Intracellular Ca²⁺ levels in CLd- or CLw-treated THP-1 macrophage-like cells.

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Raw uncropped images of western blots shown in Fig 4C.

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Solubility of the synthetic candidalysin peptide.

A: Amino acid sequence, putative tertiary structure, GRAVY index score, and hydropathy plot with Kyte-Doolittle scale of candidalysin (left) and Ece1-II (right). A positive hydropathy score reflects an enrichment of hydrophobic amino acids. B: Microscopic images of 10 μM CLd, CLw, and the Ece1-II control peptide dissolved in water. Images were taken at 200× magnification. Scale bar, 100 μm. The quantified data of particle number/mm2 in the peptide solutions is shown in the chart. Data are presented as the mean ± SD (n = 5) and are representative of three independent experiments. *P < 0.05 compared with 10 μM Ece1-II by one-way ANOVA followed by Dunnett’s test (μc < μi). C: Plots of the particles detected in 10 μM CLd, CLw, and Ece1-II by the flowcytometric forward scatter (FS) and side scatter (SS) analyses. D: The number and size of microparticles in 10 μM CLd, CLw, and Ece1-II quantified using a microparticle counter. Data are representative of three independent experiments.</p

Reference list

References that data were extracted to calculate weighted response ratio of N fertilization on microbial biomass and soil respiration, and of P fertilization on microbial biomass and soil respiration

Influence of the simultaneous inhibition of the NLPR3 pathway and cathepsin B on the IL-1β-producing activity of CLd and CLw.

LPS-primed THP-1 cells were pretreated with following inhibitors: 20 μM of the caspase-1 inhibitor Z-YVAD-fmk and/or 20 μM of the cathepsin B inhibitor Ca074Me (A and B); 2 μM of the NLRP3 inhibitor MCC950 and/or 20 μM Ca074Me (C and D); and 20 μM Z-YVAD-fmk and/or 2 μM MCC950 (E and F) or with vehicle for 1 h. Cells were then treated with 1 μM CLd (A, C, and E) or 10 μM CLw (B, D, and F) for 3 h. The vehicle controls for CLd and CLw are a medium containing 1% DMSO (A, C, and E) and a water-added medium (B, D, and F), respectively. TheIL-1β levels in the culture supernatants were assessed by ELISA. Data are presented as the mean ± SD (n = 3) and are representative of three independent experiments. *P †P  μi); ‡P  μi); §P < 0.05 compared with the single use of the second inhibitor (μc < μi) by one-way ANOVA followed by Dunnett’s test.</p

The IL-1β-producing activity of CLd and CLw in LPS-primed THP-1 macrophage-like cells.

A: Cells were treated with varied concentrations of CLd, CLw, Ece1-II, or 1 μM nigericin for 3 h. The vehicle controls for CLd and CLw are a medium containing 1% DMSO and a water-added medium (H2O), respectively. The IL-1β levels in the culture supernatants were assessed by ELISA. Data are presented as the mean ± SD (n = 3) and are representative of three independent experiments. *P †P 2O; μc P S1 Raw images.</p

Detection of nanoparticles in CLd and CLw.

Analyses of the number and size of nanoparticles in 10 μM CLd, CLw, and Ece1-II control peptide were performed using a NanoSight NS300 nanoparticle characterizer. Data are presented as the mean of five independent experiments.</p

Influence of the inhibitors on the cytotoxicity of CLd and CLw.

The differentiated THP-1 cells were pretreated with the following inhibitors: 20 μM of the pan-caspase inhibitor Z-VAD-fmk (A); 20 μM of the caspase-1 inhibitor Z-YVAD-fmk (B); 2 μM of the NLRP3 inhibitor MCC950 (C); 25 mM KCl for inhibition of K+ efflux (D); 20 μM of the cathepsin B inhibitor Ca074Me (E); and 20 μM of the phagocytosis inhibitor cytochalasin D (F) or with vehicle for 1 h. The vehicle control is a medium containing 2% DMSO. Cells were then treated with 1 μM CLd or 10 μM CLw for 3 h. Cytotoxicity was quantified by LDH release assay. Data are presented as the mean ± SD (n = 3) and are representative of three independent experiments. *P †P  μi) by one-way ANOVA followed by Dunnett’s test.</p

Cytotoxicity of CLd and CLw in differentiated THP-1 cells.

A: Cells were treated with varied concentrations of CLd, CLw, or Ece1-II for 3 h. The vehicle controls for CLd and CLw are a medium containing 1% DMSO and a water-added medium (H2O), respectively. Cytotoxicity was quantified by a LDH release assay. Data are presented as the mean ± SD (n = 3) and are representative of three independent experiments. *P †P 2O; μc P 2 of PI-positive cells) are presented as the mean ± SD (n = 4) and are representative of three independent experiments. *P < 0.05 compared with the vehicle by one-way ANOVA followed by Dunnett’s test (μc < μi).</p

Lack of P limitation on litter decomposition: a case study from long-term addition of phosphorus and nitrogen in three subtropical forests in southern China

Contrary to the widely accepted belief that elevated soil respiration after phosphorus (P) fertilization indicates P limitations for soil microorganisms in (sub)tropical forests, our previous studies have proposed that the elevated respiration results from the abiotically elevated availability of carbon (C), originating from competitive desorption from sorption sites on the surface of soil particles following P fertilization. In this study, we tested the impact of P fertilization (150 kg NaH2PO4-P ha−1 yr−1) on the decomposition of tea bags (Lipton Rooibos and Green teas) in three types of subtropical forests (primary, secondary, and planted forests) at the Dinghushan Biosphere Reserve, where previous findings reported increased soil respiration following P fertilization. If the traditional view holds true, early-stage litter decomposition should be stimulated by P fertilization, similar to the observed stimulation in soil respiration. Conversely, if this is not the case, it strongly reinforces our new hypothesis. Tea bags were employed to serve a twofold purpose: to assess the effects of P fertilization on litter decomposition within the soil, rather than on the soil surface, and to concentrate on the decomposition of early-stage litter. Our results demonstrated that P fertilization did not accelerate the decomposition of tea bags, indicating the exclusive enhancement of soil respiration without simultaneous stimulation of litter decomposition. This contradicts expectations if P were limiting microbial activity, reinforcing our hypothesis. In conclusion, our study reaffirmed the hypothesis that microbial activity in tropical forest soils is not primarily limited by P availability.</p