Figure S1 . Detection of the components of the condensin I and II complexes in human cell lines.
( A ) Cell lysates from the RPE-1 and cancer cell lines (HeLa, HCT116, and U2OS) were used for immunoblot analysis to detect the subunits of the human condensin I and II complexes. Tubulin serves as a loading control. ( B ) Whole cell lysates of RPE-1 and HeLa cells were subjected to Western blot analysis.
Expression of NCAPH2 shRNA (shH2#1) was induced by doxycycline (Dox). shRNA + and - indicate
Dox + and -, respectively. Arrows indicate the full-length hCAP-H2 and its smaller variant. Relative expression (%) of hCAP-H2 proteins after knockdown compared to expression without the shRNA induction was estimated. ( C ) OVCAR5 and OVCAR10 cells were scored for BrdU staining. ( D and E )
Rb proteins in the RPE-1, HeLa, HCT116, and U2OS cell lines (D), and the HOSE4 and ovarian cancer cell lines (E) were assessed by immunoblot analysis. Rb proteins were highly phosphorylated (P). ( F ) qRT-PCR analysis to detect NCAPH2 mRNA. RNA samples were prepared from RPE-1 cells cultured in normal and starvation media and subjected to cDNA synthesis, followed by quantitative PCR.
1

Figure S2. The hCAP-H2∆N variant is not derived from alternative splicing. ( A ) NCAPH2 mRNA in RPE-1 and HeLa cells was analyzed by RT-PCR. Open boxes and lines represent the exons and introns of the NCAPH2 gene, respectively. Arrows indicate primers used for RT-PCR. GAPDH mRNA was also analyzed as an experimental positive control. Negative control (N.C.) omits a reverse transcription step. ( B ) An alignment of the kleisin subunits of the condensin II complexes from human, mouse, bovine,
Xenopus laevis (frog), and Danio rerio (zebrafish). The first and second methionines are shown in red.
Asterisks, colons, and dots show that amino acid residues from the different species are identical, very similar, and weakly homologous, respectively. ( C ) An alignment of the N-termini of hCAP-H2 (human condensin II subunit), hCAP-H (human condensin I subunit), and Cnd2 (fission yeast condensin subunit).
Cylinders and arrows show helixes (H) and strands, respectively. Winged-helix motif consists of helixturn (T)-helix and two strands. The kleisin subunits of the condensin complexes are known to contain the winged-helix motifs at their N- and C-terminal domains.
32 ( D ) Graphic illustration for the full-length hCAP-H2, ∆N and ∆C variants.
2

3

Figure S3. hCAP-H2 proteins surround SAHF.
( A ) RPE-1 cells expressing the full-length hCAP-H2 fused to EYFP or carrying an empty vector (EYFP only) were subjected to paraformaldehyde fixation for
EYFP visualization. ( B ) Histone H3 tri-methylated Lys9 was visualized by IF in RPE-1 cells without overexpression of hCAP-H2 proteins. ( C ) RPE-1 cells expressing the full-length hCAP-H2 were subjected to IF analysis to visualize histone H3 di/tri-methylated Lys4 and DAPI signals. Inset shows enlarged views.
( D ) IMR90 cells were infected with a lentivirus encoding shRNA against NCAPH2 mRNA (shH2#1, #2, and #3). Relative expression of NCAPH2 transcripts after knockdown was estimated by qRT-PCR.
Expression level of the B2M (β -2-macrogloblin) gene was used as an internal control. ( E ) IMR90 cells were infected with a retrovirus encoding H-RasV12 as well as a lentivirus encoding shH2#2 (left) or #3
(right), and assessed for SA-β-gal activity and SAHF formation 8 days after the infection. ( F ) RPE-1 cells expressing the full-length hCAP-H2 or ∆N variant fused to the Flag epitope were subjected to IF analysis to co-visualize hCAP-H2 (red) and Rb (green). These microscopic images were captured by a Leica SP5
II laser scanning confocal microscope. Scale bars indicate 5 µm.
4

Table S1 – List of primers
Primers Sequence (5’ to 3’)
Exon1_F GGCGGGAACAGCAAAATG
Exon5_R
Exon9_R
CACGTTAGTCCGGGAGTCAG
AGCTCTACTGCCTCCTCTGC
Exon8_F AGGAGCAGCCAATGGAAGTT
Exon18_R AGCAGAGGCTGCACTGTGT
Exon12_F GACAGAAGCGCAAGAGGAAG
Exon20_R ACACACGGCTCCTCAGTACA
GAPDH_F TCACCAGGGCTGCTTTTAAC
GAPDH_R GCCCCACTTGATTTTGGAG
NCAPH2_F AGGAGCAGCCAATGGAAGTT
NCAPH2_R AGCTCTACTGCCTCCTCTGC
B2M_F GGCATTCCTGAAGCTGACA
B2M_R CTTCAATGTCGGATGGATGAAAC
These primers were used for RT-PCR and qRT-PCR to detect NCAPH2 , GAPDH, and B2M transcripts.
5