Fig. 2.

Nanosilicates lead to stress-induced MAPK signaling. (A) Nanosilicate treatment results in activation of stress-related response. A list of significant GO terms related to stress after nanosilicate treatment indicate signal propagation via MAPK/ERK signaling pathways. (B) The majority of genes involved in stress-activated kinase signaling cascade (GO:0031098) undergo a significant differential expression. (C) The change in gene expression profile of MAP4K4 and TAOK1 (aligned reads normalized by total library size). (D) Comparison of TAOK1 gene expression obtained from RNA-seq was validated using qRT-PCR. (E) Nanosilicates trigger a stress-responsive kinase cascade (Ras–Raf–MEK–ERK pathways), leading to changes in reactive oxygen species (ROS) production and subsequent RNA transcription and protein synthesis. (F) Flow-cytometric analysis was performed to measure the stress-responsive kinase cascade, by measuring ROS production with a ROS-sensitive fluorescent reporter dye. Experiments were performed in the presence or absence of a MAPK inhibitor. A significant increase in ROS-mediated fluorescent signal is observed upon exposure to nanosilicate, and this is abrogated after treatment with the MAPK inhibitor. *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001. (G) Production of p-MEK1/2 was determined using Western blot in presence of nanosilicates and MEK inhibitor, establishing the role of nanosilicate in MAPK/ERK signaling. *P < 0.05. ns, not significant.