human embryonic kidney epithelial 293t cell line  (ATCC)

Journal: Vaccines

Article Title: A Precision-Engineered DC-Targeting mRNA-LNP Neoantigen Vaccine Elicits Stronger T Cell Responses and Exhibits Superior Tumor Control

doi: 10.3390/vaccines14030239

Figure Lengend Snippet: Isolation and characterization of mCLEC9A-specific nanobodies. ( A ) The workflow for the isolation and characterization of nanobodies. ( B ) SDS-PAGE/Coomassie blue staining of purified anti-mCLEC9A nanobody 2A4. Measurement of the affinity of 2A4 to mCLEC9A by Biolayer Interferometry (BLI). Two-fold serial dilutions of 2A4 from 100 nM to 6.25 nM were used to measure binding to biotinylated mCLEC9A immobilized on the Octet BLI biosensor. The equilibrium dissociation constant ( K D ) and binding kinetic parameters are presented. ( C ) SDS-PAGE/Coomassie blue staining of purified 2A4-Fc and flow cytometry histograms showing binding of 2A4-Fc to mCLEC9A + HEK-293T cell line. Uncropped SDS-PAGE images can be found in .

Article Snippet: Human embryonic kidney epithelial 293T cell line was purchased from ATCC (CAT.CBP60439, RRID: CVCL_0063, Manassas, VA, USA).

Techniques: Isolation, SDS Page, Staining, Purification, Binding Assay, Flow Cytometry

Journal: Vaccines

Article Title: A Precision-Engineered DC-Targeting mRNA-LNP Neoantigen Vaccine Elicits Stronger T Cell Responses and Exhibits Superior Tumor Control

doi: 10.3390/vaccines14030239

Figure Lengend Snippet: Transfection, cytotoxicity, and biodistribution of mRNA-LNP complexes. ( A ) Cytotoxicity of Mal-LNP and Nb-LNP in mCLEC9A + HEK-293T cells. Cell viability evaluated using Cell Titer Luminescent Cell Viability Assay kit 24 h after treatment with mRNA Mal-LNP or Nb-LNP. ( B , C ) Time-course quantification of bioluminescence resulted from treating mCLEC9A + HEK-293T cells and FL-DCs derived from mouse bone marrow with mRNA Mal-LNP or Nb-LNP at 0.5 μg/mL ( n = 5). ( D ) Ex vivo imaging of organs was performed 4 h after injection. Shown are representative bioluminescence images and quantification of bioluminescence signals from organs extracted from C57BL/6 mice following intramuscular administration of 5 μg luciferase (Luc) formulated in either Mal-LNP or Nb-LNP. Abbreviations: LN, lymph node; Li, liver; S, spleen; Lu, lung; K, kidney. ( E , F ) Quantification of eGFP + cells in distinct cell subsets in spleen and lymph nodes 36 h after mice were treated intramuscularly with 10 μg eGFP mRNA-LNP. Statistical analyses were performed using two-way ANOVA. All results are presented as mean ± SEM. “ns”, no significant difference; * p < 0.05; ** p < 0.01; *** p < 0.001; **** p < 0.0001.

Article Snippet: Human embryonic kidney epithelial 293T cell line was purchased from ATCC (CAT.CBP60439, RRID: CVCL_0063, Manassas, VA, USA).

Techniques: Transfection, Cell Viability Assay, Derivative Assay, Ex Vivo, Imaging, Injection, Luciferase

Journal: Journal of Experimental & Clinical Cancer Research : CR

Article Title: Galectin-3 induces pathogenic immunosuppressive macrophages through interaction with TREM2 in lung cancer

doi: 10.1186/s13046-024-03124-6

Figure Lengend Snippet: Tumor derived galectin-3 is a ligand for TREM2. ( A ) Illustration of human lung cancer tissue protein precipitated by anti-TREM2 or IgG antibody ( n = 3), and peptides enriched in each complex were identified by mass spectrometry. ( B ) Venn diagram and tables showing secreted proteins concentrated in the TREM2 enriched complex. ( C ) Immunohistochemical staining of galectin-3 in adjacent normal lung and intratumor areas of lung tissues ( n = 5). Scale bars, 20 μm. ( D ) Galactin-3 levels in lung cancer or normal lung lavage were determined by ELISA. ( E ) 293T cells were transfected with pcDNA3.1-vector/pcDNA3.1-hTREM2-HA/pcDNA3.1-hGalectin-3-Flag plasmids as indicated. Anti-HA antibody was employed for exogenous CO-IP experiments. ( F ) F4/80 + macrophages were sorted from human lung cancer tissue. Anti-TREM2 antibody was employed for endogenous CO-IP experiment. ( G ) 293T cells were transfected with pcDNA3.1-TREM2-HA and pcDNA3.1-Galectin-3-Flag plasmids. The co-localization between TREM2 and Galectin-3 was examined using confocal microscopy. Scale bars, 5 μm. ( H ) 293T cells were transfected with pcDNA3.1-TREM2-HA/pcDNA3.1-TREM2-ΔIg-HA/pcDNA3.1-TREM2-ΔTm-HA/pcDNA3.1-TREM2-ΔCyto-HA/pcDNA3.1-Galectin-3-Flag/pcDNA3.1-vector plasmids as indicated. Anti-Flag antibody was employed for exogenous CO-IP experiments. ( I ) 293T cells were transfected with pcDNA3.1-vector/pcDNA3.1-TREM2-Ig-HA/pcDNA3.1-TREM2- HA/pcDNA3.1-Galectin-3-Flag plasmids as shown. Anti-Flag antibody was employed for exogenous CO-IP experiments. ( J ) 293T cells were transfected with pcDNA3.1-vector/pcDNA3.1-TREM2-HA/pcDNA3.1-Galectin-3-ΔNH2-Flag/pcDNA3.1-Galectin-3-ΔCRD-Flag/pcDNA3.1-Galectin-3- ΔRepeats-Flag plasmids as indicated. Anti-HA antibody was employed for exogenous CO-IP experiments. ( K ) Galectin-3 protein bound to TREM2 in the plate was determined using anti-Galectin-3 antibody. Data represent mean ± SD from three experiments. **, P < 0.01; ***, P < 0.001

Article Snippet: The human lung cancer cell line A549 (ATCC, RRID: CVCL_4V07) and H292 (ATCC, RRID: CVCL_0455), mouse fibroblast cell line L929 (ATCC, RRID: CVCL_AR58), mouse lung adenocarcinoma cell line LLC (ATCC, RRID: CVCL_4358), mouse mononuclear macrophage leukemia cell line RAW264.7 (ATCC, RRID: CVCL_C6 × 3), and human embryonic kidney epithelial cell line 293T (ATCC, RRID: CVCL_0063) were procured from ATCC.

Techniques: Derivative Assay, Mass Spectrometry, Immunohistochemical staining, Staining, Enzyme-linked Immunosorbent Assay, Transfection, Plasmid Preparation, Co-Immunoprecipitation Assay, Confocal Microscopy

Journal: Journal of Experimental & Clinical Cancer Research : CR

Article Title: Galectin-3 induces pathogenic immunosuppressive macrophages through interaction with TREM2 in lung cancer

doi: 10.1186/s13046-024-03124-6

Figure Lengend Snippet: Galectin-3 inhibits TREM2/DAP12 receptor complex to suppress Src/Syk signaling pathway and altered macrophage to an M2-like phenotype. (A , B ) 293T cells were transfected with plasmids as shown, and anti-HA ( B ) or anti-Flag ( B ) antibodies were employed for exogenous CO-IP experiments. ( C-E ) 293T cells were transfected with plasmids as shown and anti-HA ( C ), anti-Flag ( D ), or anti-Myc ( E ) antibodies were employed for exogenous CO-IP experiments, respectively. (F , G ) After 24 h of LLC CM treatment with the addition of GB1107 (5 µM), and 2 h of stimulation with LPS (1 ng/mL), the phosphorylation levels of Src and Syk in indicated time point were analyzed by western blot ( F ). The gray values of p-Syk and p-Src protein bands were analyzed by Image J software, and the relative gray values were standardized to the gray values of Syk and Src ( G ). (H , I ) After 24 h of LLC CM treatment with the addition of GB1107 (5 µM), and 2 h of stimulation with LPS (1 ng/mL), the phosphorylation levels of Src and Syk in WT or TREM2 KO BMDMs were analyzed by western blot ( H ). The gray values of p-Syk and p-Src protein bands were analyzed by Image J image analyses software, and the relative gray values were standardized to the gray values of Syk and Src ( I ). (J , K ) After 24 h of LLC CM treatment with the addition of rm galectin-3 (200 ng/ml), and 2 h of stimulation with LPS (1 ng/mL), the phosphorylation levels of Src and Syk in WT or TREM2 KO BMDMs were analyzed by western blot ( J ). The gray values of p-Syk and p-Src protein bands were analyzed by Image J image analyses software, and the relative gray values were standardized to the gray values of Syk and Src ( K ). ( L ) Phagocytosis assay between WT or TREM2 KO BMDMs which were pretreated with LLC CM supplemented with GB1107 (5 µM) for 24 h and LLC were detected by flow cytometry. ( M ) Following a 24 h-treatment with LLC CM supplemented with GB1107 (5 µM), the F-actin polarization of RAW264.7 cells blocked with anti-TREM2 antibody was observed by confocal microscopy. Scale bars, 5 μm. Quantitative statistics of F-actin polarization were analyzed by Image J image analyses software. ( N-P ) After 24 h of LLC CM which was supplemented with GB1107 (5 µM) treatment, the transcription levels of Ccr2 ( N ), M2-like macrophage markers ( CD206 , Arg1 ) ( O ) and M1-like macrophage markers ( Nos2 , TNFα ) ( P ) in WT or TREM2 KO BMDMs were detected by RT-qPCR assay. Data represent mean ± SD from three experiments. *, P < 0.05; **, P < 0.01; ***, P < 0.001. ns, no significance

Article Snippet: The human lung cancer cell line A549 (ATCC, RRID: CVCL_4V07) and H292 (ATCC, RRID: CVCL_0455), mouse fibroblast cell line L929 (ATCC, RRID: CVCL_AR58), mouse lung adenocarcinoma cell line LLC (ATCC, RRID: CVCL_4358), mouse mononuclear macrophage leukemia cell line RAW264.7 (ATCC, RRID: CVCL_C6 × 3), and human embryonic kidney epithelial cell line 293T (ATCC, RRID: CVCL_0063) were procured from ATCC.

Techniques: Transfection, Co-Immunoprecipitation Assay, Phospho-proteomics, Western Blot, Software, Phagocytosis Assay, Flow Cytometry, Confocal Microscopy, Quantitative RT-PCR