Non-cell autonomous and spatiotemporal signalling from a tissue organizer orchestrates root vascular development

During plant development, a precise balance of cytokinin is crucial for correct growth and patterning, but it remains unclear how this is achieved across different cell types and in the context of a growing organ. Here we show that in the root apical meristem, the TMO5/LHW complex increases active cytokinin levels via two cooperatively acting enzymes. By profiling the transcriptomic changes of increased cytokinin at single-cell level, we further show that this effect is counteracted by a tissue-specific increase in CYTOKININ OXIDASE 3 expression via direct activation of the mobile transcription factor SHORTROOT. In summary, we show that within the root meristem, xylem cells act as a local organizer of vascular development by non-autonomously regulating cytokinin levels in neighbouring procambium cells via sequential induction and repression modules

Data from: Molecular mechanisms of 2, 3′, 4, 4′, 5-pentachlorobiphenyl-induced thyroid dysfunction in FRTL-5 cells

Polychlorinated biphenyls (PCBs) can severely interfere with multiple animals and human systems. To explore the molecular mechanisms underlying 2, 3′, 4, 4′, 5- pentachlorobiphenyl (PCB118)-induced thyroid dysfunction, Fischer rat thyroid cell line-5(FRTL-5) cells were treated with either different concentrations of PCB118 or dimethyl sulfoxide (DMSO). The effects of PCB118 on FRTL-5 cells viability and apoptosis were assessed by using a Cell Counting Kit-8 assay and apoptosis assays, respectively. Quantitative real-time polymerase chain reaction was used to quantify protein kinase B (Akt), Forkhead box protein O3a (FoxO3a), and sodium/iodide symporter (NIS) mRNA expression levels. Western blotting was used to detect Akt, phospho-Akt (p-Akt), FoxO3a, phospho-FoxO3a (p-FoxO3a), and NIS protein levels. Luciferase reporter gene technology was used to detect the transcriptional activities of FoxO3a and NIS promoters. The effects of the constitutively active Akt (CA-Akt) and dominant-negative Akt (DN-Akt) plasmids on p-Akt, p-FoxO3a, and NIS levels were examined in PCB118-treated FRTL-5 cells. The effects of FoxO3a siRNA on FoxO3a, p-FoxO3a, and NIS protein levels were examined in the PCB118-treated FRTL-5 cells. The effects of pcDNA3 (plsmid vectors designed for high-level stable and transient expression in mammalian host)-FoxO3a on NIS promoter activity were examined in the PCB118-treated FRTL-5 cells. Our results indicated that relatively higher PCB118 concentrations can inhibit cell viability in a concentration- and time-dependent manner. Akt, p-Akt, and p-FoxO3a protein or mRNA levels increased significantly in PCB118-treated groups and NIS protein and mRNA levels decreased considerably compared with the control groups. FoxO3a promoter activity increased significantly, whereas NIS promoter activity decreased. These effects on p-FoxO3a and NIS could be decreased by the DN-Akt plasmid, enhanced by the CA-Akt plasmid, and blocked by FoxO3a siRNA. The overexpressed FoxO3a could reduce NIS promoter activity. Our results suggested that PCB118 induces thyroid cell dysfunction through the Akt/FoxO3a/NIS signaling pathway

Molecular mechanisms of 2, 3', 4, 4', 5-pentachlorobiphenyl-induced thyroid dysfunction in FRTL-5 cells.

Polychlorinated biphenyls (PCBs) can severely interfere with multiple animals and human systems. To explore the molecular mechanisms underlying 2, 3', 4, 4', 5- pentachlorobiphenyl (PCB118)-induced thyroid dysfunction, Fischer rat thyroid cell line-5(FRTL-5) cells were treated with either different concentrations of PCB118 or dimethyl sulfoxide (DMSO). The effects of PCB118 on FRTL-5 cells viability and apoptosis were assessed by using a Cell Counting Kit-8 assay and apoptosis assays, respectively. Quantitative real-time polymerase chain reaction was used to quantify protein kinase B (Akt), Forkhead box protein O3a (FoxO3a), and sodium/iodide symporter (NIS) mRNA expression levels. Western blotting was used to detect Akt, phospho-Akt (p-Akt), FoxO3a, phospho-FoxO3a (p-FoxO3a), and NIS protein levels. Luciferase reporter gene technology was used to detect the transcriptional activities of FoxO3a and NIS promoters. The effects of the constitutively active Akt (CA-Akt) and dominant-negative Akt (DN-Akt) plasmids on p-Akt, p-FoxO3a, and NIS levels were examined in PCB118-treated FRTL-5 cells. The effects of FoxO3a siRNA on FoxO3a, p-FoxO3a, and NIS protein levels were examined in the PCB118-treated FRTL-5 cells. The effects of pcDNA3 (plsmid vectors designed for high-level stable and transient expression in mammalian host)-FoxO3a on NIS promoter activity were examined in the PCB118-treated FRTL-5 cells. Our results indicated that relatively higher PCB118 concentrations can inhibit cell viability in a concentration- and time-dependent manner. Akt, p-Akt, and p-FoxO3a protein or mRNA levels increased significantly in PCB118-treated groups and NIS protein and mRNA levels decreased considerably compared with the control groups. FoxO3a promoter activity increased significantly, whereas NIS promoter activity decreased. These effects on p-FoxO3a and NIS could be decreased by the DN-Akt plasmid, enhanced by the CA-Akt plasmid, and blocked by FoxO3a siRNA. The overexpressed FoxO3a could reduce NIS promoter activity. Our results suggested that PCB118 induces thyroid cell dysfunction through the Akt/FoxO3a/NIS signaling pathway

The Efficacy of Shikonin on Cartilage Protection in a Mouse Model of Rheumatoid Arthritis

The potential therapeutic action of shikonin in an experimental model of rheumatoid arthritis (RA) was investigated. As a RA animal model, DBA/1J mice were immunized two times with type II collagen. After the second collagen immunization, mice were orally administered shikonin (2 mg/kg) once a day for 35 days, and the incidence, clinical score, bone mineral density (BMD), bone mineral content (BMC) and joint histopathology were evaluated. BMD in the proximal regions of the tibia largely increased in the shikonin treatment group compared with the control group. We also examined the effect of shikonin on inflammatory cytokines and cartilage protection. Shikonin treatment significantly reduced the incidence and severity of collagen-induced arthritis (CIA), markedly abrogating joint swelling and cartilage destruction. Shikonin also significantly inhibited the production of matrix metalloproteinase (MMP)-1 and up-regulated tissue inhibitors of metalloproteinase (TIMP)-1 in mice with CIA. In conclusion, shikonin exerted therapeutic effects through regulation of MMP/TIMP; these results suggest that shikonin is an outstanding candidate as a cartilage protective medicine for RA

Effect of PCB118 on FRTL-5 cell viability and apoptosis.

<p>Cells were treated with PCB118 at different concentrations (0.025–25000 nM in the Cell Counting Kit-8 (CCK8) assay and 0.025–25 nM in the apoptosis assay). The viability was measured using the CCK-8 assay (A). The apoptosis rate was measured using a FACSVantage SE flow cytometer (B). Data are presented as the mean ± SD of three independent experiments.*p < 0.05, compared with the DMSO control group. ^#p < 0.05, compared with the low PCB118-treated groups (0.025, 0.25, 2.5, and 25 nM).</p

Functional diagrams of NIS and the PI3K/Akt/FoxO3a pathway.

<p>NIS is located on the outer membrane of the thyrocyte. Its function is to pump sodium out of the follicular cells while pumping iodide into the follicular cells (A); PCB118 stimulates cells, activates the PI3K/Akt signaling pathway, and increases Akt phosphorylation levels, after which p-Akt phosphorylates its downstream target gene FoxO3a (B).</p

Exposure of FRTL-5 cells to PCB118 results in the up-regulation of FoxO3a or p-FoxO3a expression.

<p>Cells were treated with DMSO or various concentrations of PCB118 (0.025–25 nM) for 24 or 48 h. Whole cell lysates were analyzed by western blotting using antibodies recognizing FoxO3a and p-FoxO3a. GAPDH served as the loading control (A and B). Total RNA was isolated for qRT-PCR of FoxO3a (C), and the PCB118-treated FRTL-5 cells were harvested for the measurement of FoxO3a promoter activity (D). Results show sections of blots from one experiment of the three that yielded similar results (A) or represent the mean ± SD of three independent experiments (B, C, and D). GAPDH served as the loading control in western blotting.*p < 0.05, compared with the DMSO control group. The relative ratio of target mRNA/β-actin, target protein/GAPDH and promoter activity in solvent control group was set as 1.</p