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Ekareni Ufomine
Ekareni Ufomine

Against The Ice YTS

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Against The Ice YTS

CLEC16A overexpression impairs cytotoxicity, IFN-γ production and DC maturation, whereas siRNA mediated knockdown enhances the cytotoxicity in NK cells. (A) Transduced YTSeco cells were sorted via FACS into population with comparable GFP expression and cultures had consistent GFP expression over time. (B) Negative, YTS-GFP, and YTS-CLEC16A cells analyzed for CLEC16A and GFP expression in an immunoblot analysis. Membranes were stripped and re-probed for β-actin as a loading control (bottom panel). Quantitation graph depicting CLEC16A expression (top panel; n = 3 repeats). (C) Cytotoxicity of negative control (mock transduced YTS cells), YTS-GFP and YTS-CLEC16A NK cell lines against 51Cr-labeled 721.221 targets. Data represents means SE of five independent experiments. (D) Cytotoxicity of parental YTS and YTS-CLEC16A NK cells against 51Cr-labeled 721.221 target. Data represents meansSE of three independent experiments (E) Representative Western blot of YTS and YTS-CLEC16A cells for CLEC16A expression and β-actin (bottom panel). Quantitation graph depicting CLEC16A expression (top panel; n = 3 repeats). (F,I) CLEC16A overexpression limits IFN-γ production in YTS-CLEC16A. IFN-γ release measured by ELISA in YTS and YTS-CLEC16A NK cell lines at the indicated time points. Data represents means SE of three independent experiments. (F) Cell lines were cultured alone or with PMA and ionomycin (PMA+I). (G) YTS and YTS-CLEC16A cells were activated with plate bound anti-CD28 for the indicated time points shown. (H) YTS and YTS-CLEC16A cells activated with plate bound anti-NKp30 for the indicated time points shown. (I) YTS and YTS-CLEC16A cells activated with plate bound anti-CD226 for the indicated time points and IFN-γ release shown. (J) siRNA knockdown of CLEC16A in parental YTS cells. 721.221 target cell killing by YTS cells, 24 h post nucleofection of the YTS cells with either CLEC16A or Control siRNAs. Data represents means SE of three independent experiments. (K) Representative Western blot depicting CLEC16A expression after siRNA mediated knockdown in YTS cells. Quantitation graph depicting CLEC16A expression (top panel; n = 3 repeats). (l) siRNA mediated knockdown of CLEC16A in ex vivo NK cells and cytotoxicity. K562 target cell killing by ex vivo NK cells, 24 h after nucleofection of the ex vivo NK cells with either CLEC16A or Control siRNAs. Data represents means SE of three independent experiments. (M) Representative Western blot depicting CLEC16A protein expression after knockdown in ex vivo NK cells. Quantitation graphdepicting CLEC16A expression (top panel; n = 3 repeats). *P < 0.05, **P < 0.01, ***P < 0.001 (unpaired two-tailed Student's t-test).

We assessed the splenic immune cell populations by flow cytometry using a gating strategy as shown in Supplementary Figure S3. Clec16a KO mice show significant reduction in total numbers of B, T, NK cells, and macrophages (Figure 6A). Total splenocyte numbers and % immune cell types redistribution is shown in Supplementary Figure S3C. We recently demonstrated that TAM treated (Clec16a KO) mice show atrophy of thymus, spleen and significant reduction in total splenocytes numbers (19). We next evaluated NK cytotoxicity in the splenocytes of TAM treated and control mice, with and without rmIL-15 activation post 48 h in a standard 4-h 51Cr release assay. Resting murine NK cells are minimally cytotoxic against their targets and activation is required to induce cytotoxicity with IL-15. At an effector: target ratio of 50:1, resting control splenocytes exhibited only 7.6 1.8% (males) and 7.9 1.3% (females) of YAC-1 killing. In contrast, resting murine NK cells from TAM (Clec16a KO) male (14.5 1.8%) and female (14.31.8%) mice demonstrated a significant increase in cytotoxicity. Cytotoxicity dramatically increased after splenocytes were activated with IL-15. At an identical effector: target ratio of 50:1, control splenocytes demonstrated 40.6 1.0% (males) and 23.7 1.4% (females) cytotoxicity, respectively. TAM treated (Clec16a KO) mice exhibited even greater enhancement in cytotoxicity, 52.2 2.5% (males) and 39 2.1% (females). Loss of CLEC16A resulted in a significant increase in killing of YAC-1 targets by murine NK cells despite a decrease in numbers (Figures 6A,B).

Clec16a knockout mice depicts altered immune cell populations, increased splenic NK cell cytotoxicity, upregulated cytokine and chemokine secretion and an imbalance in dendritic cell subsets with altered receptor expression. (A) Reduced spleen cell numbers and altered splenic Immune cell population in Clec16a KO (TAM) mice (n = 12 mice per group). *P < 0.05, **P < 0.01, ***P < 0.001 (unpaired two-tailed Student's t-test). (B) Cytotoxicity of resting and mrIL-15 (100 ng/ml) activated male and female murine splenocytes against 51Cr-labeled YAC-1 targets. Results show mean SE of three independent repeats. *P < 0.05, **P < 0.01, ***P < 0.001 (unpaired two-tailed Student's t-test). (C) The representative graph is quantification of cytokines and chemokine from plasma of Control (Oil) and Clec16a KO (TAM) mice using the Mouse Cytokine Array panel. Data represents means SE of three independent experiments. *P < 0.05, **P < 0.01, ***P < 0.001 (unpaired two-tailed Student's t-test). (D) Percent splenic dendritic cells (pDCs and cDCs), percent DC subsets, MHC-II, CD80, and CD86 MFI on pDCs, cDCs, and subsets from Clec16a KO and control mice analyzed by flow cytometry (n = 10 mice per group). Data represents means SE of three independent experiments. *P < 0.05, **P < 0.01, ***P < 0.001 (unpaired two-tailed Student's t-test). (E) MFI quantification graph depicting adhesion, activating and inhibitory receptors expressed on NK cells of control and Clec16a KO mice. Data represents means SE of three independent experiments (n = 12 mice per group). *P < 0.05, **P < 0.01, ***P < 0.001 (unpaired two-tailed Student's t-test).

In October 2015, the YIFY website went down, with no word from any of the staff or those connected with the site, and no new YIFY releases. It was confirmed on October 30, 2015, that YIFY/YTS was shut down permanently.[15][16] The site was shut down due to a lawsuit coming from the Motion Picture Association of America (MPAA).[17][18] They filed a multi-million-dollar lawsuit against the website's operator, accusing him of "facilitating and encouraging massive copyright infringement". This news came as a surprise to some, such as a spokesman for the New Zealand Screen Association who would have expected the site to have been operating from Eastern Europe, the case with some other past websites.[19] Swery was able to settle out of court a month later, signing a non-disclosure agreement.[20] Yiftach did not resist legal action in any way, and co-operated with authorities as needed. In a 2016 Reddit AMA, Yiftach justified this saying that he never intended to "put up a fight", and had frequently told himself "When someone asks you to stop properly, you stop".[21]

In addition to the proposed consent judgment against YTS, the seven movie companies also agreed to a similar deal with the operator of This torrent site was considerably smaller and shut down months ago, however, the operator also agreed to pay $1,050,000 in damages, on paper.

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