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Supplementary Figure S1. Flow cytometry analysis showing gating strategies for the sorting
of live quiescent (GFP-negative) and replicating (GFP-positive) beta-cells, and evidence for
beta-cell specificity of sorting. A. Beta-cells were gated using TSQ-staining of dissociated
islet cells from CcnB1-GFP transgenic mice. B. Beta-cells were alternatively isolated by
sorting Tomato-positive islet cells from Insulin-Cre;Rosa-LSL-Tomato, CcnB1-GFP
transgenic mice. C. Beta-cell specificity: Tomato is exclusively expressed in Insulin-positive
cells. Pancreatic sections from 1 month-old Insulin-Cre; Rosa-LSL-Tomato transgenic mice
were immunostained for DsRed (Tomato) (red) and insulin (green) and nuclei were
counterstained with DAPI (blue). Original magnification: 400. Tomato is expressed in the
majority of insulin positive cells, and only in insulin-positive cells. D. GFP marks replicating
beta-cells in pancreatic sections. Ccnb1-GFP transgenic mice were sacrificed 3h after BrdU
injection and pancreatic sections were immunostained for BrdU (red), GFP (green) and
Insulin (white), and nuclei were counterstained with DAPI (blue). Original magnification: 400.
E. Heatmap showing the Euclidian distances between the different RNA-seq samples as
calculated from the variance stabilizing transformation of the RNA-seq read counts (DESeq)
(25).
Supplementary Figure S2. Genes repressed in replicating beta-cells significantly overlap
with genes upregulated in human beta-cell-line after excision of T-Ag and hTERT.
Conversely, genes induced in replicating beta-cells are significantly correlated with genes
downregulated in beta-cell-line after excision of T-Ag and hTERT. For each gene set (row)
(genes activated or repressed upon cell cycle exit induced by T-Ag excision), the mean
expression of the overlapping differentially expressed genes in each experiment (column) is
shown, after logarithmic transformation and row centering. The overrepresentation
significance (p-value) was computed using a statistical test based on hypergeometric
distribution.
Supplementary Figure S3. Most repressed genes in replicating cells are downregulated
roughly two-fold (Log2 (fold change) =-1). Scatter plot showing Log2 (Fold change) of
repressed genes (1245 genes) in replicating cells (Fold change was calculated by DESeq
after count normalization).
Supplementary Figure S4. Quantitative RT-PCR (qRT-PCR) analysis of the cell-cycle gene
Top2A and selected beta-cell maturation genes in sorted beta-cells isolated from Ccnb1-GFP
P7 pups. For each gene the mean expression value in GFP+ cells (n=3, FACS experiments)
+/- standard error is presented relative to GFP- cells after normalization to beta-actin (Actb). *
P<0.05 (t-test)
Supplementary Table 1. Genes upregulated in replicating beta-cells, associated with RNA
processing-GO category. Genes associated with the RNA splicing GO category are marked
in bold. log2(FC) (log2 fold change). FDR (BH) (False detection rate, Benjamini-Hochberg).
Supplementary Table 2. Genes encoding beta-cells ‘disallowed’ genes involved in glucose
metabolism are expressed at very low levels and are not significantly differentially expressed
in replicating cells. GFP- and GFP+ values are mean normalized counts (DESeq) for
quiescent and replicating beta-cells, respectively. FDR (BH) (False detection rate, BenjaminiHochberg).
Supplementary Table S3-1. Genes previously associated with cell cycle/proliferation,
upregulated in replicating beta-cells
Supplementary Table S3-2. Candidate Cancer genes with no documented function in
cell-cycle, upregulated in replicating beta-cells
Supplementary Table S3-3. Genes upregulated in replicating beta-cells, not associated
with cancer or cell cycle/proliferation
Supplementary Table 4. Cell-cycle and Proliferation-associated gene sets (MSigDB)
BIOCARTA_G1_PATHWAY
BIOCARTA_CELLCYCLE_PATHWAY
CELL_CYCLE_ARREST_GO_0007050
CELL_CYCLE_CHECKPOINT_GO_0000075
CELL_CYCLE_GO_0007049
CELL_CYCLE_PHASE
CENTROSOME_CYCLE
DNA_DAMAGE_CHECKPOINT
DNA_INTEGRITY_CHECKPOINT
KEGG_CELL_CYCLE
WHITFIELD_CELL_CYCLE_LITERATURE
S_PHASE_OF_MITOTIC_CELL_CYCLE
M_PHASE_OF_MITOTIC_CELL_CYCLE
M_PHAS
E
MITOTIC_CELL_CYCLE_CHECKPOINT
S_PHASE
INTERPHASE_OF_MITOTIC_CELL_CYCLE
INTERPHASE
REACTOME_CELL_CYCLE
REACTOME_CELL_CYCLE_CHECKPOINTS
REACTOME_REGULATION_OF_MITOTIC_CELL_CYCLE
WHITFIELD_CELL_CYCLE_G1_S
WHITFIELD_CELL_CYCLE_G2
WHITFIELD_CELL_CYCLE_G2_M
WHITFIELD_CELL_CYCLE_M_G1
WHITFIELD_CELL_CYCLE_S
STEGMEIER_PRE-MITOTIC_CELL_CYCLE_REGULATORS
BENPORATH_PROLIFERATION
Supplementary Table 5. sm-RNA-FISH probe sequences
Actb
Hars
Slc2a2
Glul
Pax6
gcagcgatatcgtcatccat
ttcggatgacttccggttac
ttccggtgatcttgtcttct
gaacttgctgaggtggccat
actcacacaaccgttggata
ggagccgttgtcgacgacca
actgacggctgtctaaaacc
agtgaagacagtgaaagcca
cttgatgcctttgttcaagt
cgtaatacctgcccagaatt
cgaagccggctttgcacatg
gacgctttctgctccttaag
aactggaaggaactcagtac
ggggcagggacatgtacatt
ttggcttactccctccgatt
acgatggaggggaatacagc
ccttcagttttaggagtttc
tgatcacaccgatgtcatag
atggcttggactttctcacc
tgcttacaacttctggagtc
atcacaccctggtgcctagg
ttgggggttttgagcacaaa
gaacacccaaaacatgtcga
tcttgcagcgcagtccttct
tcccgtttatactgggctat
gtccttctgacccattccca
ggggactatagtctctcgtg
tagttaatggcagctttccg
ggctcacagtccagggtacg
ttcccaagcaaagatggaag
tgggcctcgtcacccacata
acatcaaacaccttttcccg
atgctggtgtgactgtaagt
aggtaactcttccacacact
cggataataatctgtctcgg
ggtcaggatacctctcttgc
gcgtttgaaacagcggatga
tccttcagtctcctcttcat
tagagccatcaaagttccac
tgttatcgttggtacagacc
catgttcaatggggtacttc
ggtgtatcaatcacttctgc
agcatagtgactatgtgagc
gagccttcagactgaaaggt
ggttgcgaagaactctgttt
tcgtcccagttggtaacaat
tcagtgtttcctttagttca
aaagaatgaggcgaccattc
atggaggtacatgtcgctgt
atctgttgcttttcgctagc
gtggtgccagatcttctcca
ttgagtcttctccgtacttt
tgatccttccaagtttgtcc
ggtctcgaaacatggcaaca
ttatcatacatgccgtctgc
acacgcagctcattgtagaa
aggtcataacggagggacag
tcaatgagaggctgtttgca
ttgttggggtctttgcggaa
ctgcccgttcaacatcctta
agcacagggtgctcctcagg
ccagatagcgagcaaaaggg
ttggaacatcccatcaagag
gaaaacttcacatagcacca
aagtcccgggataccaacca
gttcaggggggcctcggtga
agcgtttaatgttggtcagt
acttcgtccagcaatgatga
ctgcaggcttccggttatac
tgttgctggcagccatcttg
tcttttcacggttggcctta
gcgatacacctttgctatat
ttagcccacaatacagtcct
cagatgtgcctcaagttggt
tgatggagttggtgttctct
gtctcaaacatgatctgggt
tagaattcacggtaacggcc
tgtacattggaaccagtcct
caccatgtccattatccgtt
tccgagtcttctccgttaga
gtacatggctggggtgttga
ccggcaatgtcaaaatcgca
taagaatgcctgtgacaagg
caaaccaggggtgctggttg
ctgaagtcgcatctgagctt
acagcacagcctggatggct
aggaatcatggggtcgaact
ataaagctgaggccagcaat
agagtatattcctgctccat
tttctttgcagcttccgctt
gtacgaccagaggcatacag
taatctttaggcactctgca
tgtgccaatgatcctgattg
tgggtggccgtctgttccca
ctgctcttgggtaaaagatg
tctccggagtccatcacaat
aagtgaactcaggatctcgc
ttctggacagaagagcagta
agccattggaaggccaacca
aaactctttctccagagcct
tagatgggcacagtgtgggt
aaacatcccatctaggatgc
ctgacttcctcttccaactt
gtctgctcccacaccgcagt
acatctggataatgggtcct
gtgagggagagcatagccct
tgctatcaggaacaccacag
cctcttagtctcttcaagct
cgatgtccctgccgtaggcc
tttggctgctagtctttccc
ccaggtccagacgcaggatg
gaggagcagatggtacggaa
cgatgcctcttccttttctt
caggcccggtagtgagcctc
ttcttgcttcaggtagatct
aggtagtctgtcaggtcccg
gaaaccttgtctagcttgtc
taattggcatccgtgaagag
gatcttgactccagcataca
cgattagaaaaccatacctg
acgctcggtcaggatcttca
accatgctgctggacatagt
ttgattcctgagaactgctg
taacctccgcatttgtcccc
tcttctcttctccatttggc
ctgtggtggtgaagctgtag
actgctccaccagagaaacc
gctgtctgaaaaatgctggt
tggaattcccactgggcagg
tcttctctggttcctcagtt
atgtcacgcacgatttccct
gagagtttaggatcctgcag
aatggttgcatacacaggct
tccaaagatgatctcccatt
atgtgactaggagtgttgct
caacatagcacagcttctct
ctctacagcctgtttgtttt
tgaagatcatgttgatggcc
cgatgcaagataaaacgggc
tactgaagctgctgctgata
atctcctgctcgaagtctag
caaagagcagctttaggtct
aatcatcccggttaggaaca
caccccaaagtcttcgcaca
gggattggctggtagacact
aggaagaggatgcggcagtg
gtcatcaatgccaaacagga
gacatgaagatggtgcagaa
gcttggggtcaaaggttgct
atgtgaaggaggagacaggt
agctcatagctcttctccag
caaggctcaggtcaaaggag
tttatccagcagcacaagtc
ccattccagttccctggaat
tgttcggcccaacatggaac
agtgatgacctggccgtcag
tggcatctgtagcagcactg
atgctcacgtaactcatcca
gaagttggtatggcagcctg
aaagcactgtacgtgttggt
atcggaaccgctcgttgcca
ttggggtcaaacatgccaac
tgacaaagaggaagatggca
cctcccgcatggccttggtg
ttgtttgccatggtgaagct
aaggctggaaaagagcctca
aatactgagcccaacgcatg
aaccatgaaccaagggattg
tcaatgcacttcagaccatt
agtatgaggaggtctgactg
caggattccatacccaagaa
aatggaaaagatccgctcca
ggtccttggctgaaaaattc
gctcagtttgtcaatggcct
atcataactccgcccattca
gaatgtagtttcatggatgc
tgaagcctctaatctttgct
aaattgcagacccagttgct
ggatgtggtactggtgcctc
tcatgtgtgtttgcatgtgc
cgtcacacttcatgatggaa
ttctgtgcagatgccacaag
aagtccgcaatgtactggaa
ccccccttgggatcgtaggc
atgagtcctgttgaagtggt
tagaggtctttacggatgtc
actctgagacaagcttcagt
aagaggaagaacacgtaagg
cagacgccgggcgttgtcca
aacttggacgggaactgaca
accagacagcactgtgttgg
aacttcgggttcttcttgta
tgtaaacagggtgaagacca
tggaggtttcgtggaatcca
agacatgtcaggttcactcc
tcagcaatgcctgggtacat
cacagtactgcaactggttt
gactttcctttggtttctgg
ccggcagaaaagtcgttgat
actgtaatcgaggccagtac
agtaatctccttctgcatcc
tcttctcttcggacatccac
attctgcagcgatttcctca
actggcaccgcggttggcaa
cttggttaaagtcttctgcc
atcttcatggtgctaggagc
tggagaggggctgatttgtt
tttgtacagcagctttgcgt
cgacagtccggggaatgcgg
agatctcacacatctgctca
ctcaggaggagcaatgatct
tactggtttgctcagctcag
aagctggacacagacagaga
aagtagcccttcttctcctg
gatatacgtccacttctaga
cgatccacacagagtacttg
ttaagatcaatcctgtttcc
agttggtgaagagtacccag
ggcagaaggccgacggtctt
aatacggggctctgagaact
SUPPLEMENTARY DATA
ac agt gaggeea ggatggag
atgc agttttgttttcgttc
c aca gcagata ggcc aagta
tc accgc ata ggggtcac aa
t gact gttct gcat gctgga
at ccac atctgctggaaggt
t gctggat ggc aaat atgca
t atctttggtgac atcctc a
cacgtgeggaegatggcttc
aagacaccaccaa gctgatt
actcatc atactcctactt a
ttttaccaataattact aat
ttctcc acaaacaacacaaa
at cacct attic altaaaaa
aactaactct acaatcttct
aa gcacttgc ggt gcacgat
ctataatcccc gac agt gta
ctctgaagacgcc aggaatt
agttcttgtattggaagggt
a gaattcgggaaat gtcgc a
©2016 American Diabetes Association. Published online at http://diabetes.diabetesjournals.org/lookup/suppl/doi:10.2337/db16-0003/-/DC1