Fig 2: Doubling time and anchorage-dependent and anchorage-independent colony formation of parental and NAT1 KO clones for MDA-MB-231, MCF-7, and ZR-75-1 cell lines. (a) Doubling time was determined in MDA-MB-231, MCF-7, and ZR-75-1 parental (P) and gRNA #2 (2) and #5 (5) NAT1 KO clones. Each bar illustrates mean ± SEM for number of replicates (N). Doubling times differed significantly between parental and NAT1 KO cell lines for MDA-MB-231 (p < 0.0001), and ZR-75-1 (p=0.0006) cell lines. (b) Anchorage-dependent growth/colony formation was determined in MDA-MB-231, MCF-7, and ZR-75-1 parental (P) and NAT1 KO cell lines. Cells (300) were plated on plastic in triplicate and allowed to grow for 14 days before staining. Anchorage-dependent growth/colony formation between parental and NAT1 KO cells were not significantly different (p > 0.05) for all cell lines, except for ZR-75-1. (c) Anchorage-independent/soft agar assays were completed in MDA-MB-231, MCF-7, and ZR-75-1 parental (P) and NAT1 KO cell lines. Cells (6000) plated in triplicate in soft agar were allowed to grow for 14 days before staining. The number of colonies formed in soft agar were significantly higher in parental MDA-MB-231 (p < 0.0001) MCF-7 (p < 0.0001) and ZR-75-1 (p < 0.05) than their respective NAT1 KO cell lines.

Fig 3: Methylation levels across the NAT1 promoter region. Representative DNA sequencing of (A) completely unmethylated and (B) completely methylated samples of the NAT1 gene. All unmethylated cytosines were changed to thymine by bisulfite treatment, but no methylated cytosines were changed. The CpG sites are indicated by underlining. (C) Methylation levels across the NAT1 promoter region. Data are presented as mean methylation of each CpG unit ± standard deviation. Significant differences between the MCF-7 and MCF-7R cells were detected at CpG3, CpG4, CpG5 and CpG6. *P<0.05 vs. corresponding MCF-7. NAT1, N-acetyltransferase 1; MCF-7R, tamoxifen-resistant MCF-7 cells.

Fig 4: In vitro and in situ PABA (N)-acetylation activity of parental and NAT1 KO clones for MDA-MB-231, MCF-7, and ZR-75-1 cell lines. (a) The in vitro PABA N-acetyltransferase activity in MDA-MB-231, MCF-7, and ZR-75-1 parental (P) and gRNA #2 (2) and #5 (5) clones NAT1 KO cell lines are shown. (b) The in situ PABA N-acetylation in MDA-MB-231, MCF-7, and ZR-75-1 parental (P) and gRNA #2 (2) and #5 (5) clones NAT1 KO cell lines are shown. Each bar illustrates mean ± SEM. Three or four separate determinations were performed in triplicate. ND is nondetectable ((a) <0.05 nmoles/min/mg; (b) <0.20 nmoles/hr/million cells).

Fig 5: NAT1 expression is decreased in MCF-7R cells and rescued by long non-coding RNA H19 knockdown. NAT1 (A) mRNA and (B) protein expression levels were detected using reverse transcription-quantitative PCR and western blot analysis, respectively, in various cell lines. Representative blots are shown with lanes as follows: lane 1, MCF-7; lane 2, MCF-7R, lane 3, MCF-7R/shH19; lane 4, MCF-7/NC; lane 5, MCF-7R/siH19; lane 6, MCF-7R/NS siRNA. All data are presented as the mean ± SEM of three independent experiments. *P<0.05 and ***P<0.05 as indicated. NAT1, N-acetyltransferase 1; MCF-7R, tamoxifen-resistant MCF-7 cells; siH19, siRNA targeting H19; NS, non-specific; siRNA, small interfering RNA; shH19, shRNA targeting H19; NC, negative control shRNA; shRNA, short hairpin RNA.