DataSheet_1_The effects of atmospheric nitrogen deposition in coral-algal phase shifts on remote coral reefs.pdf

Remote seawater has been considered a potential refuge for corals in the face of anthropogenic disturbances. However, these remote areas may receive increased atmospheric N deposition, and the ecological consequences remain unclear. This field survey revealed coral-algal phase shifts in the mid-north of the South China Sea. These shifts were observed in 44%, 13.6%, and 26.5% of the sampled reef sites at depths of 1-4 m, 5-8 m, and 10-15 m, respectively. Over 50% of sections in the deeper depths hosted fewer corals compared to shallower areas, coinciding with a higher abundance of macroalgae in the deeper layers. Furthermore, based on long-term observation of atmospheric N flux, laboratory experiments were conducted to explore the cause of coral declines. The results indicate that N supply efficiently promoted macroalgae growth. The saturation of N absorption by macroalgae occurred within 2 weeks, leading to nutrient accumulation in seawater, especially nitrate, which had a direct impact on corals. While moderate N fluxes appeared to mitigate coral bleaching, high N fluxes, even with a balanced P supply or medium level of nutrients with an imbalanced N/P ratio, can both increase the susceptibility of corals to heat bleaching. This study explains the coral-algal phase shift in remote and relatively deep seawater and improves understanding of the cause-and-effect relationship between atmospheric N deposition and coral reef decline.</p

JNK/mTOR regulates GRP78 induction through ATF4 in human CCA cells.

<p>(A) After treated with SP600125 (SP, 20 µM) for 48 h, ATF4 and phosphorylated eIF2α in QBC939, RBE and HCCC-9810 cells were analyzed using western blot. (B) After treated with rapamycin (Rap, 20 nM) for 48 h, phosphorylated eIF2α and phosphorylated p70S6K in QBC939, RBE and HCCC-9810 cells were analyzed using western blot. (C) After treated with salubrinal (Sal, 25 µM) for 30 h with or without SP600125 (SP, 20 µM) and rapamycin (Rap, 20 nM) preincubation for 1 h, ATF4 and phosphorylated eIF2α were analyzed using western blot in HepG2 cells. (D) After treated with PF-4708671 (PF, 10 µM) and 4EGI-1 (50 µM) for 48 h, GRP78 and ATF4 in QBC939, RBE and HCCC-9810 cells were analyzed using western blot. (E) QBC939, RBE and HCCC-9810 cells were treated with rapamycin (Rap, 20 nM) for 12 h, and ATF4 and GRP78 mRNA levels were analyzed using real-time RT-PCR. Values are means±S.D. Columns, mean of three individual experiments; bars, SD. *Significantly different from control value.</p

JNK Contributes to the Tumorigenic Potential of Human Cholangiocarcinoma Cells through the mTOR Pathway Regulated GRP78 Induction

<div><p>Less is known about the roles of c-Jun N-terminal kinase (JNK) in cholangiocarcinoma (CCA). Here, we report that JNK exerts its oncogenic action in human CCA cells, partially due to the mammalian target of rapamycin (mTOR) pathway regulated glucose-regulated protein 78 (GRP78) induction. In human CCA cells, the phosphorylation of eukaryotic initiation factor alpha (eIF2α) results in the accumulation of activating transcription factor 4 (ATF4) and GRP78 independent of unfolded protein response (UPR). Suppression of GRP78 expression decreases the proliferation and invasion of human CCA cells. It's notable that mTOR is required for eIF2α phosphorylation-induced ATF4 and GRP78 expression. Importantly, JNK promotes eIF2α/ATF4-mediated GRP78 induction through regulating the activity of mTOR. Thus, our study implicates JNK/mTOR signaling plays an important role in cholangiocarcinogenesis, partially through promoting the eIF2α/ATF4/GRP78 pathway.</p></div

eIF2α/ATF4 induces GRP78 accumulation in human CCA cells.

<p>(A) Western blot analysis of GRP78 in human cholangiocarcinoma cells. DMSO- and tunicamycin (Tun, 2.0 µg/ml)-treated HepG2 cells were used as negative and positive control, respectively. (B) RT-PCR analysis of spliced XBP1 mRNA in human CCA cells. Tunicamycin (Tun, 2.0 µg/ml)-treated QBC939 cells were used as positive control. (C) Western blot analysis of phosphorylated eIF2α and ATF4 in human CCA cells. Salubrinal (Sal, 25 µM)-treated HepG2 cells were used as positive control. (D) After transfected with ATF4 siRNA for 60 h, QBC939, RBE and HCCC-9810 cells were subjected to western blot analysis.</p

GRP78 promotes human CCA cells proliferation and invasion.

<p>(A) GRP78 suppression inhibits human CCA cells proliferation. QBC939, RBE and HCCC-9810 cells were transfected with siGRP78 for indicated time periods. Cell viability was determined by CCK8 assay. (B and C) GRP78 suppression inhibits human CCA cells migration and invasion. To knockdown GRP78, human CCA cells were transfected with siGRP78 for 36 h before transferring to 24-well transwell chambers. The migration (B) and invasion (C) of QBC939, RBE and HCCC-9810 cells with or without siGRP78 treatment were analyzed using transwell assay. (D) The effects of siGRP78 on GRP78 suppression were measured using western blot. Values are means±S.D. Columns, mean of three individual experiments; bars, SD. *Significantly different from control value.</p

mTOR controls GRP78 synthesis in human cholangiocarcinoma cells.

<p>(A) After treated with rapamycin (Rap, 20 nM) for indicated time periods, GRP78 in QBC939, RBE and HCCC-9810 cells was analyzed using western blot. (B) After transfected with simTOR for 60 h, GRP78 in QBC939, RBE and HCCC-9810 cells was analyzed using western blot. (C) After treated with salubrinal (Sal, 25 µM) for 30 h with or without rapamycin (Rap, 20 nM) preincubation for 1 h, GRP78 was analyzed using western blot in HepG2 cells. (D) After treated with tunicamycin (Tun, 2.0 µg/ml) for 24 h with or without rapamycin (Rap, 20 nM) preincubation for 1 h, GRP78 in HepG2 cells was analyzed using western blot.</p

JNK promotes the activity of mTOR in human CCA cells.

<p>(A) After treated with SP600125 (SP, 20 µM) for indicated time periods, phosphorylated p70S6K in QBC939, RBE and HCCC-9810 cells was analyzed using western blot. Rapamycin (Rap, 20 nM)-treated cholangiocarcinoma cells were used as positive control. (B) After transfected with siJNK for 60 h, phosphorylated p70S6K in QBC939, RBE and HCCC-9810 cells was analyzed using western blot. (C) After treated with SP600125 (SP, 20 µM) for indicated time periods, phosphorylated mTOR in QBC939, RBE and HCCC-9810 cells was analyzed using western blot. (D) After treated with SP600125 (SP, 20 µM) for indicated time periods, phosphorylated Raptor and phosphorylated 4E-BP1 in QBC939, RBE and HCCC-9810 cells were analyzed using western blot.</p

Expression of GRP78 and phosphorylated JNK in human CCA.

<p>(A) GRP78 and phosphorylated JNK in human CCA were analyzed using immunohistochemistry. (B) Summary of experimental findings. In human CCA cells, the phosphorylation of eIF2α initiates ATF4 expression, which then induces GRP78 accumulation. GRP78 plays an important role in promoting human CCA cells proliferation and invasion. Suppression of mTOR inhibits eIF2α-induced ATF4 expression, which leads to a decrease in GRP78 levels. JNK blocking decreases GRP78 levels through inhibiting the activity of mTOR. Similarly, PI3K/Akt blocking decreases GRP78 levels through inhibiting the activity of mTOR.</p

Additional file 1 of Multidomain interventions for non-pharmacological enhancement (MINE) program in Chinese older adults with mild cognitive impairment: a multicenter randomized controlled trial protocol

Supplementary Material