Journal: bioRxiv

Article Title: Quantitative proteomics and phosphoproteomics reveal glucocorticoid stimulation of TLR and Rho GTPase signaling in neutrophil-like cells

doi: 10.1101/2025.06.25.661639

Figure Lengend Snippet: A) GR mRNA levels represented as normalized transcript per million (nTPM) in human immune cell lines. RNA-seq data was acquired from the Human Protein Atlas (HPA). B-C) Cell viability assay of HL-60 cells (B) and NLCs (C) after dose-dependent titration of DEX for 24 hours. D) Schematic illustration of proteome and phosphoproteome workflow after time-course treatment of 1 µM GC treatments in HL-60 cells and NLCs. Spectronaut logo was obtained from Biognosys AG and Orbitrap Astral MS image was obtained from Thermo Fisher Scientific. Illustration was created with Biorender.com. (E-F) Venn diagrams showing overlaps between DEX and PRED treatments in proteome (E) and phosphoproteome (F) profiles. (G-H) Bar plots showing the number of proteins (G) and phosphosites (H) with statistically significant regulations compared to 0 hour control using one-way ANOVA and Tukey’s HSD correction (adjusted p -value < 0.05). (I-J) Box plots showing individual log 2 transformed label-free quantification of GR phosphorylation at Thr-8 and total form after DEX (I) and PRED (J) treatments. (K) Box plots showing log 2 transformed label-free quantification of FKBP5 protein expression. One-way ANOVA with Dunnette’s multiple comparisons test was used for statistical tests.

Article Snippet: MS quantifications were median normalized and statistical analysis was performed using one-way analysis of variance (ANOVA) followed by Tukey’s Honestly Significant Difference (HSD) post hoc test using R or one-way ANOVA with Dunnette’s multiple comparisons test using GraphPad Prism 10.

Techniques: RNA Sequencing, Viability Assay, Titration, Control, Transformation Assay, Quantitative Proteomics, Phospho-proteomics, Expressing

Journal: bioRxiv

Article Title: Quantitative proteomics and phosphoproteomics reveal glucocorticoid stimulation of TLR and Rho GTPase signaling in neutrophil-like cells

doi: 10.1101/2025.06.25.661639

Figure Lengend Snippet: A) Soft clustering analysis with statistically significant proteins (adjusted p -value < 0.05, one-way ANOVA with Tukey’s HSD correction) after DEX treatment in NLCs. Mfuzz clustering package was used with fuzzification factor m = 1.5 and cluster number 8. B) Functional enrichment of proteins in cluster 1 using string analysis after DEX treatment. Dot size represents the number of proteins and dot color represents FDR values. Heatmap represents proteins in TLR cascades with z-scored log 2 transformed label-free quantification, colors showing z scores between −1 and 1. C) Scatter plot comparing protein expression changes after 24 hours treatment of DEX (x-axis) and PRED (y-axis) relative to 0 h control. Labeled dots represent proteins significantly regulated (adjusted p -value < 0.05 and log 2 FC > |1|) after one-way ANOVA with Tukey’s HSD correction in either DEX or PRED treatment. D-F) Box plots showing individual log 2 transformed label-free quantification of TLR2 (D), PER1 (E), and FPR1 (F) protein expressions after DEX and PRED treatments. One-way ANOVA with Dunnette’s multiple comparisons test was used.

Article Snippet: MS quantifications were median normalized and statistical analysis was performed using one-way analysis of variance (ANOVA) followed by Tukey’s Honestly Significant Difference (HSD) post hoc test using R or one-way ANOVA with Dunnette’s multiple comparisons test using GraphPad Prism 10.

Techniques: Functional Assay, Transformation Assay, Quantitative Proteomics, Expressing, Control, Labeling

Journal: bioRxiv

Article Title: Quantitative proteomics and phosphoproteomics reveal glucocorticoid stimulation of TLR and Rho GTPase signaling in neutrophil-like cells

doi: 10.1101/2025.06.25.661639

Figure Lengend Snippet: A) Immunoblotting analysis of MAPK signaling proteins after 24 hours dose-dependent treatment with PRED and DEX (0.1, 0.5, 1, 5, 10 µM). B) Immunoblotting analysis of MAPK signaling proteins after time-dependent treatment of 1 µM DEX (1, 2, 4, 8 hours). C) Box plots showing individual log 2 transformed label-free quantification of ADAM17 phosphorylation at Thr-735 and protein expression. D) Kinase activity analysis using RoKAI app after treatment of DEX. Bar color represents z-scored kinase activity (0 to 5). E-F) Box plots showing individual log 2 transformed label-free quantification of phosphorylation and protein expressions of MYH9 (E) and CXCR4 (F) after DEX treatment. G) Flow cytometry analysis of 2 and 24 hours DEX treatments in NLCs. Cells were stained with CD15, CD45, and CXCR4 (CD182). CD15 neg CD45 High cell populations are highlighted under live cell gate. Bar plot shows the number of CD15 neg CD45 High cells after DEX treatment and histogram represents CXCR4 expression of CD15 neg CD45 High cells. One-way ANOVA with Dunnette’s multiple comparisons test was used.

Article Snippet: MS quantifications were median normalized and statistical analysis was performed using one-way analysis of variance (ANOVA) followed by Tukey’s Honestly Significant Difference (HSD) post hoc test using R or one-way ANOVA with Dunnette’s multiple comparisons test using GraphPad Prism 10.

Techniques: Western Blot, Transformation Assay, Quantitative Proteomics, Phospho-proteomics, Expressing, Activity Assay, Flow Cytometry, Staining