Table 1.

Features of senescent cells that can serve as hallmarks of a state of cellular senescence and/or can be used as diagnostic biomarkers of senescent cells existing in organismal tissues

Affected aspect of cell morphology and function	Feature of senescent cells	Observed		Can serve as a hallmark/diagnostic biomarker of senescent cells	References
		in vitro*	in vivo**
Cell size and shape	Cell enlargement and acquisition of a flat or spindle-like shape	✓	✓	✓	22, 139, 140
Cell cycle	Cell cycle arrest - which is an essentially irreversible in vivo, but in culture can be reversed by certain genetic manipulations	✓	✓	✓	12, 97–99, 139,141, 142
Lysosomes	Increased size and number of lysosomes	✓	✓	✓	140, 143, 145, 146
	Many lysosomes become non-functional due to accumulation of lipofuscin-like indigestible molecular aggregates	✓	✓	✓	140, 144, 146
Senescence-associated β-galactosidase (SA β-Gal)	Elevated activity of SA β-Gal detectable at pH 6 - likely due to a senescence-associated increase in the level of lysosomal β-Gal protein, which exhibits the highest activity at pH 4, but if becomes abundant can also be detected at suboptimal pH 6	✓	✓	✓	140, 147, 149–151
Mitochondria	Excessive proliferation of mitochondria that are elongated, highly interconnected to form an extensive network, and aggregated	✓	✓	✓	140, 152, 154, 157
	Depolarization of the mitochondrial inner membrane, mitochondrial dysfunction, reduced ATP synthesis in mitochondria, and accumulation of ROS (that are produced mostly in mitochondria)	✓	✓	✓	140, 153, 157–161
DNA damage foci	Permanent establishment of nuclear foci marked with a set of the DNA damage response (DDR) proteins; these stable foci are known as DNA segments with chromatin alterations reinforcing senescence (DNA-SCARS), DNA double-strands breaks (DSBs), senescence-associated DNA-damage foci (SDF) and telomere dysfunction-induced foci (TIF)	✓	✓	✓	4, 12, 22, 25, 111, 140, 162–168
Nuclear bodies	Formation of promyelocytic leukemia nuclear bodies (PML NBs) also known as PML oncogenic domains (PODs); these sub-nuclear organelles concentrate numerous DNA-binding proteins that initiate heterochromatin establishment	✓	✓	✓	103, 124, 169–171
Heterochrom atic DNA foci	PML NBs-instigated formation of senescence-associated heterochromatic foci (SAHF); these foci are enriched in methylated Lys 9 of histone H3 (a heterochromatin marker) and concentrate a set of heterochromatin-associated proteins	✓	✓	✓	4, 12, 172–177
SASP/SMS	Specific changes in pattern of gene expression at transcriptional level - which result in secretion of a distinct set of interleukins, inflammatory cytokines, chemokines, growth factors, insoluble protein components of the extracellular matrix, extracellular proteases, as well as such non-protein soluble compounds as ROS, nitric oxide and prostaglandin E2	✓	✓	✓	4, 11, 22, 28, 31, 32, 165

^*Observed in cells entered a state of senescence in culture.

^**Observed in senescent cells recovered from organismal tissues.