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. 2021 Oct 25;22(21):11505. doi: 10.3390/ijms222111505

Figure 3.

Figure 3

NCAPH was a poor prognostic factor in ER-positive breast cancer patients. (A) Representative micrographs of breast cancer tissues stained with NCAPH antibody. Strong immunoreactivity (IR) was defined as positive IR, whereas weak IR or no IR was defined as negative IR. One breast cancer tissue was applied with non-specific rabbit IgG antibody as a negative control. The scale bars represent 10 μm. (B) Disease-free survival of breast cancer patients with positive or negative NCAPH IR is shown by the Kaplan-Meier method. p-value was determined by the log-rank test. The red line represents cases with positive OCT1 IR (n = 63), and the blue line represents negative NCAPH IR (n = 45). (C) Western blot analysis for NCAPH expression in MCF-7 cells and LTED cells. β-actin protein was blotted as a loading control. IB, immunoblot. (D) Western blot analysis for NCAPH expression in MCF-7 cells and LTED cells treated with two kinds of siRNAs for NCAPH (siNCAPH #1 or #2) or siControl (siCont.). β-actin protein was blotted as a loading control. (E) DNA content of MCF-7 and LTED cells on indicated days after transfection of indicated siRNAs analyzed by Hoechst 33342 staining. Relative fluorescence intensity (FI) was shown as mean and SEM (n = 4). *** p < 0.001 compared to cells treated with siControl. (F) Proportions of cell populations in G0/G1, S and G2/M phase of cell cycle in MCF-7 and LTED cells transfected with indicated siRNAs. The results of flow cytometric analysis shown in Figure S8 were quantified. (G) DNA content of MCF-7 cells treated with indicated siRNAs and expression vectors on indicated days after transfection of siRNAs was analyzed by Hoechst 33342 staining. On the day 0, transfection with siRNAs (siControl or siOCT1 #1) was performed. On the first day (Day1), transfection with expression vector encoding NCAPH (NCAPH) or empty vector (Vector) was performed. Relative fluorescence intensity (FI) was shown as mean and SEM (n = 4). * p < 0.05, *** p < 0.001. (H) Proportions of cell populations in G0/G1, S and G2/M phase of cell cycle in MCF-7 cells transfected with indicated siRNAs and expression vectors. The results of the flow cytometric analysis shown in Figure S9 were quantified.