Fig 2: Generation of Scp4 gene knockout mice.(A) Diagram illustrating the strategy used to generate Scp4 knockout mice. Red arrows indicate the position of primers used for genotyping. (B) Genotyping of Scp4 knockout mice by PCR. Het heterozygous mouse, Mut Scp4 mutant allele.

Fig 3: SCP4 is a chromatin-associated protein and interacts with H3 directly.(A) Complex formation between endogenous H3 and SCP4. HEK293T cell lysates were subjected to IP with anti-SCP4 antibody or rabbit IgG as a negative control. The IP products were examined by WB to detect the presence of H3. WCL: whole cell lysate. IgG-HC: heavy chain of IgG. (B) SCP4 binds to H3 directly in vitro. Purified GST only, GST-SCP4-WT, or GST-SCP4-DN proteins absorbed to glutathione sepharose beads were incubated with in vitro translated Myc-tagged H3. The pulled-down products were analyzed by WB to determine H3 and SCP4 association. Input: in vitro translated Myc-tagged H3. Comparison of binding affinity to H3 between GST-SCP4-WT and GST-SCP4-DN was performed using ImageJ (Version 1.54p). The relative density ratios of Myc-H3 to GST-SCP4-WT and GST-SCP4-DN levels were quantified and presented as arbitrary units. Assays were repeated three times independently (n = 3). Results from the first experiment are shown as representative data, with all individual data points presented. All raw data are provided in the Source Data File. (C) SCP4 has a higher binding affinity for H3 than for other histones. Purified GST only or GST-SCP4 fusion proteins were incubated separately with in vitro-translated, Flag-tagged histones (H2A, H2B, H3, and H4). SCP4-bound histones were analyzed by WB. WCL: whole cell lysates. (D) H3 binds to SCP4, but not other SCP family members. Flag-tagged SCP family members (SCP1, SCP2, SCP3, and SCP4) were co-transfected with Myc-tagged H3 individually in HEK293T cells. Cell lysates were subjected to Co-IP using anti-Flag-conjugated beads. H3 was only detected in the SCP4 IP products by WB. WCL: whole cell lysate. (E) Cytosol and nucleus fractionation assay of HEK293T cells indicated that SCP4 is a nuclear protein. GAPDH: marker for cytosol fraction; Lamin A/C: marker for nucleus fraction. β-Actin: internal control for WB. (F) HEK293T cell lysates were separated into cytosol, soluble nuclear fraction, and chromatin fraction. The subcellular location of SCP4 was determined by WB, indicating the chromatin localization. GAPDH: marker for cytosol fraction; PML (promyelocytic leukemia protein): marker for soluble nuclear and chromatin fractions. H3: marker for chromatin-associated protein. WCL whole cell lysate. (G) SCP4 is a nuclear protein as determined by IF in HeLa cells. α-tubulin: marker of the cytoskeleton. Scale bars: 5 μm. (H) IF staining of mitotic HeLa cells showed that SCP4 was localized on the chromosome. Scale bars: 5 μm. Source data are available online for this figure.

Fig 4: Generation of HCT116 and HeLa stable cell lines with SCP4 overexpression or knockdown, as well as H3T3 mutant.(A, B) Dox-inducible SCP4 overexpression (A) or knockdown by shSCP4 (B) was confirmed by WB. β-Actin: internal control for WB. Source data are available online for this figure.

Fig 5: Stable HeLa and HCT116 cell lines were generated to express either a phospho-mimetic H3 mutant (H3T3D or Thr3Asp) together with SCP4 or a non-phosphorylatable H3 mutant (H3T3A or Thr3Ala) together with shSCP4, while endogenous H3 was simultaneously knocked down using shH3.(A) The knockdown efficiency of shH3#1 and shH3#2 was confirmed by WB in HeLa cells. (B) Expression of shH3-resistant H3T3D and H3T3A mutants was confirmed by WB in HeLa cells. (C, D) SCP4-overexpressing cells were established in H3T3D-expressing HeLa (C) and HCT116 cells (D). (E, F) SCP4-knockdown cells were established in H3T3A-expressing HeLa (E) and HCT116 cells (F). β-Actin: internal control for WB. Source data are available online for this figure.