Rabbit Anti-Human S6 monoclonal antibody, clone 0I9M3 [Alexa Fluor 555]

Hepatic stellate cells (HSCs) play a vital role in liver fibrosis, and a greater understanding of their regulation is required. Recent studies have focused on relationships between extracellular matrix (ECM) stiffness and gene expression or cellular metabolism, but none have provided a detailed metabolic analysis of HSC changes in spheroid cultures. Accordingly, in the present study, we created an HSC spheroid culture and analyzed changes in gene expression and metabolism. Expression of alpha -smooth muscle actin (alpha -SMA) decreased in the spheroids, suppressing proliferation. Gene expression analysis revealed the cell cycle, sirtuin signaling, mitochondrial dysfunction, and the Hippo pathway to be canonical pathways, believed to result from decreased proliferative ability or mitochondrial suppression. In the Hippo pathway, nuclear translocation of the yes-associated protein (YAP) was decreased in the spheroid, which was associated with the stiffness of the ECM. Metabolome analysis showed glucose metabolism changes in the spheroid, including glutathione pathway upregulation and increased lipid synthesis. Addition of the glycolytic product phosphoenolpyruvate (PEP) led to increased spheroid size, with increased expression of proteins such as alpha -SMA and S6 ribosomal protein (RPS6) phosphorylation, which was attributed to decreased suppression of translation. The results of our study contribute to the understanding of metabolic changes in HSCs and the progression of hepatic fibrosis.

Background: Ribosomal protein S6 (rpS6), a component of the 40S ribosomal subunit, is involved in multiple cellular bioactivities. However, its clinicopathological significance in non-small cell lung cancer (NSCLC) is poorly understood. Methods: Expressions of total rpS6 (t-rpS6) and phosphorylated rpS6 (Ser235/236, p-rpS6) were detected immunohistochemically in 316 NSCLC tissues and 82 adjacent controls, followed by statistical evaluation of the relationship between proteins expressions and patients' survivals to identify their prognostic values. Cytological experiments with overexpressing or silencing rpS6 by lentivirus in human bronchial epithelial (HBE) and NSCLC cell lines were performed to explore potential mechanisms by which rpS6 affects the clinical development of NSCLC. Additionally, specific RNA interference for Akt1, Akt2, Akt3, Akt inhibitor and subsequent cellular bioactivity tests were employed as well to investigate the upstream regulation of rpS6. Results: Positive rates of t-rpS6 and p-rpS6 were both significantly increased in NSCLC tissues, compared with controls (82.91 vs 62.20 % for t-rpS6; 52.22 vs 21.95 % for p-rpS6; both P < 0.001). However, only hyperphosphorylation of rpS6, expressed as either elevated p-rpS6 alone or the ratio of p-rpS6 to t-rpS6 (p-rpS6/t-rpS6) no less than 0.67, was greatly associated with the unfavorable survival of NSCLC patients, especially for cases at stage I (all P < 0.001). The independent adverse prognostic value of hyperphosphorylated rpS6 was confirmed by multivariate Cox regression analysis (hazard ratios for elevated p-rpS6 alone and p-rpS6/t-rpS6 no less than 0.67 were 2.403, 4.311 respectively, both P < 0.001). Overexpression or knockdown of rpS6, along with parallel alterations of p-rpS6, led to increased or decreased cells proliferations respectively, which were dependent on redistributions of cell cycles (all P < 0.05). Cells migration and invasion also changed with rpS6 interference (all P < 0.05). Furthermore, upstream overexpression or knockdown of Akt2 or Akt2 phosphorylation inhibition, rather than Akt1 or Akt3, resulted in striking hyperphosphorylation or dephosphorylation of mTOR, p70S6K and rpS6 (all P < 0.05), without any change in total proteins expressions. Further tests showed markedly accompanied variation of cells proliferation, cell cycle distribution and invasion (all P < 0.05). Conclusion: Hyperphosphorylation of rpS6, probably regulated by the Akt2/mTOR/p70S6K signaling pathway, is closely relevant to the progression of NSCLC and it might be served as a promising therapeutic target for NSCLC treatment.