Journal: eLife

Article Title: Hyperphosphatemia increases inflammation to exacerbate anemia and skeletal muscle wasting independently of FGF23-FGFR4 signaling

doi: 10.7554/eLife.74782

Figure Lengend Snippet: Blood urea nitrogen (BUN), serum creatinine ( A ), serum FGF23 and serum phosphate (Pi) levels ( B ). Quantitative polymerase chain reaction (qPCR) analysis of Il1b , Il6 , Saa1 ( C, D ) and Hamp ( E ) expression levels in liver tissue. ( F ) Complete blood count (CBC) analysis. ( G ) Representative gross pathology of Perls’ Prussian blue-stained spleen sections (scale bar, 50 μm). Larger magnification is shown in supplementary figure and legends. ( H ) Liver Pi levels. All values are mean ± standard error of the mean (SEM; n = 8–9 mice/group; *p ≤ 0.05 vs. Fgfr4 +/+ + control diet, # p ≤ 0.05 vs. Fgfr4 −/− + control diet, $ p ≤ 0.05 vs. Fgfr4 +/+ + adenine diet) where statistical analyses were calculated by two-way analysis of variance (ANOVA) followed by Tukey’s multiple comparison post hoc test. Dotted lines indicate median Fgfr4 +/+ + control diet measurements.

Article Snippet: Recombinant proteins used are mouse FGF23 (2629-FG, R&D Systems), mouse TNFα (410-MT, R&D Systems), and mouse IL6 (406 ML, R&D Systems).

Techniques: Real-time Polymerase Chain Reaction, Expressing, Staining, Control, Comparison

Journal: eLife

Article Title: Hyperphosphatemia increases inflammation to exacerbate anemia and skeletal muscle wasting independently of FGF23-FGFR4 signaling

doi: 10.7554/eLife.74782

Figure Lengend Snippet: ( A ) Serum FGF23 and serum Pi levels. ( B, C ) Quantitative polymerase chain reaction (qPCR) analysis of Il1b , Il6 , and Saa1 expression levels in liver tissue. ( D ) Scatter plots showing correlations between liver Pi and serum Pi levels. ( E ) Scatter plots showing correlations between liver Hamp expression and liver Pi levels (a = slopes are significantly different from each other). ( F ) CBC analysis. ( G ) Representative gross pathology of Perls’ Prussian blue-stained spleen sections (scale bar, 50 μm). Larger magnification is shown in supplementary figure and legends. All values are mean ± standard error of the mean (SEM; n = 8 mice/group; *p ≤ 0.05 vs. Fgfr4 +/+ + 0.7% Pi diet, # p ≤ 0.05 vs. Fgfr4 −/− + 0.7% Pi diet, $ p ≤ 0.05 vs. Fgfr4 +/+ + 2% Pi diet, @ p ≤ 0.05 vs. Fgfr4 −/− + 2% Pi diet, & p ≤ 0.05 vs. Fgfr4 +/+ + 3% Pi diet) where statistical analyses were calculated by two-way analysis of variance (ANOVA) followed by Tukey’s multiple comparison post hoc test. Dotted lines indicate median Fgfr4 +/+ + 0.7% Pi diet measurements. Scatter plot shadows indicate 95% confidence interval.

Article Snippet: Recombinant proteins used are mouse FGF23 (2629-FG, R&D Systems), mouse TNFα (410-MT, R&D Systems), and mouse IL6 (406 ML, R&D Systems).

Techniques: Real-time Polymerase Chain Reaction, Expressing, Staining, Comparison

Journal: eLife

Article Title: Hyperphosphatemia increases inflammation to exacerbate anemia and skeletal muscle wasting independently of FGF23-FGFR4 signaling

doi: 10.7554/eLife.74782

Figure Lengend Snippet: BUN, serum creatinine ( A ), serum FGF23 and serum Pi levels ( B ). Quantitative polymerase chain reaction (qPCR) analysis of Il1b , Il6 , and Saa1 ( C, D ) and Hamp ( E ) expression levels in liver tissue. ( F ) CBC analysis. ( G ) Representative gross pathology of Perls’ Prussian blue-stained spleen sections (scale bar, 50 μm). Larger magnification is shown in supplementary figure and legends. ( H ) Liver Pi levels. All values are mean ± standard error of the mean (SEM; n = 7–9 mice/group; *p ≤ 0.05 vs. Col4a3 +/+ + 0.6% Pi diet, # p ≤ 0.05 vs. Col4a3 −/− + 0.6% Pi diet) where statistical analyses were calculated by two-way analysis of variance (ANOVA) followed by Tukey’s multiple comparison post hoc test. Dotted lines indicate median Col4a3 +/+ + 0.6% Pi diet measurements.

Article Snippet: Recombinant proteins used are mouse FGF23 (2629-FG, R&D Systems), mouse TNFα (410-MT, R&D Systems), and mouse IL6 (406 ML, R&D Systems).

Techniques: Real-time Polymerase Chain Reaction, Expressing, Staining, Comparison

Journal: eLife

Article Title: Hyperphosphatemia increases inflammation to exacerbate anemia and skeletal muscle wasting independently of FGF23-FGFR4 signaling

doi: 10.7554/eLife.74782

Figure Lengend Snippet: ( A ) Immunoblot analysis of total protein extracts from primary hepatocytes ( n = 5 independent isolations). β-Actin serves as loading control. Quantitative polymerase chain reaction (qPCR) analysis of Il1b , Il6 , Saa1 ( B, C ), Hamp ( D ), and Slc20a1 ( E ) expression levels in primary hepatocytes; values are mean ± standard error of the mean (SEM; n = 4 independent isolations; *p ≤ 0.05 vs. control [Ctrl]). Dotted lines indicate median Ctrl measurements. ( F ) qPCR analysis of Slc20a1 expression levels in primary hepatocytes following stimuli, with or without phosphonoformic acid (PFA); values are mean ± standard error of the mean (SEM; n = 6 independent isolations; *p ≤ 0.05 vs. vehicle control [Ctrl], # p ≤ 0.05 vs. 1 mM PFA Ctrl). Dotted lines indicate median vehicle Ctrl measurements. ( G ) Immunoblot analysis of total and phosphorylated p65 (NFκB) protein levels from primary hepatocytes following stimuli, with or without PFA ( n = 5 independent isolations). β-Actin serves as loading control. ( H–J ) qPCR analysis of Il1b , Il6 , Saa1 ( H–I ) and Hamp ( J ) expression levels in primary hepatocytes following stimuli, with or without PFA; values are mean ± standard error of the mean (SEM; n = 6 independent isolations; *p ≤ 0.05 vs. vehicle control [Ctrl], # p ≤ 0.05 vs. 1 mM PFA Ctrl) where statistical analyses were calculated by one-way analysis of variance (ANOVA; B–E ) or by two-way ANOVA ( F, H–J ) followed by Tukey’s multiple comparison post hoc test. Dotted lines indicate median vehicle Ctrl measurements. Figure 6—source data 1. Original western blots. Original uncropped western blots of the cropped western blots shown in . The molecular weight is indicated on the right in kDa.

Article Snippet: Recombinant proteins used are mouse FGF23 (2629-FG, R&D Systems), mouse TNFα (410-MT, R&D Systems), and mouse IL6 (406 ML, R&D Systems).

Techniques: Western Blot, Control, Real-time Polymerase Chain Reaction, Expressing, Comparison, Molecular Weight

Journal: eLife

Article Title: Hyperphosphatemia increases inflammation to exacerbate anemia and skeletal muscle wasting independently of FGF23-FGFR4 signaling

doi: 10.7554/eLife.74782

Figure Lengend Snippet: Quantitative polymerase chain reaction (qPCR) analysis of Il1b , Il6 ( A ) and Hamp ( B ) expression levels in primary hepatocytes following stimuli, with or without BAY 11-7082; values are mean ± standard error of the mean (SEM; n = 4 independent isolations; *p ≤ 0.05 vs. vehicle control [Ctrl], # p ≤ 0.05 vs. 20 μM BAY 11-7082 Ctrl). Dotted lines indicate median vehicle Ctrl measurements. ( C ) qPCR analysis of Hamp expression levels in primary hepatocytes following stimuli with or without anti-IL1β, anti-IL6, or both antibodies in combination; values are mean ± standard error of the mean (SEM; n = 4 independent isolations; *p ≤ 0.05 vs. vehicle control [Ctrl], # p ≤ 0.05 vs. anti-IL1β Ctrl, $ p ≤ 0.05 vs. anti-IL6 Ctrl, @ p ≤ 0.05 vs. anti-IL1β + anti-IL6 Ctrl) where statistical analyses were calculated by two-way analysis of variance (ANOVA) followed by Tukey’s multiple comparison post hoc test. Dotted lines indicate median vehicle Ctrl measurements.

Article Snippet: Recombinant proteins used are mouse FGF23 (2629-FG, R&D Systems), mouse TNFα (410-MT, R&D Systems), and mouse IL6 (406 ML, R&D Systems).

Techniques: Real-time Polymerase Chain Reaction, Expressing, Control, Comparison

Journal: eLife

Article Title: Hyperphosphatemia increases inflammation to exacerbate anemia and skeletal muscle wasting independently of FGF23-FGFR4 signaling

doi: 10.7554/eLife.74782

Figure Lengend Snippet: Quantitative polymerase chain reaction (qPCR) analysis of primary hepatocytes shows expression levels of Saa1 ( A ), Hp ( B ), and Slc20a1 ( C ) following lipopolysaccharide (LPS) or Pi stimulation, with or without BAY 11-7082; values are mean ± standard error of the mean (SEM; n = 4 independent isolations; *p ≤ 0.05 vs. vehicle control [Ctrl], # p ≤ 0.05 vs 20 μM BAY 11-7082 Ctrl). Dotted lines indicate corresponding median measurements from vehicle Ctrl. ( D, E ) qPCR analysis of primary hepatocytes shows Saa1 and Hp expression levels following LPS or Pi stimulation, with or without anti-IL1β, anti-IL6, or both antibodies in combination; values are mean ± standard error of the mean (SEM; n = 4 independent isolations; *p ≤ 0.05 vs. vehicle control [Ctrl], # p ≤ 0.05 vs. anti-IL1β Ctrl, $ p ≤ 0.05 vs. anti-IL6 Ctrl, @ p ≤ 0.05 vs. anti-IL1β + anti-IL6 Ctrl) where statistical analyses were calculated by two-way analysis of variance (ANOVA) followed by Tukey’s multiple comparison post hoc test. Dotted lines indicate corresponding median measurements from vehicle Ctrl.

Article Snippet: Recombinant proteins used are mouse FGF23 (2629-FG, R&D Systems), mouse TNFα (410-MT, R&D Systems), and mouse IL6 (406 ML, R&D Systems).

Techniques: Real-time Polymerase Chain Reaction, Expressing, Control, Comparison

Journal: eLife

Article Title: Hyperphosphatemia increases inflammation to exacerbate anemia and skeletal muscle wasting independently of FGF23-FGFR4 signaling

doi: 10.7554/eLife.74782

Figure Lengend Snippet: Oligonucleotides used as sequence specific primers in quantitative polymerase chain reaction (qPCR) analyses.

Article Snippet: Recombinant proteins used are mouse FGF23 (2629-FG, R&D Systems), mouse TNFα (410-MT, R&D Systems), and mouse IL6 (406 ML, R&D Systems).

Techniques: Sequencing, Real-time Polymerase Chain Reaction

Journal: eLife

Article Title: Hyperphosphatemia increases inflammation to exacerbate anemia and skeletal muscle wasting independently of FGF23-FGFR4 signaling

doi: 10.7554/eLife.74782

Figure Lengend Snippet:

Article Snippet: Recombinant proteins used are mouse FGF23 (2629-FG, R&D Systems), mouse TNFα (410-MT, R&D Systems), and mouse IL6 (406 ML, R&D Systems).

Techniques: Knock-Out, Cell Culture, Recombinant, Iron Assay, Enzyme-linked Immunosorbent Assay, Reverse Transcription, SYBR Green Assay, Bicinchoninic Acid Protein Assay, Software, Control

Journal: bioRxiv

Article Title: Network analysis reveals a distinct axis of macrophage activation in response to conflicting inflammatory cues

doi: 10.1101/844464

Figure Lengend Snippet: A) Dynamics of predicted gene expression in response to stimulation with LPS+IFNγ or IL4. Stimuli were added at 0 h. B) Kinetics of selected mRNAs in response to stimulation with LPS+IFNγ or IL4. C) mRNA expression profiles predicted by the model, validated against RNA-Seq measurements from peritoneal macrophages treated with LPS+IFNγ or IL4 for 4h. For semi-quantitative comparison between model and experiment, the log2 fold change of each mRNA vs. control was normalized by the root mean square between the M1 and M2 conditions. Classic M1 (orange) and M2 (green) phenotype markers are highlighted.

Article Snippet: Macrophages were assigned to one of three treatment groups: 1) stimulated with 1 μg/mL LPS (Sigma, L2880) and 20 ng/mL IFNγ (R&D, 485-MI) for 4 h; 2) stimulated with 20 ng/mL IL4 (R&D, 404-ML) for 4 h; or 3) untreated for 4 h, serving as the negative control.

Techniques: Gene Expression, Expressing, RNA Sequencing, Comparison, Control

Journal: bioRxiv

Article Title: Network analysis reveals a distinct axis of macrophage activation in response to conflicting inflammatory cues

doi: 10.1101/844464

Figure Lengend Snippet: A) Overall network influence of node knockdowns under stimulation with either LPS+IFNγ (orange) or IL4 (green). Nodes were ranked by the overall influence of their knockdown on all other network nodes, under conditions of LPS+IFNγ stimulation. B) Predicted effect of knockdown of influential nodes on activity of highly sensitive nodes, under conditions of LPS+IFNγ or IL4 treatment.

Article Snippet: Macrophages were assigned to one of three treatment groups: 1) stimulated with 1 μg/mL LPS (Sigma, L2880) and 20 ng/mL IFNγ (R&D, 485-MI) for 4 h; 2) stimulated with 20 ng/mL IL4 (R&D, 404-ML) for 4 h; or 3) untreated for 4 h, serving as the negative control.

Techniques: Knockdown, Activity Assay

Journal: bioRxiv

Article Title: Network analysis reveals a distinct axis of macrophage activation in response to conflicting inflammatory cues

doi: 10.1101/844464

Figure Lengend Snippet: A) Model-predicted signaling module activities in response to IFNγ and IL4 treatments at 4h, column normalized. B) Experimental validation of mRNA expression predicted in response to IFNγ, IL4, or IFNγ+IL4. For both experimental data and model predictions, mRNA were independently normalized by RMS-normalized log2 fold change at 4 h. C) Predicted expression dynamics of selected mRNAs in response to IFNγ, IL4, or IFNγ+IL4. D) Context-dependent network response to node knockdowns under treatments of IFNγ, IL4, or IFNγ+IL4.

Article Snippet: Macrophages were assigned to one of three treatment groups: 1) stimulated with 1 μg/mL LPS (Sigma, L2880) and 20 ng/mL IFNγ (R&D, 485-MI) for 4 h; 2) stimulated with 20 ng/mL IL4 (R&D, 404-ML) for 4 h; or 3) untreated for 4 h, serving as the negative control.

Techniques: Biomarker Discovery, Expressing