Data_Sheet_1_Structure and assembly process of fungal communities in the Yangtze River Estuary.pdf

Marine fungi are essential for the ecological function of estuarine ecosystems. However, limited studies have reported on the structure and assembly pattern of the fungal communities in estuaries. The purpose of this study is to reveal the structure and the ecological process of the fungal community in the Yangtze River Estuary (YRE) by using the amplicon sequencing method. Phyla of Ascomycota, Basidiomycota, and Chytridiomycota were dominant in the seawater and sediment samples from YRE. The null model analysis, community-neutral community model (NCM), and phylogenetic normalized stochasticity ratio (pNST) showed that the stochastic process dominated the assembly of fungal communities in YRE. Drift and homogeneous dispersal were the predominant stochastic processes for the fungal community assembly in seawater and sediment samples, respectively. The co-occurrence network analysis showed that fungal communities were more complex and closely connected in the sediment than in the seawater samples. Phyla Ascomycota, Basidiomycota, and Mucoromycota were the potential keystone taxa in the network. These findings demonstrated the importance of stochastic processes for the fungal community assembly, thereby widening our knowledge of the community structure and dynamics of fungi for future study and utilization in the YRE ecosystem.</p

Signaling pathways cross-talk study.

<p>(A) Cardiomyocytes isolated from WT mice were incubated with SP600125 (10 µM) or its vehicle control DMSO for 1 hr, followed by 30 min of hypoxia (H), hypoxia (30 min) and 1 hr reoxygenation (HR). Cell lysates were collected for the study of phospho/total-Akt by Western blot. n = 3. (B) Cardiomyocytes isolated from RKO mice were incubated with LY294002 (10 µM) or its vehicle control DMSO for 1 hr, followed by 30 min of hypoxia (H), hypoxia (30 min) and 1 hr reoxygenation (HR). Cell lysates were collected for the study of phospho/total-JNK by western blot. n = 3. SP: SP600125; LY: LY294002.</p

Impact of RAGE ligand on key stress signaling pathways in the absence of H/R in cardiomyocytes.

<p>WT cardiomyocytes were incubated with CML-AGE (50 µg/ml) for different time points as indicated. Cells were lysed and subjected to Western blot analysis for the detection of phospho-JNK and total-JNK level (A), as well as phospho-S9-GSK3β and total- GSK3β (B). Data are representative of three independent experiments.</p

Inhibition of JNK alleviates the injury due to H/R in cardiomyocytes.

<p>WT cardiomyocytes were subjected to hypoxia/reoxygenation after 1 hr incubation with JNK inhibitor SP600125 (10 µM) or the vehicle control DMSO. (A) Cell supernatant was collected for LDH level measurement. n = 3. (B) Cleaved caspase 3 levels and (C) Cytochrome c were detected after hypoxia/reoxygenation by Western blot analysis. Data are representative of three independent experiments. (D) Cardiomyocytes isolated from WT and RKO and sRAGE-treated mice were incubated with JNK inhibitor SP600125 (10 µM) or its vehicle control DMSO for 1 hr, followed by hypoxia 30 mins/reoxygenation 1 hr treatment. Cell supernatant was collected for LDH release measurement. (E) The hearts of RKO mice were pre-perfused with JNK-specific inhibitor SP600125 or its negative control for 30 mins prior to the cardiomyocyte isolation process. Cell supernatant was collected for LDH level measurement. Pre-perfusion with the JNK inhibitor did not abrogate the protective effects of RAGE deletion in H/R. n = 3. SP: SP600125.</p

Deletion of RAGE promotes cardiomyocyte survival in H/R by enhancing phosphorylation of GSK3β.

<p>(A) Cells were collected and lysates obtained at the end of normoxia (N), 30 mins of hypoxia (H), and hypoxia (30 min) followed by 1 hr reoxygenation (HR). Lysates from WT, RKO and sRAGE-treated cardiomyocytes were subjected to Western blot analysis for the detection of phospho-S9-GSK3β and total-GSK3β. n = 3. (B) Cardiomyocytes were incubated with or without GSK3 inhibitors LiCl (12.5 mM) and SB216763 (3 µM) for 1 hr, followed by 30 min hypoxia/1 hr reoxygenation (HR), cell supernatant was collected for LDH release measurement. n = 3.</p

Deletion of RAGE modulates H/R stress via blockade of JNK signaling pathway.

<p>Cardiomyocytes were collected and lysates obtained at the end of normoxia (N), 30 min of hypoxia (H), and hypoxia (30 min) followed by 1 hr reoxygenation (HR). Lysates were subjected to Western blot analysis for the detection of (A) phospho-JNK and total-JNK. (B) phospho/total-ERK and (C) phospho/total-p38 level. n = 5. (D) JNK activity was measured by using commercially available ELISA kit; n = 3.</p