Serum withdrawal establishes a stress-dominant entry state during myogenic differentiation

Skeletal myogenesis in C2C12 cultures is typically induced by switching confluent cells from high-serum growth medium to low-serum differentiation medium. Because serum withdrawal is itself an acute physiological perturbation, early post-switch transcriptional changes may reflect varying mixtures of lineage progression and stress adaptation.

Here we performed a descriptive reanalysis of publicly available bulk RNA-seq datasets spanning (i) canonical C2C12 differentiation by serum withdrawal, (ii) bovine satellite-cell differentiation under serum-withdrawal versus serum-free or chemically defined conditions, (iii) additional bovine serum-free or defined-condition validation datasets, and (iv) a non-canonical serum-free \textit{Myod1} loss-of-function perturbation. Using a unified rank-based framework, we quantified stress-associated transcription, myogenic programme activation, and cell-cycle exit, and summarised their relationships using composite axes of stress dominance, transition abruptness, and exit--myogenic alignment.

Across bovine and murine serum-withdrawal time-course datasets, the earliest post-induction states showed relatively elevated and temporally leading stress-associated scores, together with weaker alignment between myogenic activation and durable cell-cycle withdrawal; by contrast, non-withdrawal conditions showed more gradual trajectories and stronger alignment. Independent bovine validation datasets provided directionally consistent external support for this descriptive contrast, showing progressive myogenic organisation and exit-related structure under non-withdrawal conditions or enhanced media without selective expansion of stress-dominant states. In the non-canonical serum-free \textit{Myod1} perturbation, myogenic scores decreased and cell-cycle-related scores shifted, whereas stress-associated scores remained similar, indicating a descriptive dissociation between these axes in a restricted perturbational context rather than evidence of physiological differentiation dynamics.

Together, these analyses provide operational, structure-neutral criteria for comparing induction conditions and interpreting early myogenic differentiation readouts across protocols.