Journal: Journal of Biological Chemistry

Article Title: Intracellular Interaction of Interleukin-15 with Its Receptor α during Production Leads to Mutual Stabilization and Increased Bioactivity

doi: 10.1074/jbc.m705725200

Figure Lengend Snippet: FIGURE 2. IL-15 stabilizes IL-15R. Human 293 cells were transfected with plasmids expressing 0.05 g IL-15R (A) or IL-15sR (B) alone or in combina- tion with 0.05 g of IL-15t, as indicated. After 72 h, IL-15R production in the culture supernatants and cell extracts was analyzed by Western immunoblot using a goat anti-IL-15R antibody. Sample dilutions of 1:2 and 1:4 were loaded as indicated to quantify the produced IL-15R. Mock indicates trans- fectionwithacontrolplasmidonly(GFP).Theamountofundilutedcellextract and culture supernatants loaded on the gel were 1:200 and 1:300, respec- tively. Arrows indicate the position of IL-15R bands, and arrowheads indicate the position of molecular weight markers. Similar transfection efficiencies were verified by co-transfection of GFP expression vectors. C, mRNA expres- sion levels of IL-15 and IL-15R plasmids transfected individually or together in human 293 cells. The expression of cellular gene GAPDH was used as con- trol (lower panel). The location of mRNAs was identified by Northern blot analysis and is indicated by arrows.

Article Snippet: Human IL-15 levels were measured by ELISA (Quantikine human IL-15 immunoassay; R & D Systems) or by Western immunoblot (using the polyclonal goat anti-human IL-15 antibody AF315, R & D Systems).

Techniques: Transfection, Expressing, Western Blot, Produced, Molecular Weight, Cotransfection, Northern Blot

Journal: Journal of Biological Chemistry

Article Title: Intracellular Interaction of Interleukin-15 with Its Receptor α during Production Leads to Mutual Stabilization and Increased Bioactivity

doi: 10.1074/jbc.m705725200

Figure Lengend Snippet: FIGURE4.IntracellularassociationofIL-15withtheIL-15R.A,human293 cells were transfected with IL-15-expressing plasmid (IL-15t) alone or in com- bination with IL-15R-expressing plasmid producing either full-length (IL- 15R) or soluble form (IL-15sR). Complexes were immunoprecipitated from cell extracts or culture medium with an anti-IL-15R antibody and were sub- sequently examined by Western immunoblot analysis using an anti-IL-15 antibody. The three bands corresponding to the unglycosylated, partially, and fully N-glycosylated forms of IL-15 are indicated. B, human 293 cells were transfected with 0.05 g of IL-15FLAG DNA alone, IL-15R DNA alone, or IL-15FLAGIL-15RDNAsincombination.Cellstransfectedwiththeindivid- ual plasmids were harvested and washed separately at 4 °C and divided into two tubes. Recombinant human IL-15 protein was added to the cells as indi- cated using 50 excess of the expected IL-15 production. The cells were lysed, and IL-15 was immunoprecipitated using anti-FLAG antibody. The complexes were separated on SDS-PAGE, and the IL-15R was visualized by Western immunoblot. The positions of the glycosylated and unglycosylated IL-15R are indicated.

Article Snippet: Human IL-15 levels were measured by ELISA (Quantikine human IL-15 immunoassay; R & D Systems) or by Western immunoblot (using the polyclonal goat anti-human IL-15 antibody AF315, R & D Systems).

Techniques: Transfection, Expressing, Plasmid Preparation, Immunoprecipitation, Western Blot, Recombinant, SDS Page

Journal: Cancer research

Article Title: IL15RA drives antagonistic mechanisms of cancer development and immune control in lymphocyte-enriched triple-negative breast cancers.

doi: 10.1158/0008-5472.CAN-14-0637

Figure Lengend Snippet: Figure 4. IL15 is expressed by TNBCs and basal BCCLs and its silencing specifically impairs growth in high IL15RA–expressing cell lines. Correlation of gene- expression levels between IL15RA and IL15 across A, 579 TNBC (GSE31519) and B, 51 BCCLs. The Pearson r values and P values for significance are shown. C, relative gene expression of IL15 in 51 BCCL dichotomized into the basal- like (triangles) and luminal-like (dots) phenotypes, whereby each symbol represents a cell line. D, ELISA analysis of IL15 levels in a subset of basal or luminal BCCLs. HaCat cells were included as a positive control. IL15 levels were assessed in whole cells (lysate). E to G, cell population growth assays of MDA-MB-231 (E), HCC1143 (F), (high levels of both IL15 and IL15RA), and SKBR3 (G) cells (low levels of both IL15 and IL15RA) following IL15 knockdown by two different shRNA species (IL15 sh26 and IL15 sh27). H, caspase-3/7 activation was assessed in HCC1143 cells transduced with a control shRNA (nontargeting) or shRNAs targeting IL15, 24 hours after seeding the cells for the cell population growth assay, equivalent to 7 days posttransduction. I, quantification of shRNA-mediated IL15 knockdown in MDA-MB-231 cells by qPCR. Data representative of at least two independent experiments; values are average SD; sample size, 3; , P < 0.05; , P < 0.01; , P < 0.001.

Article Snippet: When indicated, a blocking anti-IL15 monoclonal mouse antibody (MAB2471; R&D Systems) was added at 10 mg/mL to BCCLs 30 minutes before coculture with PBMC.

Techniques: Expressing, Gene Expression, Enzyme-linked Immunosorbent Assay, Positive Control, Knockdown, shRNA, Activation Assay, Transduction, Control, Growth Assay

Journal: Cancer research

Article Title: IL15RA drives antagonistic mechanisms of cancer development and immune control in lymphocyte-enriched triple-negative breast cancers.

doi: 10.1158/0008-5472.CAN-14-0637

Figure Lengend Snippet: Figure 5. IL15 signals through IL15RA to promote phosphorylation of ERK1/2, JAK1, STAT1, STAT2, AKT, and PRAS40 in BCCLs. A, HCC1143 cells (high IL15RA), were stimulated with 100 ng/mL of IL15 for 15 minutes, then lysed and protein phosphorylation assessed using a human phospho- protein array. Phospho-protein patterns were consistent in two independent experiments; for a full description of the phospho- antibodies included in the array please refer to Supplementary Fig. S1. B, IL15 induced increased ERK1/2, AKT, STAT2, PRAS40, and STAT1 phosphorylation as assessed by densitometry in one representative experiment; error bars, SD of the densitometry readings from the experimental replicates. C, phospho-protein array results were validated by Western blotting using a different set of phospho-antibodies and a wider set of BCCLs: either high IL15RA, expressing such as HCC1954 and HCC1143, or low IL15RA, expressing such as SKBR3. D, Western blot analysis of JAK1 and JAK2 phosphorylation in response to IL15 stimulation in HCC1954, HCC1143, and MDA- MB-231 (high IL15RA) or SKBR3 (low IL15RA) cell lines. E, Western blot analysis of STAT3, STAT5, and STAT6 phosphorylation in response to IL15 stimulation in HCC1143 cells. F, Western blot analysis of JAK1 and ERK1/2 phosphorylation in response to IL15 stimulation following siRNA- mediated IL15RA knockdown (IL15R siRNA #0, #1, and #2) in HCC1954 cells; nontargeting siRNAs used as negative control.

Article Snippet: When indicated, a blocking anti-IL15 monoclonal mouse antibody (MAB2471; R&D Systems) was added at 10 mg/mL to BCCLs 30 minutes before coculture with PBMC.

Techniques: Phospho-proteomics, Protein Array, Western Blot, Expressing, Knockdown, Negative Control

Journal: Cancer research

Article Title: IL15RA drives antagonistic mechanisms of cancer development and immune control in lymphocyte-enriched triple-negative breast cancers.

doi: 10.1158/0008-5472.CAN-14-0637

Figure Lengend Snippet: Figure 6. IL15RA knockdown impairs motility of basal BCCLs by a JAK1/STAT1–dependent, ERK1/ 2–independent mechanism. A, MDA-MB-231 and HCC1954 cell migration was investigated using a standard Transwell assay in cells transfected with a control siRNA (nontargeting) and siRNAs targeting IL15RA (IL15R siRNA#0, IL15R siRNA#2). B, MDA-MB-231 cells were seeded on a thin layer of collagen type I and adhesion assessed following transfection with a control siRNA (nontargeting) and siRNAs targeting IL15RA (IL15R siRNA#0, IL15R siRNA#2). C, Western blot analysis showing efficacy of JAK1 knockdown 72 hours posttransfection. D, MDA- MB-231 and HCC1954 cell migration was investigated using a standard Transwell assay in cells transfected with a control siRNA (nontargeting) and siRNAs targeting JAK1 (JAK1 siRNA#47, JAK1 siRNA48). E, HCC1954 cell population growth was assessed in cells transfected with a control siRNA (nontargeting) and siRNAs targeting JAK1 (JAK1 siRNA#47, JAK1 siRNA48). F, the impact of JAK1 knockdown on IL15-induced phosphorylation of STAT1 and ERK1/2 was assessed in HCC1954 cells by Western blot analysis. G, quantitation by densitometry of the IL15-induced phosphorylation of STAT1 as showed in F. H, quantitation by densitometry of the IL15-induced phosphorylation of ERK1/2 as showed in F. Data are representative of at least two independent experiments; values are average SD; sample size, 3; , P < 0.05; , P < 0.01; , P < 0.001.

Article Snippet: When indicated, a blocking anti-IL15 monoclonal mouse antibody (MAB2471; R&D Systems) was added at 10 mg/mL to BCCLs 30 minutes before coculture with PBMC.

Techniques: Knockdown, Migration, Transwell Assay, Transfection, Control, Western Blot, Phospho-proteomics, Quantitation Assay

Journal: Cancer research

Article Title: IL15RA drives antagonistic mechanisms of cancer development and immune control in lymphocyte-enriched triple-negative breast cancers.

doi: 10.1158/0008-5472.CAN-14-0637

Figure Lengend Snippet: Figure 7. IL15RA/IL15–expressing BCCLs elicit STAT5 phosphorylation in PBMC upon cocultivation. Flow-cytometry analysis of STAT5 phosphorylation in PBMC cocultured 1:1 with BCCLs or stimulated with recombinant IL15 (rIL15; 100 pg/mL) for 30 minutes. IL15-blocking antibody (aIL15) effects were assessed by adding 30 minutes before coculture. The effect of IL15RA knockdown was also assessed following transduction and selection of MDA-MB-231 cells with shRNAs targeting IL15RA (IL15R sh3) or IL15 (IL15 sh26 and IL15 sh27) as well as a control shRNA (nontargeting). Data are representative of at least two independent experiments; values are average SD; sample size, 3; , P < 0.05; , P < 0.01; , P < 0.001.

Article Snippet: When indicated, a blocking anti-IL15 monoclonal mouse antibody (MAB2471; R&D Systems) was added at 10 mg/mL to BCCLs 30 minutes before coculture with PBMC.

Techniques: Expressing, Phospho-proteomics, Flow Cytometry, Recombinant, Blocking Assay, Knockdown, Transduction, Selection, Control, shRNA