pstat3 tyr705 pe  (Revvity)

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