Journal: Pharmacological research

Article Title: Ectopic expression of NKG7 enhances CAR-T function and improves the therapeutic efficacy in liquid and solid tumors.

doi: 10.1016/j.phrs.2024.107506

Figure Lengend Snippet: Fig. 4. NKG7 promotes the proliferation of CAR-T cells by enhancing the IL-2 signaling pathway. (A and B) GSEA of DNA replication and cell cycle between B7H3- CAR-TNKG7 and B7H3-CAR-T cells following B7H3 protein stimulation for 6 h. (C) Volcano plot of T cell proliferation-related genes between B7H3-CAR-TNKG7 and B7H3-CAR-T cells. (D) Heatmaps of significant T cell proliferation-related genes. (E and F) IL-2 expression of CAR-T cells was measured by FCM analysis following target cell stimulation for 6 h. (G) Statistics for (E and F). (H) The proliferation of CAR-T cells was detected by a CFSE assay. (I and J) GSEA of the Jak-STAT and PI3K- Akt pathways between B7H3-CAR-TNKG7 and B7H3-CAR-T cells following B7H3 protein stimulation for 6 h. (K) The IL-2 signaling pathway (p-AKT (Ser473), AKT, p- ERK (Thr202/Tyr204), ERK, p-STAT5 (Thr694) and STAT5) in CAR-T cells were explored by WB assays. ***P < 0.001.

Article Snippet: After blocking with 5 % nonfat milk at room temperature for 1 h, the membranes were incubated with primary antibodies against NKG7 (Cat No. 84835, 1:1000, CST), AKT (Cat No. A11016, 1:1000, ABclonal), p-AKT (Ser473) (Cat No. AP0140, 1:1000, ABclonal), ERK (Cat No. 4695, 1:1000, CST), p-ERK (Thr202/Tyr204) (Cat No. 4370, 1:1000, CST), STAT5 (Cat No. 25656, 1:1000, CST), pSTAT5 (Thr694) (Cat No. 4322, 1:1000, CST) and β-actin (Cat No. 66009–1-Ig, 1:5000, Proteintech) overnight at 4◦C.

Techniques: Expressing, Cell Stimulation, CFSE Assay

Journal: International Journal of Molecular Sciences

Article Title: Different Lipid Regulation in Ovarian Cancer: Inhibition of the Immune System

doi: 10.3390/ijms19010273

Figure Lengend Snippet: Normal expression and phosphorylation of the IL-2 receptor and its downstream signaling components. PBLs of healthy individuals or sorted ascites-derived CD4 + CD25 − T cells (5 × 10 5 cells/well) were stimulated with 20,000 αCD3/28 beads/well for three days. Subsequently, samples were stained for flow cytometry analysis. ( A ) Cells were stained for isotype control, CD25, CD122, and CD132. Plots are representative for eight controls and three patients. ( B ) Control ( n = 3) and ascites-derived T cells ( n = 3) were stained for JAK3, STAT5, phospho-STAT5, phosphor-AKT and phosphor-ERK. One representative example is shown.

Article Snippet: The following antibodies were used in this study: CD4-APC (300514, Beckman Coulter, Brea, CA, USA), CD4-FITC (A07750, Beckman Coulter), CD25-PE (555432, BD Biosciences, San Jose, CA, USA), CD122-PE (IM1978, Beckman Coulter), CD132-APC (338608, BioLegend, San Diego, CA, USA), cyclin D (2936P, Cell Signaling, Danvers, MA, USA), cyclin E (551159, Cell Signaling, Danvers, MA, USA), JAK3 (ab45141, Abcam, Cambridge, UK), STAT5 (9363P, Cell Signaling, Danvers, MA, USA), pSTAT5 (9314S, Cell Signaling, Danvers, MA, USA), pAKT (4075S, Cell Signaling, Danvers, MA, USA), and pERK1/2 (4284S, Cell Signaling).

Techniques: Expressing, Phospho-proteomics, Derivative Assay, Staining, Flow Cytometry, Control

Journal: PLoS ONE

Article Title: Interleukin-15 Plays a Central Role in Human Kidney Physiology and Cancer through the γc Signaling Pathway

doi: 10.1371/journal.pone.0031624

Figure Lengend Snippet: A) Scatchard's plot analysis: Effects of anti-IL-15Rβ and γc mAbs on IL-15 binding to RCC. For the IL-15 binding experiments, RCC7 cells were incubated with increasing concentrations of radioiodinated rIL-15 in presence or not of the following neutralizing mAbs: anti-IL-2Rβ and IL-2Rγ. The nonspecific cell binding was determined in the presence of radioiodinated rhIL-15 and a 100-fold excess of unlabeled rhIL-15. Cell-bound (B) and unbound (free, F) fractions were measured, and the specific bound fraction was calculated by subtracting the nonspecific binding from the cell-bound fraction. On the ordinate is plotted the ratio of the specific bound fraction (expressed in sites per cell) over the total concentration (bound plus free) of radioiodinated rIL-15 (expressed in pM). On the abscissa bound fraction (expressed in sites per cell). The high affinity specific IL-15 binding (Kd = 375 pM, 413 IL-15 binding sites per cell), which was completely abrogated by neutralizing antibody against the IL-2Rβ (inset) but not the γc chain, suggested the presence on RCC of an IL-15Rα/IL-2Rβ complex. B ) Detection of IL-15Rαβ complex by immunoprecipitation (IP) with anti-IL-15Rα (M161) or mouse IgG protein G-Sepharose-conjugate on total lysate (TL) of RCC7. Immunoprecipitated complexes were blotted either with anti-IL-2Rβ (sc-1046) and anti-IL-15Rα (sc-9172). C ) Stimulation for 10 and 40 min with physiologic (10 pg/mL) and supra-physiologic (10 ng/mL) concentrations of rhIL-15 induces the phosphorylation of MAPK ERK1/2 and IκBα in RPTEC and RCC7, whereas STAT5 activation was only observed in RPTEC. Histograms represent densitometry comparison of each factor normalized to β-actin in 3 different RCC (RCC5, RCC7, RCC8) and 3 RPTEC batches. * P<0.05 versus control, Mann-Whitney test. One experiment representative of a total of three is shown.

Article Snippet: Antibodies against phosphorylated ERK (4377), phosphorylated IκB (4921), STAT5 (9358) phosphorylated STAT5 (9356), and the Alexa fluor-conjugated rabbit monoclonal antibody against phosphorylated STAT5 (3939) were obtained from Cell Signaling (Beverly, MA).

Techniques: Binding Assay, Incubation, Concentration Assay, Immunoprecipitation, Phospho-proteomics, Activation Assay, Comparison, Control, MANN-WHITNEY

Journal: PLoS ONE

Article Title: Interleukin-15 Plays a Central Role in Human Kidney Physiology and Cancer through the γc Signaling Pathway

doi: 10.1371/journal.pone.0031624

Figure Lengend Snippet: The γc neutralization, as well as JAK3 or STAT5 inhibition, hamper the maintenance of E-cadherin surface expression induced by rhIL-15 on RPTEC without interfering on the E-cadherin down-regulation on rhIL-15-treated RCC7. Cells were pretreated with 1 µg/ml of neutralizing anti-IL2Rγ antibody mAb2842, 0.25 µM of JAK3 inhibitor (CP-690, 550, Calbiochem) or 100 µM of STAT5 inhibitor (STAT5 Inh., 573108, Calbiochem) for 1 h before adding the recombinant cytokine (10 pg/mL) for 5 days. Treatment with rhIL-15 and STAT5 inhibitor was renewed at day 3. White histograms refer to isotype-matched control. Mean fluorescence intensity values for each marker are shown in each histogram. The data are representative of 3 separate experiments performed using different RCC (RCC5, RCC8) and RPTEC batches.

Article Snippet: Antibodies against phosphorylated ERK (4377), phosphorylated IκB (4921), STAT5 (9358) phosphorylated STAT5 (9356), and the Alexa fluor-conjugated rabbit monoclonal antibody against phosphorylated STAT5 (3939) were obtained from Cell Signaling (Beverly, MA).

Techniques: Neutralization, Inhibition, Expressing, Recombinant, Control, Fluorescence, Marker

Journal: PLoS ONE

Article Title: Interleukin-15 Plays a Central Role in Human Kidney Physiology and Cancer through the γc Signaling Pathway

doi: 10.1371/journal.pone.0031624

Figure Lengend Snippet: RCC7 were transiently transfected for 48 hours with vectors containing IL-2Rγ and/or JAK3 Human cDNA. A ) Transient expression of IL-2Rγ and JAK3 was analyzed by immunoblotting in each transfected RCC. Immunoblotting for β-actin was used as a control for equal protein loading and transfer. B ) Flow cytometry shows that 40 min rhIL-15 treatment did not induce STAT5 phosphorylation in IL-2Rγ- or JAK3-transfected RCC while rhIL-15 treatment induced STAT5 phosphorylation in co-transfected cells. C ) After 48 h, transfected RCC were treated for an additional 48 h with 10 pg/mL of rhIL-15 before evaluating E-cadherin expression by flow cytometry. The introduction of either IL-2Rγ chain, JAK3 or both molecules do not modify E-cadherin expression on untreated rhIL-15 cells, while the E-cadherin down-regulation observed after 48 hours of rhIL-15 treatment was counterbalanced only in co-transfected cells. Mean fluorescence intensity values for each marker are shown in each histogram. One experiment representative of a total of three is shown.

Article Snippet: Antibodies against phosphorylated ERK (4377), phosphorylated IκB (4921), STAT5 (9358) phosphorylated STAT5 (9356), and the Alexa fluor-conjugated rabbit monoclonal antibody against phosphorylated STAT5 (3939) were obtained from Cell Signaling (Beverly, MA).

Techniques: Transfection, Expressing, Western Blot, Control, Flow Cytometry, Phospho-proteomics, Fluorescence, Marker

Journal: BioMed Research International

Article Title: Phloretin Modulates Human Th17/Treg Cell Differentiation In Vitro via AMPK Signaling

doi: 10.1155/2020/6267924

Figure Lengend Snippet: Phloretin influences the differentiation of Th17 and Treg cells in vitro. (a) The frequency of Th17 cells generated under Th17 polarization conditions in the presence or absence of phloretin (25, 50, and 100 μ M) using fluorescent antibody staining and flow cytometry. ∗ p < 0.05 and ∗∗∗∗ p < 0.0001 versus the control group; #### p < 0.0001 versus the 25 μ M phloretin treatment group; ns means no significant difference between the 50 μ M and 100 μ M phloretin treatment group (mean ± SD, n = 4, one-way ANOVA followed by Tukey's multiple comparison test). (b) The frequency of Treg cells generated under Treg polarization conditions in the presence or absence of phloretin (25, 50, and 100 μ M) using fluorescent antibody staining and flow cytometry. ∗∗ p < 0.01 and ∗∗∗∗ p < 0.0001 versus the control group; #### p < 0.0001 versus the 25 μ M phloretin treatment group; ns means no significant difference between the 50 μ M and 100 μ M phloretin treatment group (mean ± SD, n = 4, one-way ANOVA followed by Tukey's multiple comparison test). (c) Expression levels of phospho-Stat3 or phospho-Stat5 were examined under Th17 or Treg polarization conditions in the presence or absence of phloretin (50 μ M) using fluorescent antibody staining and flow cytometry. ∗∗ p < 0.01 versus the control group (mean ± SD, n = 4, Student's unpaired t -test). (a–c) The control group was treated with DMSO.

Article Snippet: PE anti-phospho-Stat3 antibody (8119, CST) and Alexa Fluor® 647 anti-phospho-Stat5 antibody (9365, CST) were used for staining T cells after polarization.

Techniques: In Vitro, Generated, Staining, Flow Cytometry, Control, Comparison, Expressing

Journal: mAbs

Article Title: Highly potent anti-CD20-RLI immunocytokine targeting established human B lymphoma in SCID mouse

doi: 10.4161/mabs.28699

Figure Lengend Snippet: Figure 4. Cytokine-dependent functional effects of anti-CD20-RLI. (A) Kit225 or (B) 32Dβ cell proliferation induced by increasing concentrations of human IL-15 (▲), RLI (large-black-square) or anti-CD20-RLI (black-square) was assessed by Alamar blue reduction assay. (C) Phosphorylation of STAT5 was evaluated in 32Dβ cells stimulated during 30min by increasing concentrations of human IL-15 (▲), RLI (large-black-square) or anti-CD20-RLI (black-square). Data are means ± SEM of three experiments.

Article Snippet: ScreenSureFire STAT5 (p-Tyr694/699) Assays Kit (TGRS5S500) was from PerkinElmer.

Techniques: Functional Assay, Phospho-proteomics