High autophagosome concentration is consumed during early immortalized human mesenchymal stem cell differentiation. (A) Immortalized human mesenchymal stem cells were differentiated under osteogenic conditions (see Materials and methods) and assayed for changes in LC3I and LC3II during a 72-hour window. Cells were differentiated under standard conditions (top) or with addition of 5 μM rapamycin (middle) or 5 nM bafilomycin (bottom) for the first 3 hours of differentiation to modulate autophagy. Immunoblots were performed for LC3 at the indicated time points to assess autophagosome degradation via relative changes in LC3II (lower band; 17 kDa). Studies were repeated three times with similar trends seen consistently. (B) Average standardized densities normalized by the sum of replicates were quantified via densitometry to measure autophagosome accumulation (LC3II bands) across three separate differentiations. Average values as standardized to β-actin are reported here. LC3, light chain 3.