Article Figures & Data
Figures
- FIG 1
HBO1 is not essential for DNA replication or sustained cell proliferation. (A) 293T, MCF7, and HeLa parental cells were treated with Dox for 3 days to induce sgRNA expression and assessed for HBO1 protein levels 1 week later. HeLa parental cells were also assessed for HBO1 protein levels 4 weeks after the initial Dox treatment. Each lane represents one parental cell line transduced with HBO1 sgRNA1 or sgRNA2. (B) 293T, MCF7, and HeLa clonal cell lines were assessed for HBO1 protein levels approximately 4 to 6 weeks after the initial Dox treatment. Each lane represents one clonal cell line transduced with HBO1 sgRNA1 or sgRNA2. (C to E) Flow cytometry quantification of BrdU and 7-AAD in cells, which distinguishes phases of the cell cycle in 293T, MCF7, and HeLa clonal cell lines. (F) Cumulative proliferation curves of 293T, MCF7, and HeLa clonal cell lines. Thirty thousand cells were seeded during the start of each passage interval. Clonal cell lines are the same as the ones presented in panel B and Fig. 3F to K. *, P < 0.05; **, P < 0.01; ***, P < 0.001. Data are presented as means ± standard errors of the means for 4 clonal cell lines, i.e., 2 each transduced with HBO1 sgRNA1 or sgRNA2. Numerical cell cycle phase assessment and cell proliferation rates per passage interval for individual clonal cell lines are shown in Fig. S2 in the supplemental material.
- FIG 2
Acute reduction in HBO1 does not lead to perturbation of HeLa cell cycle parameters. (A) HBO1 siRNAMiotto and HBO1 siRNAs253 are effective in reducing HBO1 protein levels. (B) Flow cytometry quantification of BrdU and 7-AAD in HeLa cells 72 h after HBO1 knockdown using siRNA. Note that there is no difference in the rates of the nucleotide analogue BrdU during S phase comparing untreated cells, cells treated with Lipofectamine alone, or cells with Lipofectamine-mediated transfection of nonsilencing RNA, HBO1 siRNAMiotto, and HBO1 siRNAs253. (C) Treatment of HeLa cells with siRNAs targeting HBO1 mRNA does not impair proliferation. KD, knockdown.
- FIG 3
Cytoplasmic, soluble nuclear, and chromatin-bound protein levels in HBO1-deleted HeLa cells compared to control cells. HeLa cells were synchronized for 16 h with 50 ng/ml nocodazole and released from nocodazole-induced cell cycle arrest for 2, 4, or 8 h or harvested without release at 0 h, as indicated, and fractionated into chromatin-enriched, soluble nuclear, and soluble cytoplasmic fractions. Lysed proteins were separated by SDS-PAGE, blotted, and subjected to immunodetection of specific proteins, as indicated. (A to C) Representative images of Western blots. (D to F) Densitometry results of MCM6 protein levels normalized to values for wild-type cells at 0 h and relative to loading controls. (G to I) Densitometry results of ORC2 protein levels normalized to values for wild-type cells at 0 h and relative to loading controls. Data are displayed as means ± standards errors of the means for eight experiments (incorporating 2 control cell lines for each of 2 sgRNAs [sgRNA1 and sgRNA2] and 4 HBO1-deleted cell lines for each of the 2 sgRNAs) and were analyzed by Student’s t test. Related data are displayed in Fig. S3 in the supplemental material, including protein levels of other proteins affected by the loss of HBO1.
- FIG 4
HBO1 depletion alters cellular morphology. (A to F) Cell morphology of 293T, MCF7, and HeLa single-cell clonal lines. (G to K) 293T cells were cultured for 6 h before removal of nonadherent cells and then counted. ****, P < 0.0001.
- FIG 5
HBO1 is essential for H3K14ac. (A to D) 293T, MCF7, and HeLa parental cells were treated with Dox for 3 days and assessed for histone acetylation levels 1 week (A to C) or 4 weeks (D) after Dox treatment. (E) HeLa parental cell acetylation levels were quantified by densitometry. Ponceau S staining was conducted as a loading control for the respective Western blots (see Fig. S4 in the supplemental material). Each lane represents one parental cell line transduced with HBO1 sgRNA1 or sgRNA2. HBO1 protein levels in the respective parental cell lines are shown in Fig. 1A. (F to K) 293T, MCF7, and HeLa clonal cell lines were assessed for histone acetylation levels approximately 4 to 6 weeks after the initial Dox treatment. Histone acetylation levels were quantified by densitometry in panels G, I, and K. Ponceau S staining was conducted as a loading control for the respective Western blots (Fig. S4). Each lane represents one clonal cell line transduced with HBO1 sgRNA1 or sgRNA2. HBO1 protein levels in the respective clonal cell lines are shown in Fig. 1B. *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001. Data are presented as means ± standard deviations from 2 parental cell lines, each transduced with HBO1 sgRNA1 or -2, in panel E. Data are presented as means ± standard errors of the means from 4 clonal cell lines, i.e., 2 each transduced with HBO1 sgRNA1 or sgRNA2, in panels G, I, and K.
- FIG 6
HBO1 siRNA-mediated knockdown reduces H3K14ac levels. Untreated HeLa cells, HeLa cells treated with Lipofectamine alone, or HeLa cells with Lipofectamine-mediated transfection of nonsilencing RNA, HBO1 siRNAMiotto, and HBO1 siRNAs253 were assayed for histone lysine acetylation. Transfection of HBO1 siRNAMiotto and HBO1 siRNAs253 resulted in reduced levels of H3K14ac but no changes in H3K9ac or H4K5ac, H4K8ac, H4K12ac, or H4K16ac. Western blots were performed 72 h after treatment. Western blots for the 24-h and 48-h time points are displayed in Fig. S5 in the supplemental material.
- FIG 7
Cell adhesion genes are downregulated in the absence of HBO1. Shown are RNA-Seq results for HeLa cells with an HBO1 deletion using sgRNA1 and sgRNA2 versus control cells (A to F) and combined analysis of sgRNA1 and sgRNA2 effects (B to F). (A) Barcode plot showing a high correlation between genes up- and downregulated as a consequence of the HBO1 deletion using sgRNA1 compared to sgRNA2 in HeLa cells. log FC, log fold change. (B) Heat map of genes differentially expressed between HBO1 knockout (KO) (guide 1, clones 1 and 2; guide 2, clones 1 and 2) and control HeLa cell (WT) clones induced for the expression of guide 1 or guide 2 in the absence of Cas9. (C) Biological process gene ontology terms enriched in genes downregulated in HBO1-null and control HeLa cells. (D) Volcano plot displaying log10 P values over log2 fold expression differences between HBO1-null and control HeLa cells, with downregulated (DR) “cell adhesion genes” indicated. UR, upregulated. (E) Cell adhesion genes >6-fold downregulated in HBO1-null and control HeLa cells. Two clonal control cell lines (one each for sgRNA1 and -2) and 4 clonal HBO1-null cell lines (two each for sgRNA1 and -2) were used. Data were analyzed by RNA sequencing as described in Materials and Methods. Genes differentially expressed with an FDR of <0.05 were considered significant. Related data on 293T and HeLa cells are displayed in Fig. S6 and S7 in the supplemental material. All data are provided in Tables S4 and S5. (F) Histone acetyltransferase (HAT) gene expression, apart from HBO1, is not changed.
Additional Files
Supplemental material
- Supplemental file 1 -
Fig. S1 to S7 and Tables S1 to S3
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- Supplemental file 2 -
Table S4
XLSX, 637K
- Supplemental file 3 -
Table S5
XLSX, 1.6M
- Supplemental file 1 -
