Fig 1: Cenpa and Hjurp are down-regulated upon p53 activation. (A) RT-qPCR analysis of CENP-A and HJURP mRNA levels in p53-null, wild-type (WT), p53Δ31/Δ31 (Δ31), or p21-null MEFs untreated or treated with 10 µM nutlin for 24 h. Results from three (or two for p21-null) independent experiments are shown, each quantified in triplicate. (B) RT-qPCR analysis of CENP-A and HJURP mRNA levels in normal human fibroblasts (MRC5) or their SV40 transformed derivative cells (SVM for SV40 MRC5) in which p53 is inactive untreated or treated with 10 µM nutlin for 24 h. Results from three experiments are shown, each quantified in triplicate. (C) Putative CDE/CHR motifs identified near the transcription start sites (TSSs) of Cenpa and Hjurp using the positional frequency matrix (shown at the top; for details see Supplemental Fig. 2). Candidate CDE/CHR motifs were found close to the TSS of each gene; numbers in parentheses are positions relative to TSS. For Cenpa the CHR element perfectly matches the consensus sequence, whereas the CDE element perfectly matches the consensus sequence for Hjurp. (D,E) A 2-kb-long fragment centered around the Hjurp and Cenpa TSS, respectively, containing a wild-type or mutant CHR motif (for Cenpa) or CDE motif (for Hjurp) was cloned upstream of a luciferase gene and transfected into NIH/3T3 cells. The putative CDE/CHR and its mutated counterpart are shown. Nutlin treatment for 24 h induces a strong decrease in luciferase activity only with the construct containing a wild-type CDE/CHR motif. Data from two independent experiments (each in duplicates) were normalized, and the average luciferase activity in untreated cells transfected with the wild-type promoter construct was assigned a value of 1. For all graphs, means + SEM are shown. (***) P ≤ 0.001; (*) P ≤ 0.05; (NS) not significant, by ANOVA or Student's t-tests.
Fig 2: Transformed cells require HJURP for growth and survival. (A) Western blot of HJURP and CENP-A levels in wild-type, p53-null, or p53-null E1A and HRas-V12 transformed MEFs 6 d after transduction with CRISPR lentiviral particles against GFP (control) or two sgRNA constructs targeting Hjurp (Hjurp #1 and Hjurp #2) following puromycin selection. γ-Tubulin is used as a loading control. An asterisk marks a nonspecific band detected with the HJURP antibody. A twofold dilution series of each extract is represented by 4X, 2X, and 1X. Molecular weight protein markers are indicated at the right. (B) Proliferation assays in MEFs of the indicated genotypes following CRISPR-mediated depletion of Hjurp. MEFs (3 × 104) were seeded in triplicate in 1 mL of growth medium on 24-well plates 4 d after lentiviral transduction of CRISPR constructs. Hjurp CRISPR-resistant clones begin to emerge in E1A/HRas-V12 transformed p53-null MEFs 12 d after transduction. Results represent mean cell number ± SEM of triplicates relative to day 1 of the experiment and are shown in log scale. (C) Soft agar assay of p53-null E1A HRas-V12 transformed MEFs transduced with control (GFP) or Hjurp CRISPR constructs. We stained colonies with Sytox Green 4 wk after cell seeding. Bars represent quantification of visible colony numbers ± SEM. n = 3. P < 0.05, t-test. (D) Quantification of cell cycle analysis by flow cytometry (Edu/PI staining) in wild-type, p53-null, or p53-null E1A HRas-V12 transformed MEFs transduced with CRISPR constructs. Mean percentages of cells ± SEM for G1, S, G2/M, and tetraploid/aneuploid (>4N) populations (n = 3) are shown. Statistical significance is shown where relevant. (*) P < 0.05; (**) P < 0.005, t-test. See also Supplemental Figure S4D for corresponding flow cytometry plots. (E) Immunofluorescence images of p53-null E1A HRas-V12 transformed MEFs at day 6 after transduction with CRISPR constructs following puromycin selection. We stained nuclei with DAPI. See also Supplemental Figure S4F for full images. Individual magnified nuclei are shown. Micronuclei are highlighted by arrows. (F) Quantification of apoptosis by flow cytometry (Annexin V/PI staining) in wild-type, p53-null, or p53-null E1A HRas-V12 transformed MEFs transduced with CRISPR constructs. Mean percentages of cells ± SEM in early or late apoptosis are shown. Statistical significance is shown where relevant. (*) P < 0.05; (**) P < 0.005, t-test. See also Supplemental Figure S4E for corresponding flow cytometry plots.
Fig 3: The expression level of HJURP is a predictive factor for radiotherapy sensitivity. Kaplan-Meier survival curves for breast cancer patients according to radiotherapy treatment are presented. Part (a) shows the survival curves for disease-free survival, while (b) shows survival curves for overall survival. The P-values shown were obtained from a long-rank test.
Fig 4: Correlation between HJURP and CENPA in mRNA levels. There is a highly significant and positive correlation between HJURP and CENPA in mRNA levels within human breast cancer cell lines (a), Primary breast tumors (b), Dataset 1 (c), Dataset2 (d), Dataset4 (e), and Dataset5 (f). R shown is Spearman's rho correlation coefficient.
Fig 5: Association of HJURP mRNA levels with clinic and pathological factors in patients with breast cancer. There was no significant association between HJUPR mRNA levels and (a) ERBB2 (erythroblastic leukemia viral oncogene homolog 2) status, or (b) lymph node status, or (c) pathological stage or (d) tumor size. There were significant higher mRNA levels of HJURP in (e) estrogen receptor (ER) negative patients, (f) progesterone receptor (PR) negative patients; higher mRNA levels of HJURP were significantly associated with (g) high SBR grade, (h) younger age, and (i) Ki67 proliferation indices. HJURP expression is measured as log2 (probe intensities) by Affymetrix microarray. The two-tailed P-values were obtained by Mann-Whitney U test for ERBB2, lymph node, ER and PR status, Kruskal-Wallis H test for pathological stage and SBR grade, and Spearman correlation for size, age, and Ki67 proliferation indices.
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