Serum starvation: caveat emptor

Abstract

Serum starvation is one of the most frequently performed procedures in molecular biology and there are literally thousands of research papers reporting its use. In fact, this method has become so ingrained in certain areas of research that reports often simply state that cells were serum starved without providing any factual details as to how the procedure was carried out. Even so, we quite obviously lack unequivocal terminology, standard protocols, and perhaps most surprisingly, a common conceptual basis when performing serum starvation. Such inconsistencies not only hinder interstudy comparability but can lead to opposing and inconsistent experimental results. Although it is frequently assumed that serum starvation reduces basal activity of cells, available experimental data do not entirely support this notion. To address this important issue, we studied primary human myotubes, rat L6 myotubes and human embryonic kidney (HEK)293 cells under different serum starvation conditions and followed time-dependent changes in important signaling pathways such as the extracellular signal-regulated kinase 1/2, the AMP-activated protein kinase, and the mammalian target of rapamycin. Serum starvation induced a swift and dynamic response, which displayed obvious qualitative and quantitative differences across different cell types and experimental conditions despite certain unifying features. There was no uniform reduction in basal signaling activity. Serum starvation clearly represents a major event that triggers a plethora of divergent responses and has therefore great potential to interfere with the experimental results and affect subsequent conclusions.

Serum deprivation elevates the levels of microvesicles with different size distributions and selectively enriched proteins in human myeloma cells in vitro

“…Additionally, we observed that 24 h of SD caused an increase in the number of RPMI 8226 cells in the G 0 /G 1 phase (50.75%±1.13% vs 62.23%±1.26%) (P<0.01) and a decrease in the number of cells in the S and G 2 /M phases (18.24%±0.65% vs 9.43%±0.40%) (P<0.01) ( Figure 1B), which is consistent with the current standard approach to the synchronization and arrest of cells in the G 0 /G 1 state through SD treatment [15] .…”

“…SARM and COBL, the other two enriched proteins in SD MVs that were identified in our study, also play key roles in the regulation of target tumor cells. SARM has been demonstrated to be an inhibitor of the mTOR signaling pathway, and mTOR activity has been demonstrated to be reduced following SD in many cell types, which results in a decrease in protein synthesis [7] . The selective excretion of SARM reflects a reorganization of metabolic pathways and the production of biosynthetic intermediates in myeloma cells under SD.…”

“…Cellular stress has been identified as one of the key factors responsible for initiating the carcinogenic process and treatment resistance [1] . SD reduces basal cellular activity and induces apoptosis in various cells [7] . While in the early stage of SD, in contrast to normal cells, tumor cells initiate complicated processes against apoptosis [2,8] .…”

Hepatocellular carcinoma redirects to ketolysis for progression under nutrition deprivation stress

“…As reported previously [24][25][26][27], we observed that phosphorylation levels of AMPK, AKT1 and ERK1/2 were increased rapidly in response to serum starvation in HepG2 cells ( Figure 3A). Consistent with previous reports that serum starvation stimulated NF-κB activity [28], we found accelerated degradation of IκBα shRNAs or non-targeting control (NTC) were cultured under normal (Nor) or SS conditions for 48 h followed by incubating with 5mM of [2, 4-13 C 2 ] β-HB for 24 h and subsequent isotope tracing of 13 C-labeled metabolites by GC-MS. OXCT1 expression was determined by western blot.…”

Differential phosphorylation of the phosphoinositide 3-phosphatase MTMR2 regulates its association with early endosomal subtypes

“…In a variety cell types, serum starvation has also been shown to inhibit MAPK activation (Hu et al, 2003;Levin et al, 2010;Pirkmajer and Chibalin, 2011). Notably, we observed a significant decrease in MTMR2 Ser58 phosphorylation levels Fig.…”