Senescence Reversal

NANOG in Human Myoblasts

Gene Expression Clusters

Co-expression modules identified through unsupervised clustering

Cluster Selection Criteria

💪

Strong Response

|FC| >2

🔄

Youth Reversal

Pattern-based

📊

Consistent

Response >0.4, variance <0.3

⚠️

Senescence-Enriched

Pattern-based selection

🎯

Timepoint-Specific

Pattern-based selection

Selection approach: Clusters were manually selected based on biological patterns, prioritizing reversal signatures, specificity, and pluripotency markers rather than automated criteria alone.

Selected Clusters for Analysis

Cluster 1

736 genes • 0.0% unknown

💪 Strong Response

• 2.2% strong responders (16 genes with |FC|>2.0)

Silhouette: 0.417

Strong responders: 2.2% (16 genes)

F2RL2, EREG, TRHDE

Cluster 4

711 genes • 0.0% unknown

🔄 Youth Reversal

• Youth-enriched pattern (S→Y = -0.35)

• 15-day Nanog response: 0.39

Silhouette: 0.307

Strong responders: 0.7% (5 genes)

GPAT3, L1CAM, LAMA5

Cluster 6

1402 genes • 0.0% unknown

📊 Consistent💪 Strong Response

• Consistent response across conditions (variance=0.068)

• 2.1% strong responders (29 genes with |FC|>2.0)

Silhouette: 0.500

Strong responders: 2.1% (29 genes)

C12orf56, SLAMF9, Metazoa_SRP

Cluster 7

426 genes • 0.0% unknown

⚠️ Senescence-Enriched

• Senescence-enriched pattern (S→Y = +0.35)

• 30% response to 15-day treatment

Silhouette: 0.566

Strong responders: 0.2% (1 genes)

TIMP3, H19, FLNB

Cluster 9

836 genes • 0.0% unknown

🎯 Timepoint-Specific

• 15-day vs 5-day treatment difference: 0.18 (primary=0.46, tertiary=0.28)

Silhouette: 0.343

Strong responders: 1.3% (11 genes)

PDZRN3, FCRLB, COL28A1

Cluster 11

743 genes • 0.0% unknown

🔄 Youth Reversal

• Youth-enriched pattern (S→Y = -0.42, strongest reversal)

• 15-day Nanog response: 0.35

Silhouette: 0.235

Strong responders: 0.5% (4 genes)

MAP3K9, KLF4, TMEM200A

Cluster 14 ⭐

1000 genes • 0.0% unknown

🎯 Timepoint-Specific💪 Strong Response

• Contains NANOG (the intervention gene)

• 15-day vs 5-day treatment difference: 0.20 (primary=0.59, tertiary=0.39)

Silhouette: 0.342

Strong responders: 3.8% (38 genes)

NANOGP8, NANOG, NANOGP1

Cluster 15

759 genes • 0.0% unknown

📊 Consistent

• Consistent response across conditions (variance=0.062)

Silhouette: 0.437

Strong responders: 1.8% (14 genes)

GPX3, KIAA1755, SCUBE3

Cluster Comparison

Cluster	Genes	% Unknown	% Type B Dual function genes: strong senescence markers + high NANOG response	% Early Early responders: genes showing significant response within 5 days of NANOG treatment	Silhouette	% Strong	Specificity	Selection Criteria
Cluster 1	736	0.0% (0)	23.5% (173)	79.8% (587)	0.417	2.2% (16)	0.17	💪 Strong Response
Cluster 4	711	0.0% (0)	12.4% (88)	69.6% (495)	0.307	0.7% (5)	0.16	🔄 Youth Reversal
Cluster 6	1402	0.0% (0)	32.0% (449)	83.0% (1164)	0.500	2.1% (29)	0.16	📊 Consistent💪 Strong Response
Cluster 7	426	0.0% (0)	11.3% (48)	70.0% (298)	0.566	0.2% (1)	0.07	⚠️ Senescence-Enriched
Cluster 9	836	0.0% (0)	20.5% (171)	72.8% (609)	0.343	1.3% (11)	0.18	🎯 Timepoint-Specific
Cluster 11	743	0.0% (0)	7.7% (57)	65.8% (489)	0.235	0.5% (4)	0.14	🔄 Youth Reversal
Cluster 14	1000	0.0% (0)	31.0% (310)	77.7% (777)	0.342	3.8% (38)	0.20	🎯 Timepoint-Specific💪 Strong Response
Cluster 15	759	0.0% (0)	18.4% (140)	76.4% (580)	0.437	1.8% (14)	0.15	📊 Consistent

Top Pathways by Cluster

Cluster 1 736 genes

Neuroactive ligand-receptor interaction

p-adj: 5.49e-2 Genes: 12

C5, P2RY11, THRB, LTB4R2, CHRNA5 ...

Cluster 4 711 genes

Herpes simplex virus 1 infection

p-adj: 3.69e-3 Genes: 41

ZNF155, ZNF551, ZNF671, ZNF790, ZNF2 ...

Cluster 6 1402 genes

Hematopoietic cell lineage

p-adj: 2.22e-1 Genes: 11

CSF1R, IL11, CD4, CSF2, IL7 ...

Cluster 7 426 genes

Focal adhesion

p-adj: 1.35e-19 Genes: 35

ITGB1, ITGB5, SHC1, LAMC1, THBS1 ...

Cluster 9 836 genes

Viral protein interaction with cytokine and cytokine receptor

p-adj: 1.77e-1 Genes: 8

CXCL10, CXCL11, CXCL12, CCL20, TNFSF10 ...

Cluster 11 743 genes

DNA replication

p-adj: 1.63e-4 Genes: 11

POLD3, POLD4, POLA1, RNASEH2B, RNASEH2A ...

Cluster 14 1000 genes

Taste transduction

p-adj: 2.26e-1 Genes: 6

PDE1A, ADCY4, GNB3, TAS1R1, PLCB1 ...

Cluster 15 759 genes

Herpes simplex virus 1 infection

p-adj: 1.67e-2 Genes: 41

ZNF793, ZNF154, ZNF230, ZNF324B, ZNF273 ...