Download Supplementary Materials and Methods and Supplementary Figure

Supplementary methods
Genotyping
PCR was used to genotype the Zbtb4-/- mice using genomic DNA extracted from the tail or the
brain. The genomic DNA was extracted by Proteinase K (Roche) digestion followed by an
isopropanol precipitation. Genomic DNA was used for PCR with the following primers using
GoTaq
(Promega)
according
to
the
manufacturer's
instructions;
PAD882:
5’-
TGCCCTTTGCTGTGGCCTGA-3 and PAD884: 5’-GCACCCTGGCTCCTTCCAGC-3’.
Immunofluorescence and confocal imaging
All coverslips were incubated 1 hour at room temperature with 0.1% bovine serum albumin
(BSA) and next incubated 1 hour at RT with the antibodies directed against the following
antigens: mouse anti-phospho-histone H3-Ser10 (Cell Signaling, 9706, 1:100), rat anti-BrdU
(Abcam, ab6326, 1:200), rabbit anti-cleaved caspase 3 (Cell Signalling, 9661, 1:500), mouse
anti-alpha-Tubulin (Abcam, ab7291, 1:2000), mouse anti-gamma-Tubulin (Abcam, ab11316,
1:500), rabbit anti-LAMIN B1 (Abcam, ab16048, 1:500), mouse anti-BUBR1 (Thermo
Scientific, MA1-16577, 1:200). DNA was visualized with Hoechst 33342 (Sigma, 0.5 µg/ml) or
DAPI in Vectashield (Vector Laboratories).
Images were collected with either a LEICA DMI6000 B or a ZEISS LSM 710 Laser Scanning
Confocal microscope with a 63×-immersion oil objective. Image capture was controlled by
MetaMorph® Microscopy Automation & Image Analysis Software (LEICA) or Zen Software
(ZEISS). For all samples, an optimal setting of the laser power and PMT voltage was chosen to
avoid pixel saturation and minimize photobleaching. The settings were kept constant so that valid
1
comparisons could be made between measurements from different samples. FiJi (ImageJ)
software was used for the 3D quantification analysis.
RT-qPCR primers
ZBTB4
:
Forward
(F)
5’-AGGAAGTACCCCTGCCGCTA-3’,
Reverse
(R)
5’-
TTGTAGCCTCCATTGGGTGT-3’. BUB1B : (F) 5’-TAGGGCGTTTATGCAATGAGC-3’, (R)
5’-TCCTGAAATATCGCATCTGCTTT-3’.
:
MAD2L1
(F)
5’-
GTTCTTCTCATTCGGCATCAACA-3’, (R) 5’-GAGTCCGTATTTCTGCACTCG-3’. BUB3 :
(F) 5’-CTGCGCCTTCTACGATCCAA-3’, (R) 5’-GCATCATGGGTCCCAACAAGAT-3’.
:
CCNB1
(F)
5’-AATAAGGCGAAGATCAACATGGC-3’,
(R)
5’-
TTTGTTACCAATGTCCCCAAGAG-3’. B2M : (F) 5’-GGCTATCCACGTACTCCAAA-3’, (R)
5’-CGGCAGGCATACTCATCTTTTT-3’. RPLP0 : (F) 5’-TCCAGAGGCACCATTGAAATT3’, (R) 5’-TCGCTGGCTCCCACCTT-3’. TBP: (F) 5’-CCACTCACAGACTCTCACAAC-3’,
(R) 5’-CTGCGGTACAATCCCAGAACT-3’.
Ccnb1: (F) 5’-GCGTGTGCCTGTGACAGTTA-3’, (R) 5’-CCTAGCGTTTTTGCTTCCCTT-3’.
Ccnb2 :
(F)
5’-GCCAAGAGCCATGTGACTATC-3’,
(R)
5’-
CAGAGCTGGTACTTTGGTGTTC-3’. Bub1 : (F) 5’-AGAATGCTCTGTCAGCTCATCT-3’,
(R) 5’-TGTCTTCACTAACCCACTGCT-3’. Bub1b : (F) 5’-GAGGCGAGTGAAGCCATGT-3’,
(R)
5’-TCCAGAGTAAAAGCGGATTTCAG-3’.
Mad2L1:
(F)
5’-
GTGTTCTCCGTTCGATCTAGTG-3’, (R) 5’-CCACGCTGATACAAAATGCTG-3’. Cdc25c :
(F) 5’-GGCAAACCTAAGCATTCTGTCG-3’, (R) 5’-CCAGAGGTCCAGATGAATCCA-3’.
Chk1 : (F) 5’-ATCGTGAACGCTTACTGAACAA-3’, (R) 5’-CTGACAGCTATCACTGGGCT3’.
Plk1
:
(F)
5’-TGTAGTTTTGGAGCTCTGTCG-3’,
2
(R)
5’-
TCCCTGTGAATGACCTGATTG-3’. Plk4 : (F) 5’-GGATCGAGGACTTTAAGGTTGG-3’, (R)
5’-TCTCTGTACCATTCCAGCTTTG-3’. Cenpn : (F) 5’-CACAGTATTCACAGCCAAACC-3’,
(R) 5’-CAATCTGATGGTGTTTGCTGG-3’.
Vector Virus Production and Infection
ZBTB4 shRNA plasmid vectors (Sigma) contain the following hairpin sequences:
shZBTB4#1 (clone NM_020899.2-1293s1c1)
CCGGCGCTATTGTGAGAAAGTGTTTCTCGAGAAACACTTTCTCACAATAGCGTTTTT
shZBTB4 #2 (clone NM_020899.2-1389s1c1)
CCGGGAGACCTTTGTCACTTACTATCTCGAGATAGTAAGTGACAAAGGTCTCTTTTT
Western blotting
Western blots were performed using the following antibodies: rabbit anti-ZBTB4 (homemade
#120), mouse anti-BUB1B (Thermo Scientific, MA1-16577, 1:2000), rabbit anti-MAD2L1
(Abcam, ab97777, 1:500), mouse anti-Cyclin B1 (Thermo Scientific, MA5-14327, 1:500), rabbit
anti-Hitone H3 (Abcam, ab1791, 1:10000), rabbit anti-p53 (Santa Cruz, sc-6243, 1:500), mouse
anti-p21Cip1 (Santa Cruz, sc-6246, 1:1000), rabbit anti-p19ARF (Novus Biologicals, NB200-106
1:500), rabbit anti-Phospho Rb (Cell Signaling, 9308, 1 :1000), rabbit anti-p16Ink4a (Cell
Signaling, 4824, 1:1000), mouse anti-CDK4 (Cell Signaling, 2906, 1:2000). Secondary HRP
donkey anti-mouse IgG (Jackson ImmunoResearch, 715-035-150, 1 :10000) and HRP donkey
anti-rabbit IgG (Jackson ImmunoResearch , 711-035-152, 1 :10000) Bands were visualized by
horseradish peroxidase labeled antibodies and SuperSignal West Dura Extended Duration
Substrate (Thermo Scientific). FiJi (ImageJ) software was used for the intensity quantification
analysis.
3
Supplementary Figure Legends
Figure S1. Controls related to Figure 1.
A, Boxplots showing the percentage of the genome affected by Copy Number Alterations (CNA)
in the indicated tumor types, comparing the lowest decile for ZBTB33 expression ("ZBTB33
low") to the remaining tumors ("Others"); p-value calculated by T-test, n.s: no statistically
significant. B, Western blotting on whole-cell protein lysates from U2OS and HeLa expressing
shCTRL, shZBTB4 #1 or shZBTB4 #2. H3: Histone H3. C, Quantification of panel B D,
Quantification of anaphase U2OS cells showing lagging chromosomes after ZBTB4 knockdown
with shZBTB4 #1 and #2. E, ZBTB4 knockdown in HCT116 cells leads to increased
polynucleation.
Figure S2. Illustrations and controls related to Figure 2.
A, Representative mitosis of HeLa cells stably expressing EB3-GFP, H2B-mCherry and shCTRL
or shZBTB4 captured by time-lapse videomicroscopy at 40X. Scale bar = 10 m. B,
Representative gating of flow cytometry cell cycle analysis by PI and Br-dUTP staining, for cells
of the indicated genotypes, before, during, and after nocodazole block. C, Summary of cell cycle
analysis by PI and Br-dUTP staining showing percentage of cells in G1, S and G2/M (non gating
cells excluded).
Figure S3. Controls related to Figure 3.
A, Diagram of the ZBTB4 protein indicating the BTB/POZ domain and the DNA binding domain,
composed of 3 zinc fingers (ZF). The exons of the Zbtb4 gene are indicated in yellow and the
coding exons appear in dark yellow. The portion of ZBTB4 protein encoded by exon 3 is
4
indicated. Exon 3 was replaced by a cassette introducing LoxP sites and the neo gene to generate
the targeted allele. The knock-out allele of Zbtb4 lacks the entire exon 3. B, Primers used in PCR
for genotyping are indicated on top. The figure shows a representative PCR on genomic DNA
purified from brain of wild-type, Zbtb4+/- and Zbtb4-/- mice. The top band of the PCR is derived
from the wild-type allele, whereas the lower band is derived from the targeted allele. C, Primers
used in PCR for mRNA expression analysis are indicated on top. The relative expression level of
Zbtb4 mRNA in the brain of wild-type, Zbtb4+/- and Zbtb4-/- adult mice is shown below.
Expression values are normalized to a housekeeping gene and expression level in wild-type set
up to 1 (n=3). D, Zbtb4+/+ and Zbtb4-/- MEF cultures at passages 5 were stained for senescenceassociated-beta-galactosidase activity (SA-β-gal) and quantified. E, Quantification of BrdUpositive cells in Zbtb4+/+ and Zbtb4-/- MEF cultures at passage 3. F, Flow cytometry analysis of
the TUNEL-positive population in Zbtb4+/+ and Zbtb4-/- MEFs. G, Western blotting for p53 on
total cellular extracts from Zbtb4+/+ and Zbtb4-/- MEFs and the indicated controls.
Figure S4. Additional data related to Figure 4.
A, Volcano plot, Log2FC against pvalue, of Zbtb4-/- vs WT MEFs, red dots indicates
differentially expressed genes (pvalue ≤ 0.01). B, Gene sets significantly deregulated in Zbtb4-/MEFs, as determined using available genesets and GSEA analysis.
Figure S5. Additional data related to Figure 5.
Representative figures and quantification of -tubulin foci per cells.
Figure S6. Additional data related to Figure 6.
5
A, The relative expression level of Zbtb4 mRNA in papillomas from Zbtb4+/+, Zbtb4+/- and
Zbtb4-/- mice. Expression values are normalized to a housekeeping gene and expression level in
wild-type set up to 1. B, As in A., but for keratoacanthomas.
6