NKp46 deficiency does not affect ILC2s and ILC3s.

(A) Gating strategy for ILC2s and ILC3s. ILC2s were gated on CD45+Lin─CD127+Gata3+, and ILC3s were gated on CD45+Lin─CD127+RORγt+. (B) Percentages of ILC2s or ILC3s were analyzed by flow cytometry in SI in Ncr1gfp/gfp mice and Ncr1+/+ littermates. ILC2s were gated on CD45+Lin─CD127+Gata3+RORγt─ lymphocytes. ILC3s were gated on CD45+Lin─CD127+RORγt+Gata3─ lymphocytes. (C) Quantities of ILC2s or ILC3s were determined in SI of Ncr1gfp/gfp mice and their Ncr1+/+ littermates (n = 4). (D) Lin─(or CD3─CD19─)NK1.1+NKp46+(or GFP+ for Ncr1gfp/gfp mice)CD49b+ NK cells were sorted from the spleen of Ncr1gfp/gfp mice or Ncr1+/+ littermates and were co-stimulated with IL-12 (10 ng/ml) and IL-18 (10 ng/ml) for 16 h, followed by the measurement of IFN-γ production by intracellular flow cytometric analysis (D, left panel, n = 3) or ELISA assays (D, right panel, n = 3). Golgi Plug was added at a 1:1,000 dilution to the culture 4 h prior to cell harvesting. (E) Homogenized SI cells isolated from Ncr1gfp/gfp mice or Ncr1+/+ littermates were stimulated with IL-23 (10 ng/ml) for 4 h, followed by the measurement of IL-22 production by flow cytometric analysis after gating ILC3s on CD45+Lin─CD127+RORγt+. Golgi Plug was added at a 1:1,000 dilution to the culture 3 h prior to cell harvesting. Error bars, standard deviations. The numerical data for panel C and D can be found in S1 Data. ELISA, Enzyme-linked immunosorbent assay; FSC-A, forward scatter area; FSC-H, FSC height; IFN, interferon; IL, interleukin; ILC, innate lymphoid cell; Ncr1, natural cytotoxicity receptor 1; NK, natural killer; NS, no significance; RORγt, retinoic acid receptor (RAR) related orphan receptor gamma t; SI, small intestine; SSC-A, side scatter area; SSC-H, SSC height; SSC-W, SSC width.</p

Dependence of innate lymphoid cell 1 development on NKp46

<div><p>NKp46, a natural killer (NK) cell–activating receptor, is involved in NK cell cytotoxicity against virus-infected cells or tumor cells. However, the role of NKp46 in other NKp46⁺ non-NK innate lymphoid cell (ILC) populations has not yet been characterized. Here, an NKp46 deficiency model of natural cytotoxicity receptor 1 (<i>Ncr1</i>)^gfp/gfp and <i>Ncr1</i>^gfp/+ mice, i.e., homozygous and heterozygous knockout (KO), was used to explore the role of NKp46 in regulating the development of the NKp46⁺ ILCs. Surprisingly, our studies demonstrated that homozygous NKp46 deficiency resulted in a nearly complete depletion of the ILC1 subset (ILC1) of group 1 ILCs, and heterozygote KO decreased the number of cells in the ILC1 subset. Moreover, transplantation studies confirmed that ILC1 development depends on NKp46 and that the dependency is cell intrinsic. Interestingly, however, the cell depletion specifically occurred in the ILC1 subset but not in the other ILCs, including ILC2s, ILC3s, and NK cells. Thus, our studies reveal that NKp46 selectively participates in the regulation of ILC1 development.</p></div

NKp46 is required for ILC1 development.

<p>(A) Gating strategy for ILC1s. ILC1s were gated on lymphocytes and then were further defined as Lin⁻NK1.1⁺NKp46⁺CD49b^─CD49a⁺, with the exception that GFP⁺ was used to replace NKp46⁺ when <i>Ncr1</i> ^gfp/+ or <i>Ncr1</i>^gfp/gfp mice were used. (B) Percentages or quantities of ILC1s were determined by flow cytometric analysis in the liver of <i>Ncr1</i>^gfp/gfp, <i>Ncr1</i>^gfp/+, and <i>Ncr1</i>^+/+ mice. (C) NK cells were gated on Lin^─NK1.1⁺NKp46⁺(or GFP⁺ for <i>Ncr1</i>^gfp/gfp mice)CD49b⁺CD49a^─ among lymphocytes. ILC1s were gated on Lin^─NK1.1⁺NKp46⁺(or GFP⁺ for <i>Ncr1</i>^gfp/gfp mice)CD49b^─CD49a⁺ among lymphocytes. (D) Quantification of ILC1s in different organs or tissues in <i>Ncr1</i>^gfp/gfp mice and <i>Ncr1</i>^+/+ littermates, i.e., summary data for (C). Each line demonstrates percentages of ILC1s for a pair of <i>Ncr1</i>^gfp/gfp and <i>Ncr1</i>^+/+ littermates. (E) Quantities of NK cells or ILC1s in different organs of <i>Ncr1</i>^gfp/gfp mice and their <i>Ncr1</i>^+/+ littermates (<i>n</i> = 4). Error bars, standard deviations; ***, <i>p</i> < 0.001; **, <i>p</i> < 0.01; *, <i>p</i> < 0.05. The numerical data for panels B, D and E can be found in <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.2004867#pbio.2004867.s007" target="_blank">S1 Data</a>. Lin^─, CD3^─CD19^─; BM, bone marrow; FSC-A, forward scatter area; FSC-H, FSC height; FSC-W, FSC width; GFP, green fluorescent protein; ILC1, innate lymphoid cell 1; NK, natural killer; <i>Ncr1</i>, natural cytotoxicity receptor 1; SI, small intestine; SSC-A, side scatter area; SSC-H, SSC height; SSC-W, SSC width.</p

NKp46 plays a cell-intrinsic role in regulating ILC1 development.

<p>(A) Scheme of BM transplantation using BM cells of CD45.2 <i>Ncr1</i>^gfp/gfp mice or <i>Ncr1</i>^+/+ littermate controls as donor cells to inject into CD45.1 recipients via tail vein. Development of ILC subsets was analyzed 2 weeks after transplantation. (B) Percentages of CD45.2⁺ NK cells or CD45.2⁺ ILC1s were analyzed by flow cytometric analysis in the liver of CD45.1 recipients, which were engrafted with BM cells of <i>Ncr1</i>^gfp/gfp mice (<i>n</i> = 5) or <i>Ncr1</i>^+/+ littermates (<i>n</i> = 4). (C) Percentages of CD45.2⁺ NK cells or CD45.2⁺ ILC1s were analyzed in the spleen or BM of CD45.1 recipient mice, which were engrafted with BM cells of <i>Ncr1</i>^gfp/gfp mice (<i>n</i> = 5) or their <i>Ncr1</i>^+/+ littermates (<i>n</i> = 4). (D and E) Data shown are representative dot plots of flow cytometric analysis (left panel) and summary data (right panel) of CD45.2⁺ILC2 (D) or CD45.2⁺ ILC3 (E) in SI in CD45.1 recipients, which were engrafted with BM cells of <i>Ncr1</i>^gfp/gfp mice (<i>n</i> = 4) or their <i>Ncr1</i>^+/+ littermates (<i>n</i> = 4). Error bars, standard deviations; ***, <i>p</i> < 0.001; **, <i>p</i> < 0.01; *, <i>p</i> < 0.05. The numerical data for panels B, C, D and E can be found in <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.2004867#pbio.2004867.s007" target="_blank">S1 Data</a>. Lin^─, CD3^─CD19^─; BM, bone marrow; ILC, innate lymphoid cells; <i>Ncr1</i>, natural cytotoxicity receptor 1; NK, natural killer; RORγt, retinoic acid receptor (RAR) related orphan receptor gamma t; SI, small intestine.</p

Absence of TRAIL⁺ ILC1s in NKp46-deficient mice.

<p>(A) Percentages of TRAIL⁺ ILC1s were analyzed by flow cytometric analysis in the liver of <i>Ncr1</i>^gfp/gfp mice and their <i>Ncr1</i>^+/+ littermates. ILC1s were gated on Lin^─NK1.1⁺NKp46⁺(or GFP⁺ for <i>Ncr1</i>^gfp/gfp mice) TRAIL⁺CD49b^─ among lymphocytes. (B) Quantification of TRAIL⁺ ILC1s in the liver of <i>Ncr1</i>^gfp/gfp mice and their <i>Ncr1</i>^+/+ littermates for (A) (<i>n</i> = 5). (C) Quantification of TRAIL⁺ ILC1s in other organs (spleen, <i>n</i> = 5; BM, <i>n</i> = 5; SI, <i>n</i> = 4) of <i>Ncr1</i>^gfp/gfp mice and their <i>Ncr1</i>^+/+ littermates. **, <i>p</i> < 0.01; *, <i>p</i> < 0.05. The numerical data for panels B and C can be found in <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.2004867#pbio.2004867.s007" target="_blank">S1 Data</a>. Lin^─, CD3^─CD19^─; BM, bone marrow; GFP, green fluorescent protein; ILC1, innate lymphoid cell 1; <i>Ncr1</i>, natural cytotoxicity receptor 1; NK, natural killer; SI, small intestine; TRAIL, tumor necrosis factor–related apoptosis-inducing ligand.</p

Data_Sheet_2_CD163ΔSRCR5 MARC-145 Cells Resist PRRSV-2 Infection via Inhibiting Virus Uncoating, Which Requires the Interaction of CD163 With Calpain 1.zip

Porcine alveolar macrophages without the CD163 SRCR5 domain are resistant to porcine reproductive and respiratory syndrome virus (PRRSV) infection. However, whether the deletion of CD163 SRCR5 in MARC-145 cells confers resistance to PRRSV and interaction of which of the host proteins with CD163 is involved in virus uncoating remain unclear. Here we deleted the SRCR5 domain of CD163 in MARC-145 cells using CRISPR/Cas9 to generate a CD163ΔSRCR5 MARC-145 cell line. The modification of CD163 had no impact on CD163 expression. CD163ΔSRCR5 cells were completely resistant to infection by PRRSV-2 strains Li11, CHR6, TJM, and VR2332. The modified cells showed no cytokine response to PRRSV-2 infection and maintained normal cell vitality comparable with the WT cells. The resistant phenotype of the cells was stably maintained through cell passages. There were no replication transcription complexes in the CD163ΔSRCR5 cells. SRCR5 deletion did not disturb the colocalization of CD163 and PRRSV-N in early endosomes (EE). However, the interaction of the viral proteins GP2a, GP3, or GP5 with CD163, which is involved in virus uncoating was affected. Furthermore, 77 CD163-binding cellular proteins affected by the SRCR5 deletion were identified by LC–MS/MS. Inhibition of calpain 1 trapped the virions in EE and forced then into late endosomes but did not block viral attachment and internalization, suggesting that calpain 1 is involved in the uncoating. Overall, CD163ΔSRCR5 MARC-145 cells are fully resistant to PRRSV-2 infection and calpain 1 is identified as a novel host protein that interacts with CD163 to facilitate PRRSV uncoating.</p

Data_Sheet_1_CD163ΔSRCR5 MARC-145 Cells Resist PRRSV-2 Infection via Inhibiting Virus Uncoating, Which Requires the Interaction of CD163 With Calpain 1.pdf

Porcine alveolar macrophages without the CD163 SRCR5 domain are resistant to porcine reproductive and respiratory syndrome virus (PRRSV) infection. However, whether the deletion of CD163 SRCR5 in MARC-145 cells confers resistance to PRRSV and interaction of which of the host proteins with CD163 is involved in virus uncoating remain unclear. Here we deleted the SRCR5 domain of CD163 in MARC-145 cells using CRISPR/Cas9 to generate a CD163ΔSRCR5 MARC-145 cell line. The modification of CD163 had no impact on CD163 expression. CD163ΔSRCR5 cells were completely resistant to infection by PRRSV-2 strains Li11, CHR6, TJM, and VR2332. The modified cells showed no cytokine response to PRRSV-2 infection and maintained normal cell vitality comparable with the WT cells. The resistant phenotype of the cells was stably maintained through cell passages. There were no replication transcription complexes in the CD163ΔSRCR5 cells. SRCR5 deletion did not disturb the colocalization of CD163 and PRRSV-N in early endosomes (EE). However, the interaction of the viral proteins GP2a, GP3, or GP5 with CD163, which is involved in virus uncoating was affected. Furthermore, 77 CD163-binding cellular proteins affected by the SRCR5 deletion were identified by LC–MS/MS. Inhibition of calpain 1 trapped the virions in EE and forced then into late endosomes but did not block viral attachment and internalization, suggesting that calpain 1 is involved in the uncoating. Overall, CD163ΔSRCR5 MARC-145 cells are fully resistant to PRRSV-2 infection and calpain 1 is identified as a novel host protein that interacts with CD163 to facilitate PRRSV uncoating.</p

11-Mercaptoundecanoic Acid-Functionalized Carbon Dots As a Ratiometric Optical Probe for Doxorubicin Detection

A ratiometric optical dual-mode probe was first designed for the detection of doxorubicin (DOX) based on 11-mercaptoundecanoic acid-functionalized carbon dots (MUA-CDs). The carbon dots (CDs) were synthesized via hydrothermal carbonization of m-phenylenediamine and p-aminobenzoic acid, and then it was reacted with 11-mercaptoundecanoic acid by the amide reaction to obtain the MUA-CDs. The as-synthesized MUA-CDs have a high fluorescence quantum yield (59.4%) with bright green emission. Based on a specific electrostatic interaction and the fluorescence resonance energy transfer (FRET) between MUA-CDs and DOX, a ratiometric optical dual-mode probe was constructed for the detection of DOX with good selectivity, high sensitivity, and excellent anti-interference performance. When DOX was mixed the MUA-CDs, the fluorescence of the MUA-CDs at 513 nm decreased, while the fluorescence at 590 nm increased. With this information, a ratiometric fluorescent method was established to determine DOX with a linear range and limit of detection (LOD) of 0.25–19.96 and 0.66 μM and 50.33–80.88 and 0.23 μM, respectively. Meanwhile, the color of the MUA-CDs varied from colorless to dark yellow in the presence of DOX, and therefore, a ratiometric colorimetric method was applied to determine DOX in a range of 2.50–29.80 μM with a LOD of 0.75 μM. Finally, the as-constructed probe was successfully used to determine DOX in serum, urine, and live cells with satisfactory results. This work will contribute to the specific design of functional CDs and broaden the application of functional CDs in analytical detection and biological imaging

Additional file 5: Figure S3. of Naringenin prevents TGF-β1 secretion from breast cancer and suppresses pulmonary metastasis by inhibiting PKC activation

Showing the effects of naringenin (Nar) on the expression of TGF-β1 and Foxp3 in vivo. A, B TGF-β1 concentrations in the homogenates of tumor, spleen, lung, and serum from mice after treatment for 14 and 28 days. Mice bearing 4T1/RFP tumors or 4T1/TGF-β1 tumors were treated with Nar for 14 and 28 days. The tissues were homogenated and collected for TGF-β1 detection by ELISA kit. C Immunohistochemical analysis of TGF-β1 expression and location in lung tissue sections of mice bearing 4T1/RFP tumors or 4T1/TGF-β1 tumors after treatment with Nar for 14 and 28 days. D Expression of TGF-β1 and Foxp3 proteins in lung tissues of mice bearing 4T1/RFP tumors or 4T1/TGF-β1 tumors after treatment with Nar for 14 and 28 days by western blot analysis. *P <0.05, **P <0.01, ***P <0.001. Error bars indicate SE. (TIF 1071 kb

Additional file 8: Figure S6. of Naringenin prevents TGF-β1 secretion from breast cancer and suppresses pulmonary metastasis by inhibiting PKC activation

Showing the effects of naringenin (Nar) on the transcription and decay of Tgf-β1 mRNA. A Secreted TGF-β1 concentrations in the media of cultured 4T1 cells with naringenin treatment for different times. B Analysis of mRNA levels of Tgf-β1 in cultured 4T1 cells with or without naringenin treatment for 24 and 48 hours. C Analysis of the stability of Tgf-β1 mRNA in 4T1 cells after naringenin treatment. Cells were pretreated with 100 μM naringenin or diluent for 2 hours, followed by addition of 10 μg/ml actinomycin D to inhibit the synthesis of new RNA. The levels of remained TGF-β1 mRNA were determined by qPCR. Data are from at least three independent experiments. *P <0.05, **P <0.01, ***P <0.001. Error bars indicate SE. (TIF 139 kb