Fig 1: S100A6 signaling is modulated by eicosapentaenoic acid.a Neuritogenesis in cortical neurons (prepared at E18.5) treated with EPA at either 1 μM or 5 μM concentration, and monitored by dynamic live-cell imaging for 7 days in vitro (DIV). Data were normalized to neurite length prior to drug exposure on 1 DIV. Neurite outgrowth was determined by using a manual overlay for each neuron (n = 7 independent experiments). b Cultured mouse astrocytes were stimulated with EPA (30 μM) vs. control. Data were normalized to cell density prior to EPA exposure on 1 DIV (n = 8 independent experiments). c ELISA of S100A6 in culture media upon EPA (30 μM) applied for 24 h (n = 3 biological replicates). d mRNA levels for Cacybp in cortical neurons after exposure to a medium pre-conditioned for 24 h (n = 3 biological replicates). e mRNA levels for Grp78, Atf4, Atf6a, Chop (p = 0.067), Xbp1u, and Xbp1s (p = 0.122) in E18.5 cortical neurons after exposure to astrocyte-conditioned medium for 24 h (n = 3 biological replicates). Mixed-sex embryos were used where relevant. Data were expressed as means ± s.d. throughout. Data were statistically evaluated using ANOVA (a–c) or two-tailed Student’s t-test (d, e), with only post-hoc comparisons indicated (/p < 0.05, *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001. Unprocessed data and statistics were included in the Source Data File.
Fig 2: S100A6 availability modulates neuronal differentiation.a Co-culture experiments schema. b S100A6 (10 kDa) WB in astrocyte cytosol after 96 h exposure to scrambled or targeting siRNA, followed by 6 h EPA (30 μM) treatment. Representative blots are shown; lane numbers correspond to subsequent panels for clarity. c S100A6 ELISA in culture media upon treatment as in (b) with either n = 4 biological replicates [control] or n = 3 biological replicates [siRNA; EPA; EPA + siRNA]. d S100A6 detection in the cytosol of astrocytes after treatments as in (b) (n = 3 biological replicates). e Representative imagines of cortical neurons exposed to conditioned media for 24 h or EPA (30 μM), and double-labeled for F-actin (phalloidin; magenta) and MAP2 (green). Lead neurites and growth cones were marked by dashed lines and solid arrowheads, respectively. Scale bars = 12 μm (top), 5 μm (bottom). f Primary neurite length after experimental manipulations (biological replicates: n = 7 [control]; n = 5 [cond. siRNA; cond. siRNA + EPA; EPA]; n = 9 [cond. EPA]). g Neuritogenesis after genetic and pharmacological manipulations and monitored for 24 h. Data were normalized to neurite length prior to treatment and expressed in percentage. Neurite outgrowth was determined by using a manual overlay (biological replicates: n = 7 [control; cond. siRNA], n = 4 [cond. EPA], n = 5 [cond. siRNA + EPA], n = 8 [EPA]). Data were expressed as means ± s.d. throughout. Samples were mixed for sex where relevant. Data were statistically evaluated by one-way ANOVA followed by Tukey’s multiple comparisons with post-hoc comparisons indicated (*p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001. Unprocessed data and detailed statistics were included in the Source Data File.
Fig 3: S100A4/A6/A10/A11 proteins directly activate HSCs and induce fibrogenic responses. (A) HSCs were individually stimulated with recombinant S100A4/A6/A10/A11 proteins at a concentration of 500 ng/mL for 12 h. Following treatment, RNA and protein samples were collected from the HSCs for analysis. RT-qPCR analysis revealed that all four S100A proteins significantly upregulated the mRNA expression of α-SMA, collagen I, collagen III, Fn1, and MMP1 in HSCs. Regarding TIMP1 mRNA expression, S100A6 upregulated its expression, whereas S100A10 and S100A11 downregulated it. (B) WB analysis demonstrated that stimulation with S100A4/A6/A10/A11 proteins consistently increased the protein levels of α-SMA, collagen I, and collagen III in HSCs. WB also revealed activation of the TGF-β/SMAD2 signaling pathway components. Data are presented as mean ± SD (n = 3 independent experiments). *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001
Fig 4: S100A4/A6/A10/A11 proteins activate the TGF-β/SMAD2 pathway in HSCs via KCs. (A) KCs were individually stimulated with recombinant S100A4/A6/A10/A11 proteins. Subsequently, the conditioned media (supernatants) from these stimulated KCs were mixed 1:1 with DMEM and used to treat HSCs for 24 h. Following treatment, RNA and protein samples were collected from the HSCs for downstream analysis. RT-qPCR analysis revealed that conditioned media from KCs stimulated with S100A4 or S100A6 significantly upregulated the mRNA expression of α-SMA, collagen I, collagen III, Fn1, and MMP1 in HSCs, while concurrently downregulating TIMP1 mRNA expression. In contrast, conditioned media from KCs stimulated with S100A10 or S100A11 significantly upregulated the mRNA expression of α-SMA, collagen I, collagen III, and Fn1 in HSCs, while similarly downregulating TIMP1 mRNA expression. (B) WB analysis demonstrated that conditioned media from KCs stimulated with S100A4/A6/A10/A11 proteins collectively upregulated the protein levels of α-SMA, collagen I, and collagen III in HSCs, primarily through the activation of the TGF-β/SMAD2 pathway. Data are presented as mean ± SD (n = 3 independent experiments). *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001
Fig 5: The S100A4/A6/A10/A11 proteins modulate the inflammatory response in KCs via the MAPK signaling pathway. (A) Following 2 h stimulation of KC with appropriate concentrations of S100A4/A6/A10/A11 proteins, cellular RNA and protein were collected for analysis. RT-qPCR results showed that S100A4 and S100A6 protein stimulation significantly upregulated the mRNA levels of various inflammatory cytokines (TNF-α, IL-1β, IL-6, IL-17, IL-12, IFN-γ) and chemokines (CCL2, CCL4). S100A10 protein stimulation significantly upregulated the mRNA levels of TNF-α, IL-17, and IL-12. Similarly, S100A11 protein stimulation significantly upregulated the mRNA levels of TNF-α, IL-6, IL-17, IL-12, IFN-γ, CCL2, and CCL4. (B) WB analysis revealed that S100A4 and S100A11 activated the p38, JNK, and ERK signaling pathways. In contrast, S100A6 and S100A10 predominantly activated the p38 and JNK signaling pathways. Data are presented as mean ± SD (n = 3 independent experiments). *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001
Supplier Page from Abcam for Recombinant Mouse S100 alpha 6/PRA protein (His tag N-Terminus)