Journal: Cancer immunology research

Article Title: Pharmacological inhibition of FGFR modulates the metastatic immune microenvironment and promotes response to immune checkpoint blockade

doi: 10.1158/2326-6066.CIR-20-0235

Figure Lengend Snippet: (A) Survival analysis of BALB/c mice bearing pulmonary 4T07 tumors following treatment with the indicated therapies (n=5 mice per group). The combination group of mice received both FIIN4 (100mg/kg/day for 14 days via oral gavage) and anti-PD-L1 (four doses of 200 μg once every three days via intraperitoneal injections) simultaneously. The 14-day FIIN4 treatment is indicated by the solid black line and each anti-PD-L1 administration is denoted by an arrowhead. (B) 4T07 cells were cocultured in vitro with splenocytes from 4T07 tumor bearing mice in the presence or absence of FIIN4 (100 nM) and tumor cell lysis was quantified as described in the materials and methods. Data are mean of triplicate experiments and data were compared using a paired T-test resulting in the indicated p value. (C) Jurkat T-cells pretreated with either DMSO or 1μM FIIN4 overnight were stimulated with CD3-specific antibody for the indicated time points, and cell lysates were collected and assayed via immunoblot for phosphorylation of p-Lck, p-PLCγ1, p-SLP76, p-ZAP70(Y319), p-LAT, and β-tubulin (β-Tub) as a loading control. Data in panel C are representative of at least two independent experiments. (D) Survival analyses of BALB/c mice bearing pulmonary 4T07 tumors treated with indicated therapies (n=10 mice per group). As in panel A, FIIN4 treatment duration (100 mg/kg/day) is indicated by the solid line and anti-PD-L1 doses (200 μg) are indicated by the arrowheads. Survival data were analyzed by a log rank test. (E) Bioluminescent images of the different treatment groups described in panel D at Day 17 post tail vein injections.

Article Snippet: Jurkat (clone e6–1) cells were obtained from Kazemian lab (Purdue University) in 2018.

Techniques: In Vitro, Lysis, Western Blot, Phospho-proteomics, Control

Journal: Molecules

Article Title: The Proteolytic Activity of Neutrophil-Derived Serine Proteases Bound to the Cell Surface Arming Lung Epithelial Cells for Viral Defense

doi: 10.3390/molecules29184449

Figure Lengend Snippet: MHC I cell surface expression increases after treatment with proteases. A549, H1299, and Jurkat cells were incubated with CatG [10 μg/mL], NE [10 μg/mL], or both for 6 h in PBS, and the MHC I cell surface expression was analyzed using flow cytometry. Data were normalized to isotype control and considered significant at p < 0.05 (*), p < 0.01 (**), or p < 0.001 (***) by using an unpaired one-way ANOVA and Sidak post hoc test. n = 3.

Article Snippet: The following cell lines were used in this study: A549 cells (CCL-185, American Type Culture Collection, ATCC, Manassas, VA, USA), an adenocarcinoma human alveolar basal epithelial cell line; NCI-H1299 cells (H1299, CRL-5803, American Type Culture Collection, ATCC, Manassas, VA, USA), a human epithelial-like, non-small cell lung carcinoma cell line derived from the lymph node; and Jurkat E6-1 cells (Jurkat, human T cell lymphoblasts, ARP-177, BEI Resources; NIAID, NIH, Bethesda, MD, USA [ ]).

Techniques: Expressing, Incubation, Flow Cytometry, Control

Journal: The Journal of Biological Chemistry

Article Title: DNA-PKcs kinase activity stabilizes the transcription factor Egr1 in activated immune cells

doi: 10.1016/j.jbc.2021.101209

Figure Lengend Snippet: Protein and RNA expression patterns associated with Egr1 in multiple cell types. Egr1 expression pattern was analyzed by Western blotting in ( A ) Jurkat T cells, ( B ) total mouse splenocytes (WT indicates WT mouse and KO indicates PRKDC functional knockout mouse), and ( C ) HEK293 kidney cells, which were chemically stimulated for 3 h and treated with NU7441 as indicated. D , real-time qPCR analysis of EGR1 transcripts in Jurkat cells. Error bars represent standard error of the mean. NS indicates no significant difference. E , Jurkat cells were treated as in ( A ) with the addition of the proteasome inhibitor MG132 and Egr1 was detected by Western blot. Error bars = s.d. of the mean of biological replicates.

Article Snippet: Jurkat E6.1 cells and mouse splenocytes from BALB/c and NOD.CB17-Prkdc scid (#001303, #000651 respectively, Jackson Laboratory) were cultured in RPMI media supplemented with Penstrep antibiotics and 10% fetal bovine serum.

Techniques: RNA Expression, Expressing, Western Blot, Functional Assay, Knock-Out

Journal: The Journal of Biological Chemistry

Article Title: DNA-PKcs kinase activity stabilizes the transcription factor Egr1 in activated immune cells

doi: 10.1016/j.jbc.2021.101209

Figure Lengend Snippet: Effect of Egr1 phosphorylation on protein stability. A , four plasmid-based variants of Egr1-3xFlag at amino acid 301, as indicated by the single-letter amino acid abbreviation ( A , D , and E ), were expressed in stimulated HEK293 cells. B , endogenous EGR1 S301A and knockout mutants along with the wild type S301S strain were generated in Jurkat cells using CRISPR. C , degradation of Egr1 or one of the Egr1 S301A variants over time following cycloheximide treatment and quantification of relative EGR1 protein levels from three independent experiments normalized to GAPDH. Δ = EGR1 CRISPR knockout, A = mutation generating S301A mutant (three separate clones are represented), S = WT EGR1 . Error bars = standard error.

Article Snippet: Jurkat E6.1 cells and mouse splenocytes from BALB/c and NOD.CB17-Prkdc scid (#001303, #000651 respectively, Jackson Laboratory) were cultured in RPMI media supplemented with Penstrep antibiotics and 10% fetal bovine serum.

Techniques: Phospho-proteomics, Plasmid Preparation, Knock-Out, Generated, CRISPR, Mutagenesis, Clone Assay

Journal: The Journal of Biological Chemistry

Article Title: DNA-PKcs kinase activity stabilizes the transcription factor Egr1 in activated immune cells

doi: 10.1016/j.jbc.2021.101209

Figure Lengend Snippet: Effect of Egr1 phosphorylation on IL2 expression. IL2 concentrations were measured by ELISA in Jurkat EGR1Δ cells transfected by electroporation with plasmids expressing the indicated variant of Egr1. Control indicates transfection with a plasmid containing GFP in place of EGR1 . Variability is represented by standard deviation of four replicates. ∗ indicates p < 0.01 evaluated by t test.

Article Snippet: Jurkat E6.1 cells and mouse splenocytes from BALB/c and NOD.CB17-Prkdc scid (#001303, #000651 respectively, Jackson Laboratory) were cultured in RPMI media supplemented with Penstrep antibiotics and 10% fetal bovine serum.

Techniques: Phospho-proteomics, Expressing, Enzyme-linked Immunosorbent Assay, Transfection, Electroporation, Variant Assay, Control, Plasmid Preparation, Standard Deviation

Journal: Clinical and Experimental Immunology

Article Title: Cultured lymphocytes’ mitochondrial genome integrity is not altered by cladribine

doi: 10.1093/cei/uxad112

Figure Lengend Snippet: Proliferation rates of the T-cell lines and effects of cladribine on cell viability. (a) The growth rate of Jurkat E6.1 is faster than the CCRF-CEM cell line ( n = 3). (b) A cladribine dose response curve in Jurkat E6.1 cell line for cell proliferation with 48 hours ( n = 5) and 96 hours culture ( n = 5). (c) The effect of cladribine on cell proliferation in Jurkat with 12 days ( n = 5) and 16 days in culture ( n = 5). (d) The effect of cladribine on cell proliferation in CCRF-CEM with 2 days ( n = 5), 8 days ( n = 4), and 12 days ( n = 4) in culture ( n = 5).

Article Snippet: The human leukemic Jurkat E6.1 cell line (pseudodiploid, modal No 46) and the CCRF-CEM (ECACC 85112105) cell line (2 n = 46) were obtained from the European Collection of Authenticated Cell Cultures (ECACC) general cell collection and cultured according to the recommended instructions (Jurkat E6.1: RPMI 1640; 2 mM glutamine; 10% FBS) (CCRF-CEM: RPMI 1640; 2 mM glutamine; 20% FBS).

Techniques:

Journal: Clinical and Experimental Immunology

Article Title: Cultured lymphocytes’ mitochondrial genome integrity is not altered by cladribine

doi: 10.1093/cei/uxad112

Figure Lengend Snippet: Effect of cladribine on mitochondrial protein synthesis. (a) Immunoblot of MT-ATP6, MT-CO1, MRPL11, MRPS35, CASPASE3, SDHA, and CANX after 12 days culture with cladribine. In this experiment, 10 nM cladribine in CCRF cells resulted in extensive cell death, and immunoblot was not performed. (b) A pulse metabolic labeling of mitochondrial protein synthesis. The effect on mitochondrial protein synthesis in the CCRF-CEM and Jurkat cells after treatment with cladribine for 8 days ( n = 3) or 12 days ( n = 3).

Article Snippet: The human leukemic Jurkat E6.1 cell line (pseudodiploid, modal No 46) and the CCRF-CEM (ECACC 85112105) cell line (2 n = 46) were obtained from the European Collection of Authenticated Cell Cultures (ECACC) general cell collection and cultured according to the recommended instructions (Jurkat E6.1: RPMI 1640; 2 mM glutamine; 10% FBS) (CCRF-CEM: RPMI 1640; 2 mM glutamine; 20% FBS).

Techniques: Western Blot, Labeling