulin K40A were kind gifts from Dr. Saudou Frederic (Institut Curie). pHR-pTRE-iCre-mCherry and pHR-rtTA (Tet-ON) were generously provided by Wilson Wong.

Techniques: Western Blot, Staining

Journal: The Journal of Neuroscience

Article Title: The Autism Protein Ube3A/E6AP Remodels Neuronal Dendritic Arborization via Caspase-Dependent Microtubule Destabilization

doi: 10.1523/JNEUROSCI.1511-17.2017

Figure Lengend Snippet: E6AP overexpression reduces the complexity of dendritic arborization. A, Hippocampal neurons were transfected with surfGFP together with vector cDNA (Control) or E6AP cDNA at DIV 11 and imaged for morphology 24 h after transfection. Scale bar, 50 μm. B, Sholl analysis of dendritic branch numbers. Overexpression of E6AP resulted in a decrease in dendritic complexity; n = 40 neurons for each condition. C, Total dendritic branch number and total dendritic length were reduced in E6AP-transfected neurons; n = 40. D, E, Dendrites were characterized as either primary, secondary, tertiary, or quaternary based on their arborization pattern. Representative images of neurons were traced with primary dendrites in blue, secondary in red, tertiary in cyan, and quaternary in magenta. E6AP overexpression led to a decrease in secondary and tertiary dendritic branch length; n = 10. F, Images of dendritic spines from neurons transfected at DIV 11 with surfGFP or together with E6AP for 24 h. Scale bar, 10 μm. G, Mean spine density decreased in E6AP neurons; n = 10 cells. H, Spines were categorized as either mushroom, stubby, thin, or filopodia. Increased E6AP expression led to a decrease in mushroom-type spines and an increase in filopodia; n = 10 cells. Error bars represent SEM, *p < 0.05, **p < 0.01, ****p < 0.0001 (Fig. 1-1, ; cellular distribution of E6AP in hippocampal neurons; Fig. 1-2, ; overexpression of E6AP causes reduction in mEPSCs).

Article Snippet: The following cDNA plasmids were obtained from Addgene: p4054-E6AP (#8658), E6AP C820A (#37602), pEBB-XIAP (#11558), and pCDNA3-caspase-3 C163A (#11814). mCherry-tubulin wild-type (WT) and mCherry-tubulin K40A were kind gifts from Dr. Saudou Frederic (Institut Curie). pHR-pTRE-iCre-mCherry and pHR-rtTA (Tet-ON) were generously provided by Wilson Wong.

Techniques: Over Expression, Transfection, Plasmid Preparation, Expressing

Journal: The Journal of Neuroscience

Article Title: The Autism Protein Ube3A/E6AP Remodels Neuronal Dendritic Arborization via Caspase-Dependent Microtubule Destabilization

doi: 10.1523/JNEUROSCI.1511-17.2017

Figure Lengend Snippet: E6AP overexpression triggers active dendrite retraction and elimination. A, GFP images of neurons transfected from DIV 10 to 11 or from DIV 11 to 12. Scale bar, 50 μm. B, Diagram of the experimental design. C, Sholl analysis showed reduced dendritic arborization in E6AP neurons at DIV 12 compared with either DIV 11 or 12 control cells; n = 10 cells per condition. D, DIV 11 hippocampal neurons were transfected with pTRE-mCherry (Control) or pTRE-E6AP-mCherry (E6AP) and their expression was induced by the addition of doxycycline (Dox, 1 μg/ml) 24 h after transfection (Fig. 2-1, ). Live imaging was performed immediately after Dox treatment (time 0) and every 6 h for 24 h. Colored tracings represent dendrites that increased in length (green), decreased in length (red), or remained the same (yellow). A portion of dendrites was enlarged (bottom row) to show dendrite retraction and fragmentation in E6AP neurons. Scale bar, 50 μm. Scale bar for bottom inset, 10 μm. E, Analysis of live-imaging dendritic events. A significant increase in retraction events was detected in E6AP-expressing neurons compared with the control neurons. F, Percentage of total length of retracted dendrites at 24 h. G, Total length of dendritic growth after 24 h. H, Total length of dendritic retraction after 24 h; n = 9 cells for control and n = 7 cells for E6AP for live-imaging experiments. Error bars represent SEM, *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.

Article Snippet: The following cDNA plasmids were obtained from Addgene: p4054-E6AP (#8658), E6AP C820A (#37602), pEBB-XIAP (#11558), and pCDNA3-caspase-3 C163A (#11814). mCherry-tubulin wild-type (WT) and mCherry-tubulin K40A were kind gifts from Dr. Saudou Frederic (Institut Curie). pHR-pTRE-iCre-mCherry and pHR-rtTA (Tet-ON) were generously provided by Wilson Wong.

Techniques: Over Expression, Transfection, Expressing, Imaging

Journal: The Journal of Neuroscience

Article Title: The Autism Protein Ube3A/E6AP Remodels Neuronal Dendritic Arborization via Caspase-Dependent Microtubule Destabilization

doi: 10.1523/JNEUROSCI.1511-17.2017

Figure Lengend Snippet: Activation of caspase-3 is required for E6AP-dependent dendritic remodeling. A, Neurons were transfected with surfGFP (green; Control) or together with E6AP, and the cleaved (activated) caspase-3 (red) was immunostained 24 h later. Scale bar, 50 μm. B, Quantification of the cleaved caspase-3 immunofluorescence signals; n = 10. E6AP expression resulted in higher levels of cleaved caspase-3. C, D, DIV 2 hippocampal neurons were infected with AAV9 GFP virus or AAV9 E6AP virus for 10 d and cleaved caspase-3 levels were measured by Western blot. Neurons infected with E6AP virus showed higher levels of cleaved caspase-3; n = 3 independent experiments. E, Dendritic arborization reduction in E6AP neurons was blocked by inhibiting caspase-3 cleavage with the caspase-9 inhibitor Ac-LEHD-CMK (150 nm) at the time of transfection, as shown by Sholl analysis; n = 10. F, G, Neurons were transfected with surfGFP (control) or together with E6AP or E6AP + Casp3 C163A (E6AP + C3 mutant), a catalytic caspase-3 mutant. Scale bar, 50 μm. Sholl analysis revealed a rescue of the E6AP-induced dendritic remodeling by Casp3 C163A; n = 10. Error bars represent SEM, **p < 0.01, ***p < 0.001, ****p < 0.0001.

Article Snippet: The following cDNA plasmids were obtained from Addgene: p4054-E6AP (#8658), E6AP C820A (#37602), pEBB-XIAP (#11558), and pCDNA3-caspase-3 C163A (#11814). mCherry-tubulin wild-type (WT) and mCherry-tubulin K40A were kind gifts from Dr. Saudou Frederic (Institut Curie). pHR-pTRE-iCre-mCherry and pHR-rtTA (Tet-ON) were generously provided by Wilson Wong.

Techniques: Activation Assay, Transfection, Immunofluorescence, Expressing, Infection, Western Blot, Mutagenesis

Journal: The Journal of Neuroscience

Article Title: The Autism Protein Ube3A/E6AP Remodels Neuronal Dendritic Arborization via Caspase-Dependent Microtubule Destabilization

doi: 10.1523/JNEUROSCI.1511-17.2017

Figure Lengend Snippet: E6AP targets XIAP for ubiquitination and degradation. A, XIAP ubiquitination assay. HEK293 cells were transfected with FLAG-XIAP, HA-ubiquitin, and either a vector control, E6AP, or the E3 ligase dead mutant E6AP C820A for 2 d. XIAP was immunoprecipitated and probed for ubiquitin (ubi). Cell lysates (input) were also probed to detect total protein levels. B, Quantification of Western blot intensities. E6AP, but not E6AP C820A, caused an increase in XIAP ubiquitination and a decrease in XIAP protein levels; n = 3 independent experiments. C, D, XIAP ubiquitination assays using lysates of neurons infected with AAV9 GFP or AAV9 E6AP virus for 10 d. Increased intensity of ubiquitination signals on XIAP was detected; n = 3 independent experiments. E, Immunostaining of endogenous XIAP (red) in neurons transfected with surfGFP (green) or together with E6AP. Nuclei were indicated by DAPI staining (blue). Scale bar, 50 μm. F, Quantification of the XIAP signal intensity relative to the control; n = 10 cells per condition. G, H, Neurons were infected with AAV9 GFP virus or AAV9 E6AP virus for 10 d, and XIAP levels were measured by Western blot. Quantification showed a reduced level of XIAP in E6AP-infected neurons; n = 3 independent experiments. I, Degradation assay of XIAP with or without E6AP. Transfected HEK cells were treated without cycloheximide (CHX) for various time points and cell lysates were collected to examine XIAP levels by Western blot. J, Quantification of the degradation rate of XIAP over time; n = 4 independent experiments. K, Morphology imaging of primary neurons transfected with surfGFP alone or together with E6AP or E6AP + XIAP. The effect on dendritic arborization markedly decreased in neurons expressing E6AP C820A (Fig. 4-1, ). Scale bar, 50 μm. L, Sholl analysis showing a blockade of the E6AP effect in dendritic remodeling by XIAP overexpression; n = 10 cells per condition. Error bars represent SEM, *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.

Article Snippet: The following cDNA plasmids were obtained from Addgene: p4054-E6AP (#8658), E6AP C820A (#37602), pEBB-XIAP (#11558), and pCDNA3-caspase-3 C163A (#11814). mCherry-tubulin wild-type (WT) and mCherry-tubulin K40A were kind gifts from Dr. Saudou Frederic (Institut Curie). pHR-pTRE-iCre-mCherry and pHR-rtTA (Tet-ON) were generously provided by Wilson Wong.

Techniques: Ubiquitin Assay, Transfection, Plasmid Preparation, Mutagenesis, Immunoprecipitation, Western Blot, Infection, Immunostaining, Staining, Degradation Assay, Imaging, Expressing, Over Expression

Journal: The Journal of Neuroscience

Article Title: The Autism Protein Ube3A/E6AP Remodels Neuronal Dendritic Arborization via Caspase-Dependent Microtubule Destabilization

doi: 10.1523/JNEUROSCI.1511-17.2017

Figure Lengend Snippet: Microtubule cleavage and retraction in E6AP-induced dendritic remodeling. A, Neurons were transfected with surfGFP and pTRE-E6AP for 24 h, and loaded with SiR-Tubulin, a fluorogenic and cell-permeable dye for tubulin labeling, before being treated with doxycycline (Dox) to induce E6AP expression. Tubulin and surfGFP images were obtained every 20 min for 12 h following Dox application. Representative images show that retraction of microtubule (red; hollow arrowhead) occurred before that of the GFP-positive dendritic branch (green; solid arrowhead). The original position of the dendritic tip is indicated by a dashed line. Scale bar, 5 μm. B, Neurons were infected with AAV9 GFP virus or AAV9 E6AP virus for 10 d, and cleaved tubulin levels were measured by Western blot with an antibody specifically against the cleaved microtubule (ΔTubulin). C, Quantification showed an increased level of microtubule cleavage in E6AP-infected neurons; n = 3 independent experiments. D, Representative image of E6AP neurons immunostained with ΔTubulin. Scale bar, 10 μm. E, Quantification of ΔTubulin immunointensity in neurons transfected with vector control, E6AP, or E6AP + Casp3 C163A (E6AP + C3 mutant), compared with control; n = 10. F, Morphology of neurons transfected with surfGFP, tubulin WT, E6AP, or E6AP + tubulin WT. Scale bar, 50 μm. G, Sholl analysis of dendritic arborization; n = 10 cells per condition. H, Quantification of total dendritic length; n = 10 cells per condition. Changes in tubulin stabilization also affected the E6AP-dependent dendritic remodeling (Fig. 5-1, . Error bars represent SEM, *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.

Article Snippet: The following cDNA plasmids were obtained from Addgene: p4054-E6AP (#8658), E6AP C820A (#37602), pEBB-XIAP (#11558), and pCDNA3-caspase-3 C163A (#11814). mCherry-tubulin wild-type (WT) and mCherry-tubulin K40A were kind gifts from Dr. Saudou Frederic (Institut Curie). pHR-pTRE-iCre-mCherry and pHR-rtTA (Tet-ON) were generously provided by Wilson Wong.

Techniques: Transfection, Labeling, Expressing, Infection, Western Blot, Plasmid Preparation, Mutagenesis