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: Figure 1. FGF23-FGFR4 signaling does not contribute to functional iron deficiency in adenine-induced CKD. 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

Article Snippet: Reagent type (species) or resource Designation Source or reference Identifiers Additional information Strain, strain background (Mus musculus; 129/SvJ) Col4a3tm1Dec Jackson Laboratory Stock No. 000691 Referred to as Col4a3−/− Strain, strain background (Mus musculus; C57Bl/6) Global FGFR4 knockout Gift from Dr. Chu- Xia Deng, NIDDK, Bethesda, USA Weinstein et al., 1998, 125, 3615- 23 Referred to as Fgfr4−/− Antibody IL6 (rat monoclonal) R&D Systems MP5- 20F3 For cell culture treatment (6 μg/ml) Antibody IL1β (goat polyclonal) R&D Systems AF- 401- NA For cell culture treatment (6 μg/ml) Antibody ERK1/2 (rabbit monoclonal) Cell Signaling 4695 For WB (1:1000) Antibody STAT3 (rabbit monoclonal) Cell Signaling 4904 For WB (1:1000) Antibody NFkB (rabbit monoclonal) Cell Signaling 8242 For WB (1:1000) Antibody β-Actin (rabbit monoclonal) Cell Signaling 4970 For WB (1:1000) Antibody Phosphorylated ERK1/2 (rabbit polyclonal) Cell Signaling 9101 For WB (1:1000) Antibody Phosphorylated STAT3 (rabbit monoclonal) Cell Signaling 9145 For WB (1:1000) Antibody Phosphorylated NFκB (rabbit monoclonal) Cell Signaling 3033 For WB (1:1000) Antibody Mouse IgG, HRP conjugate (goat monoclonal) Promega W4021 For WB (1:2500) Antibody Rabbit IgG, HRP conjugate (goat monoclonal) Promega W4011 For WB (1:2500) Peptide, recombinant protein FGF23; mouse R&D Systems 2629- FG For cell culture treatment (25 ng/ml) Peptide, recombinant protein TNFα; mouse R&D Systems 410- MT For cell culture treatment (100 ng/ml) Peptide, recombinant protein IL6; mouse R&D Systems 406 ML For cell culture treatment (50 ng/ml) Commercial assay or kit Colorimetric Pi assay Abcam Ab65622 Czaya et al. eLife 2022;11:e74782.

Techniques: Functional Assay, Real-time Polymerase Chain Reaction, Expressing, Staining

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: Figure 2. FGF23-FGFR4 signaling does not contribute to hypoferremia following dietary Pi overload. (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

Article Snippet: Reagent type (species) or resource Designation Source or reference Identifiers Additional information Strain, strain background (Mus musculus; 129/SvJ) Col4a3tm1Dec Jackson Laboratory Stock No. 000691 Referred to as Col4a3−/− Strain, strain background (Mus musculus; C57Bl/6) Global FGFR4 knockout Gift from Dr. Chu- Xia Deng, NIDDK, Bethesda, USA Weinstein et al., 1998, 125, 3615- 23 Referred to as Fgfr4−/− Antibody IL6 (rat monoclonal) R&D Systems MP5- 20F3 For cell culture treatment (6 μg/ml) Antibody IL1β (goat polyclonal) R&D Systems AF- 401- NA For cell culture treatment (6 μg/ml) Antibody ERK1/2 (rabbit monoclonal) Cell Signaling 4695 For WB (1:1000) Antibody STAT3 (rabbit monoclonal) Cell Signaling 4904 For WB (1:1000) Antibody NFkB (rabbit monoclonal) Cell Signaling 8242 For WB (1:1000) Antibody β-Actin (rabbit monoclonal) Cell Signaling 4970 For WB (1:1000) Antibody Phosphorylated ERK1/2 (rabbit polyclonal) Cell Signaling 9101 For WB (1:1000) Antibody Phosphorylated STAT3 (rabbit monoclonal) Cell Signaling 9145 For WB (1:1000) Antibody Phosphorylated NFκB (rabbit monoclonal) Cell Signaling 3033 For WB (1:1000) Antibody Mouse IgG, HRP conjugate (goat monoclonal) Promega W4021 For WB (1:2500) Antibody Rabbit IgG, HRP conjugate (goat monoclonal) Promega W4011 For WB (1:2500) Peptide, recombinant protein FGF23; mouse R&D Systems 2629- FG For cell culture treatment (25 ng/ml) Peptide, recombinant protein TNFα; mouse R&D Systems 410- MT For cell culture treatment (100 ng/ml) Peptide, recombinant protein IL6; mouse R&D Systems 406 ML For cell culture treatment (50 ng/ml) Commercial assay or kit Colorimetric Pi assay Abcam Ab65622 Czaya et al. eLife 2022;11:e74782.

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

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: Figure 4. Low Pi feeding limits functional iron deficiency in Col4a3−/− (Alport ) mice. 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

Article Snippet: Reagent type (species) or resource Designation Source or reference Identifiers Additional information Strain, strain background (Mus musculus; 129/SvJ) Col4a3tm1Dec Jackson Laboratory Stock No. 000691 Referred to as Col4a3−/− Strain, strain background (Mus musculus; C57Bl/6) Global FGFR4 knockout Gift from Dr. Chu- Xia Deng, NIDDK, Bethesda, USA Weinstein et al., 1998, 125, 3615- 23 Referred to as Fgfr4−/− Antibody IL6 (rat monoclonal) R&D Systems MP5- 20F3 For cell culture treatment (6 μg/ml) Antibody IL1β (goat polyclonal) R&D Systems AF- 401- NA For cell culture treatment (6 μg/ml) Antibody ERK1/2 (rabbit monoclonal) Cell Signaling 4695 For WB (1:1000) Antibody STAT3 (rabbit monoclonal) Cell Signaling 4904 For WB (1:1000) Antibody NFkB (rabbit monoclonal) Cell Signaling 8242 For WB (1:1000) Antibody β-Actin (rabbit monoclonal) Cell Signaling 4970 For WB (1:1000) Antibody Phosphorylated ERK1/2 (rabbit polyclonal) Cell Signaling 9101 For WB (1:1000) Antibody Phosphorylated STAT3 (rabbit monoclonal) Cell Signaling 9145 For WB (1:1000) Antibody Phosphorylated NFκB (rabbit monoclonal) Cell Signaling 3033 For WB (1:1000) Antibody Mouse IgG, HRP conjugate (goat monoclonal) Promega W4021 For WB (1:2500) Antibody Rabbit IgG, HRP conjugate (goat monoclonal) Promega W4011 For WB (1:2500) Peptide, recombinant protein FGF23; mouse R&D Systems 2629- FG For cell culture treatment (25 ng/ml) Peptide, recombinant protein TNFα; mouse R&D Systems 410- MT For cell culture treatment (100 ng/ml) Peptide, recombinant protein IL6; mouse R&D Systems 406 ML For cell culture treatment (50 ng/ml) Commercial assay or kit Colorimetric Pi assay Abcam Ab65622 Czaya et al. eLife 2022;11:e74782.

Techniques: Functional Assay, Real-time Polymerase Chain Reaction, Expressing, Staining

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: Figure 6. Pi targets hepatocytes and increases expression of inflammatory cytokines and hepcidin. (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

Article Snippet: Reagent type (species) or resource Designation Source or reference Identifiers Additional information Strain, strain background (Mus musculus; 129/SvJ) Col4a3tm1Dec Jackson Laboratory Stock No. 000691 Referred to as Col4a3−/− Strain, strain background (Mus musculus; C57Bl/6) Global FGFR4 knockout Gift from Dr. Chu- Xia Deng, NIDDK, Bethesda, USA Weinstein et al., 1998, 125, 3615- 23 Referred to as Fgfr4−/− Antibody IL6 (rat monoclonal) R&D Systems MP5- 20F3 For cell culture treatment (6 μg/ml) Antibody IL1β (goat polyclonal) R&D Systems AF- 401- NA For cell culture treatment (6 μg/ml) Antibody ERK1/2 (rabbit monoclonal) Cell Signaling 4695 For WB (1:1000) Antibody STAT3 (rabbit monoclonal) Cell Signaling 4904 For WB (1:1000) Antibody NFkB (rabbit monoclonal) Cell Signaling 8242 For WB (1:1000) Antibody β-Actin (rabbit monoclonal) Cell Signaling 4970 For WB (1:1000) Antibody Phosphorylated ERK1/2 (rabbit polyclonal) Cell Signaling 9101 For WB (1:1000) Antibody Phosphorylated STAT3 (rabbit monoclonal) Cell Signaling 9145 For WB (1:1000) Antibody Phosphorylated NFκB (rabbit monoclonal) Cell Signaling 3033 For WB (1:1000) Antibody Mouse IgG, HRP conjugate (goat monoclonal) Promega W4021 For WB (1:2500) Antibody Rabbit IgG, HRP conjugate (goat monoclonal) Promega W4011 For WB (1:2500) Peptide, recombinant protein FGF23; mouse R&D Systems 2629- FG For cell culture treatment (25 ng/ml) Peptide, recombinant protein TNFα; mouse R&D Systems 410- MT For cell culture treatment (100 ng/ml) Peptide, recombinant protein IL6; mouse R&D Systems 406 ML For cell culture treatment (50 ng/ml) Commercial assay or kit Colorimetric Pi assay Abcam Ab65622 Czaya et al. eLife 2022;11:e74782.

Techniques: Expressing, Western Blot, Control, 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: Figure 7. Pi induces hepcidin expression via paracrine IL1β and IL6 signaling. 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: Reagent type (species) or resource Designation Source or reference Identifiers Additional information Strain, strain background (Mus musculus; 129/SvJ) Col4a3tm1Dec Jackson Laboratory Stock No. 000691 Referred to as Col4a3−/− Strain, strain background (Mus musculus; C57Bl/6) Global FGFR4 knockout Gift from Dr. Chu- Xia Deng, NIDDK, Bethesda, USA Weinstein et al., 1998, 125, 3615- 23 Referred to as Fgfr4−/− Antibody IL6 (rat monoclonal) R&D Systems MP5- 20F3 For cell culture treatment (6 μg/ml) Antibody IL1β (goat polyclonal) R&D Systems AF- 401- NA For cell culture treatment (6 μg/ml) Antibody ERK1/2 (rabbit monoclonal) Cell Signaling 4695 For WB (1:1000) Antibody STAT3 (rabbit monoclonal) Cell Signaling 4904 For WB (1:1000) Antibody NFkB (rabbit monoclonal) Cell Signaling 8242 For WB (1:1000) Antibody β-Actin (rabbit monoclonal) Cell Signaling 4970 For WB (1:1000) Antibody Phosphorylated ERK1/2 (rabbit polyclonal) Cell Signaling 9101 For WB (1:1000) Antibody Phosphorylated STAT3 (rabbit monoclonal) Cell Signaling 9145 For WB (1:1000) Antibody Phosphorylated NFκB (rabbit monoclonal) Cell Signaling 3033 For WB (1:1000) Antibody Mouse IgG, HRP conjugate (goat monoclonal) Promega W4021 For WB (1:2500) Antibody Rabbit IgG, HRP conjugate (goat monoclonal) Promega W4011 For WB (1:2500) Peptide, recombinant protein FGF23; mouse R&D Systems 2629- FG For cell culture treatment (25 ng/ml) Peptide, recombinant protein TNFα; mouse R&D Systems 410- MT For cell culture treatment (100 ng/ml) Peptide, recombinant protein IL6; mouse R&D Systems 406 ML For cell culture treatment (50 ng/ml) Commercial assay or kit Colorimetric Pi assay Abcam Ab65622 Czaya et al. eLife 2022;11:e74782.

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