Penicillin–Streptomycin Treatment Rewires Core Metabolic and Ribosomal Programs in HepG2 Cells

Antibiotics are routinely
added to mammalian cell culture
media
to prevent bacterial growth. However, the use of antibiotics in a
cell culture can confound downstream experimental results. While genomic
and transcriptomic differences between cell cultures treated with
and without antibiotics are well-documented, far fewer, if any, comprehensive
proteomic comparisons on the use of antibiotics in cell culture have
been performed. Here, we present a study on the proteome-wide differences
of culturing HepG2 cells in antibiotic (i.e., penicillin/streptomycin)
and nonantibiotic-containing media. Using a longitudinal and crossover
treatment study design, we analyzed 119 samples across nine passages
and four conditions. On average, 9,374 proteins were detected per
sample, and we identified 383 proteins that were differentially abundant
between conditions. These changes included ribosomal and mitochondrial
proteins, demonstrating that off-target effects of antibiotics on
mammalian cells occur at the protein level. Linear mixed-effect modeling
suggested that the proteomic impact of antibiotic treatment is strongest
in the first passage after treatment and stabilizes after approximately
three passages. Furthermore, initiating antibiotic treatment induced
a greater number of differentially abundant proteins than discontinuing
treatment. Lastly, we compared our results to existing literature
on the use of common antibiotics in mammalian cell culture. We identified
proteins and pathways conserved across studies, omics layers, and
cell types. We hope that this detailed proteomic survey of the ubiquitous
pencillin–streptyomcin-treated HepG2 in vitro model will aid researchers in comparing cross-study or cross-condition
results from antibiotic-treated mammalian cells and inform appropriate
experimental designs for the use of antibiotics in cell culture.