one-way anova with dunnette’s multiple comparisons test  (GraphPad Software Inc)

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

Journal: Nature Communications

Article Title: Modularity of Zorya defense systems during phage inhibition

doi: 10.1038/s41467-025-57397-2

Figure Lengend Snippet: a Efficiency of plating (EOP) measurement for E. coli MT56 carrying empty vector (VC, pGM39, a pT12 backbone vector with no insert ) or the same plasmid encoding Zorya I, ZorA I, ZorB I, ZorABC, ZorABD, ZorBDC, ZorAB I, ZorCD, ZorB D24N, ZorA IΔaa 237-696 , and ZorB IΔaa 43-287 when infected with ɸAlma. Points show mean ± SEM ( n = 3 biological replicates). Statistical significance for each panel was calculated with Graphpad applying a one-way ANOVA with Dunnett’s multiple comparison test. No significance was detected, unless indicated ( * p ≤ 0.05). For the VC vs Zorya I comparison, p -value = 0.0028. b Efficiency of plating (EOP) measurement for E. coli MT56 carrying the empty vector (VC, pGM39) or the same plasmid encoding Zorya II, ZorA II, ZorB II, ZorAB II, ZorE, ZorB D24N, ZorA IΔaa 106-550 and ZorB IΔaa 45-235 when infected with phage ɸT7. Points show mean ± SEM ( n = 3 biological replicates). Points show mean ± SEM ( n = 3 biological replicates). Statistical significance for each panel was calculated with Graphpad applying a one-way ANOVA with Dunnett’s multiple comparison test. No significance was detected, unless indicated ( * p ≤ 0.05). For VC vs Zorya II comparison, p -value = 0.00001852. Induction of each construct in panels ( a , b ) was performed by the addition of 0.02% l - Rhamnose. c Overlay of the ZorB PG-binding domain of type I ZorA 5 B 2 from Shewanella sp. strain ANA-3 (gray) with type II ZorA 5 B 2 from Sulfuricurvum kujiense (blue) and H. influenza peptidoglycan-associated lipoprotein (Pal; green) with bound peptidoglycan in yellow (PDB 2AIZ). Cα RMSD between Shewanella sp. strain ANA-3 ZorB PG-binding domain and 2AIZ is 2.43 Å; Cα RMSD between Sulfuricurvum kujiense ZorB PG-binding domain and 2AIZ is 2.78 Å; Cα RMSD between both species ZorB PG-binding domains is 1.93 Å. d Conserved residues cluster proximal to the proposed PG-binding site in Shewanella sp. strain ANA-3 ZorB (i) and S. kujiense ZorB (ii). Models are colored based on conservation scores; indicated residues diminished phage infection when mutated to alanine. Black residue labels are numbered according to the corresponding residues for Serratia marcescens ATCC 274 (type I) or E. coli ATCC 8739 (type II) used in the infection assays; gray and blue residue numbers correspond to the numbering of the modeled Shewanella and Sulfuricurvum complexes, respectively. e , f Peptidoglycan pull-down assay when incubated with purified ZorB I 165–287 ( e ) and ZorB II 115–235 ( f ) . g , h Peptidoglycan pull-down assay when incubated with purified ZorB I 165–287 and its mutants as indicated in panel ( g ), or of and ZorB II 115–235 and its mutants as shown in panel ( h ) . i Efficiency of plating (EOP) measurement for E. coli MT56 harboring empty vector (VC, pGM39), the same plasmid encoding wild-type Zorya I or a version of Zorya I where single point mutations were introduced in ZorA I or ZorB I, as indicated in panel ( j ) when infected with ɸAlma. The full set of mutations tested is reported in Supplementary Fig. . For panels e – h images are representative of three independent experiments. j Efficiency of plating (EOP) measurement for E. coli MT56 expressing empty vector (VC, pGM39), the same plasmid encoding wild-type Zorya II or a version of Zorya II carrying point mutations in ZorA II or ZorB II, as indicated in panel ( i ), when infected with ɸT7. The full set of mutations tested is reported in Supplementary Fig. . Points show mean ± SEM (n = 3 biological replicates). Induction of each construct in panels ( i , j ) was performed by the addition of 0.02% l - Rhamnose. Statistical significance for each panel was calculated with Graphpad applying a one-way ANOVA with Dunnett’s multiple comparison test. No significance was detected, unless indicated ( * p ≤ 0.05). For panels i , j the statistical analysis results are reported in Supplementary Data .

Article Snippet: Statistical significance was calculated with Graphpad applying a one-way ANOVA with Dunnett’s multiple comparison test.

Techniques: Plasmid Preparation, Infection, Comparison, Construct, Binding Assay, Residue, Pull Down Assay, Incubation, Purification, Expressing

Journal: Nature Communications

Article Title: Modularity of Zorya defense systems during phage inhibition

doi: 10.1038/s41467-025-57397-2

Figure Lengend Snippet: a The anti-phage activity of Zorya I and Zorya II under the control of their native promoter was evaluated by calculation of their fold protection against a suite of newly isolated environmental coliphages. As a control, ɸAlma, ɸMav, ɸT7, and ɸCS16F were included. Fold protection was calculated by dividing the value of efficiency of plating (EOP) for strains expressing Zorya I or Zorya II by the EOP value of a strain carrying the empty vector (pSUPROM), when infected with phages as shown in panel ( a ) . b Efficiency of plating (EOP) measurement for E. coli MT56 carrying the empty vector (VC, pSUPROM) or the same vector carrying Zorya II, ZorAE, ZorBE, Zor AB II or ZorE under the control of their native promoter, when infected with ɸphAvM. Points show mean ± SEM ( n = 3 biological replicates). Statistical significance was calculated with Graphpad applying a one-way ANOVA with Dunnett’s multiple comparison test. No significance was detected, unless indicated ( * p ≤ 0.05). For VC vs Zorya II comparison, p -value = <0.0001. c – h E. coli MT56 carrying the empty vector (VC, pSUPROM) or the same vector expressing Zorya II under its native promoter was infected with ɸphAvM at MOI 5 or 0.05. The c , f , titer (PFU/mL), d , g cell counts (CFU/mL) and e , h , and g , the growth rate (OD 600nm ) of each culture was measured at several time points, as shown in panels ( c – h ), over the course of 12 h post-infection. For panels c – h , points show mean ± SEM ( n = 3 biological replicates). Statistical significance was calculated with Graphpad applying a two-way ANOVA comparison test. No significance was detected, unless indicated ( * p ≤ 0.05). c The p -values for the VC vs Zorya II comparisons are as follows: for the 3-h time point, p = 0.0066; for the 6-h time point, p = 0.045; for the 9-h time point, p = 0.049; and for the 12-h time point, p = 0.0006. f The p -values for the VC vs Zorya II comparisons are as follows: for the 3-h time point, p = 0.013; for the 6-h time point, p = 0.019; for the 9-h time point, p = 0.007; and for the 12-h time point, p = 0.0028.

Article Snippet: Statistical significance was calculated with Graphpad applying a one-way ANOVA with Dunnett’s multiple comparison test.

Techniques: Activity Assay, Control, Isolation, Expressing, Plasmid Preparation, Infection, Comparison