Journal: Frontiers in Immunology

Article Title: MicroRNA-1 Negatively Regulates Peripheral NK Cell Function via Tumor Necrosis Factor-Like Weak Inducer of Apoptosis (TWEAK) Signaling Pathways During PPRV Infection

doi: 10.3389/fimmu.2019.03066

Figure Lengend Snippet: Regulation of NK cell function by miR-1 is mediated by TWEAK signaling. (A) Goat CD16 + CD14 − NK cells were transfected with control miRNA (MC) or miR-1 mimics, or miR-1 mimics and incubated with recombinant TWEAK at the final concentration of 50 nM. Forty-eight hours later, the cells were infected with PPRV at an MOI of 1, and 24 h later, the protein expression of NKG2D, perforin, IFN-γ, and TNF-α was measured by Western blot. Histograms of the right panel showed densitometric analysis of indicated protein normalized to GAPDH to correct for protein loading. (B) Goat CD16 + CD14 − NK cells were transfected with lentivirus TWEAK, si-TWEAK, or respective negative control. Forty-eight hours later, the cells were infected with PPRV at an MOI of 1, and 24 h later, the protein levels of SOCS-1, MyD88, STAT3, pSTAT3, and NF-κB p65 in NK cells were determined by Western blot and normalized to the levels of GAPDH. Results are expressed as means ± standard error of mean (SEM). P -values were calculated using Student's t -test. An asterisk indicates a comparison with the indicated control. * P < 0.05; ** P < 0.01. n.s., not significant.

Article Snippet: For downstream signaling pathway detection, membranes were probed overnight with rabbit anti-SOCS1 (Sangon Biotech, Shanghai, China), rabbit anti-NF-κB p65 (Bioss, Beijing, China), rabbit anti-MyD88 (Bioss, Beijing, China), rabbit anti-STAT3 (Bioss, Beijing, China), or rabbit anti-pSTAT3 (Bioss, Beijing, China).

Techniques: Cell Function Assay, Transfection, Incubation, Recombinant, Concentration Assay, Infection, Expressing, Western Blot, Negative Control

Journal: bioRxiv

Article Title: An IL-1β driven neutrophil-stromal cell axis fosters a BAFF-rich microenvironment in multiple myeloma

doi: 10.1101/2023.03.03.530773

Figure Lengend Snippet: A) Transcription of STAT3, SOCS3 and BCL3 in cells in cluster MatNeu2 comparing control individuals and NDMM patients B) Transcription of genes associated with STAT3-signaling in neutrophils cultured on MSC or on iMSC (n = 3 donors, collected in single experiment). HD, healthy donor C) Representative histogram of expression of phosphorylated STAT3 (pSTAT3) from CD10 + neutrophils cultured in the presence of G-CSF (positive control), cultured alone, cultured on non-inflammatory stroma (‘MSC’), or cultured on iMSC, as determined by flow cytometry. Fold change in mean fluorescent intensity (MFI) compared to neutrophils cultured alone (dotted line) was quantified per individual of each condition. Lines depict paired samples (n = 6, collected over 6 experiments) D) Frequencies of CD10 + neutrophils expressing C3AR, CD11b(act), and CD11c, or mean fluorescent intensity (MFI) of CD66b and CD45 on CD10 + neutrophils cultured on non-inflammatory stroma (‘+ MSC’) or on iMSC in the presence of DMSO or Stattic. Lines depict paired samples (n = 6, collected over 3 experiments) E) BAFF protein in supernatant of neutrophils cultured on non-inflammatory stroma (‘+ MSC’) or on iMSC in the presence of DMSO or Stattic. Lines depict paired samples (n = 6, collected over 3 experiments). F) IL-1β protein in supernatant of neutrophils cultured on non-inflammatory stroma (‘+ MSC’) or on iMSC in the presence of DMSO or Stattic. Lines depict paired samples (n = 5, collected over 3 experiments) G) Volcano plot depicting differentially expressed genes of MSC cultured with iMSC-conditioned neutrophils versus MSC cultured with MSC-conditioned neutrophils. Log 2 FoldChange cutoff, 1; adjusted p-value (padj) cutoff 10 -3 . Genes in red are related to iMSCs. (n = 5, collected over 3 experiments) H) Transcription (transcripts per million, TPM) of iMSC-related genes in MSC cultured with MSC-conditioned neutrophils (green) and MSC cultured with iMSC-conditioned neutrophils (blue). Dotted lined depict paired samples. (n = 5, collected over 3 experiments) I) Enrichment plots for the iMSC-signature (generated from DEGs of single cell RNA sequencing results in 4 ), and iMSC-like cells signature (generated from data in ) comparing MSC cultured with iMSC-conditioned neutrophils and MSC-conditioned neutrophils J) Transcription of iMSC-related genes in MSC cultured with iMSC-conditioned neutrophils in the presence of anti-IL-1β (grey) and in the presence of an isotype control (orange). Dotted lined depict paired samples (n = 5, collected over 3 experiments) K) Enrichment plots for the iMSC-signature (generated from DEGs of single cell RNA sequencing results in 4 ), and iMSC-like cells signature (generated from data in ) comparing MSC cultured with iMSC-conditioned neutrophils in the presence of anti-IL-1β or in the presence of an isotype control. Data are presented as mean ± SEM. Significance was calculated in B , G , H and J using the Wald test (two-tailed) followed by a Benjamini– Hochberg correction, and in C , D , E and F using the Wilcoxon Rank Sum test (two-tailed); *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001, ****P ≤ 0.0001, NS P > 0.05.

Article Snippet: After permeabilisation, cells were washed twice and stained with pSTAT3[Tyr705]-PE (1:10; 13A3-1, BioLegend) at room temperature.

Techniques: Control, Cell Culture, Expressing, Positive Control, Flow Cytometry, Generated, RNA Sequencing, Two Tailed Test

Journal: Frontiers in Immunology

Article Title: Dietary phytate primes epithelial antibacterial immunity in the intestine

doi: 10.3389/fimmu.2022.952994

Figure Lengend Snippet: Epithelial STAT3 activation in the intestine is induced by phytate. (A) Interaction network of phytate-induced IEC defense genes. (B) Top hub genes in (A) . (C) Heatmap of relative mRNA expression of IEC STAT3-target genes upregulated in phytate-treated. (D) Representative flow cytometry plots from IECs of vehicle- or 2% phytate-treated mice, gated on live EpCAM + cells. (E, F) Mean Fluorescence Intensity (MFI) of pSTAT3 (Y705), normalized to vehicle (E) or control (F) . Data are representative of 2-3 independent experiments. n=4 per group. Results are mean ± SEM. ** p < 0.01.

Article Snippet: Cells were stained using the following fluorescence-conjugated monoclonal antibodies diluted in FACS buffer (2% FBS, 0.01% sodium azide, PBS): Brilliant Violent 711 anti-CD326 (EpCAM) (Clone: G8.8, BD Biosciences), PE anti-pSTAT3 (Tyr 705) (Clone: LUVNKLA, Invitrogen), PE anti-Mouse IgG2bκ isotype (Clone: eBMG2b, Invitrogen).

Techniques: Activation Assay, Expressing, Flow Cytometry, Fluorescence

Journal: Frontiers in Immunology

Article Title: Dietary phytate primes epithelial antibacterial immunity in the intestine

doi: 10.3389/fimmu.2022.952994

Figure Lengend Snippet: Phytate-mediated epithelial STAT3 activation requires HDAC3. (A) Intestinal epithelial HDAC activity of vehicle- or phytate-treated mice. (B) Representative flow cytometry plots from IECs of vehicle- or 2% phytate-treated mice, gated on live EpCAM + cells. (C) Mean Fluorescence Intensity (MFI) of pSTAT3 (Y705), normalized to FF. (D) MFI of IEC pSTAT3 of vehicle- or 2% phytate-treated HDAC3 FF or HDAC3 ΔIEC mice, normalized to FF-vehicle. (E) Through phytate metabolism by microbial phytases, rice bran and its component phytate activate IEC STAT3 and downstream defense mechanisms against enteric infection. Data are representative of 2-3 independent experiments. n=4 per group. Results are mean ± SEM. * p < 0.05, ** p < 0.01, *** p < 0.001.

Article Snippet: Cells were stained using the following fluorescence-conjugated monoclonal antibodies diluted in FACS buffer (2% FBS, 0.01% sodium azide, PBS): Brilliant Violent 711 anti-CD326 (EpCAM) (Clone: G8.8, BD Biosciences), PE anti-pSTAT3 (Tyr 705) (Clone: LUVNKLA, Invitrogen), PE anti-Mouse IgG2bκ isotype (Clone: eBMG2b, Invitrogen).

Techniques: Activation Assay, Activity Assay, Flow Cytometry, Fluorescence, Infection