Fig 1: Expression levels of ABAT and relative enzyme activity of GABAT in SKM-1 and THP-1 cells. Relative mRNA expression levels of ABAT in (A) SKM-1 and (B) THP-1 cells 72 h post-infection, as determined by reverse transcription-quantitative polymerase chain reaction. Normal control cells were not infected with a lentiviral vector. Western blot analysis of (C) SKM-1 and (D) THP-1 cells in the normal control, NC and shRNA lentivirus groups. Gray value analysis of ABAT protein expression in (E) SKM-1 and (F) THP-1 cells infected with NC and shRNA lentiviruses. Relative enzyme activity of GABAT in (G) SKM-1 and (H) THP-1 cells with ABAT silencing. Relative activity of GABAT in (I) SKM-1 and (J) THP-1 cells treated with various concentrations of GABAT inhibitor. Data are presented as the means ± standard deviation. *P<0.05, **P<0.01 and ***P<0.001 vs. the normal control or 0 µM group. ABAT, 4-aminobutyrate aminotransferase; GABAT, ?-aminobutyrate aminotransferase; NC, negative control; ns, not significant; sh/shRNA, short hairpin RNA.
Fig 2: Cell cycle distribution and associated protein expression in SKM-1 and THP-1 cells following ABAT gene silencing. Cell cycle distribution of (A) SKM-1 and (B) THP-1 cells in the normal control, negative control and sh-ABAT groups. Expression levels of cell cycle proteins in (C and D) SKM-1 cells and (E and F) THP-1 cells with ABAT gene silencing, including cyclin D1, cyclin D3, CDK4, CDK6, p16 and p21. (D and F) Gray value analysis of cell cycle-associated proteins was conducted. *P<0.05, **P<0.01 and ***P<0.001 vs. the negative control group or normal control group. ABAT, 4-aminobutyrate aminotransferase; CDK, cyclin-dependent kinase; ns, not significant; sh, short hairpin RNA.
Fig 3: Cell cycle distribution and associated protein expression in SKM-1 and THP-1 cells treated with various concentrations of a ?-aminobutyrate aminotransferase inhibitor (0, 100, 200 and 400 µM). Cell cycle distribution in (A) SKM-1 and (B) THP-1 cells following treatment with various drug concentrations. Expression levels of cell cycle-associated proteins in (C and D) SKM-1 and (E and F) THP-1 cells following treatment with various drug concentrations. (D and F) Gray value analysis of cell cycle-associated proteins was conducted. Data are presented as the means ± standard deviation. *P<0.05 and **P<0.01 vs. 0 µM. ABAT, 4-aminobutyrate aminotransferase; CDK, cyclin-dependent kinase; ns, not significant.
Fig 4: Functional analysis of THP-1 and HL-60 cells with ABAT gene overexpression and HMAs treatment.A mRNA and protein expression of ABAT gene in THP-1 and HL-60 cells with ABAT overexpression. B GABAT enzyme activity in both cell lines with ABAT overexpression. C Total cell number after ABAT overexpression and HMAs treatment. Representative figures (D) and percentage (E) of apoptosis in ABAT overexpressed cells treated with HMAs. F Cell cycle analysis after ABAT overexpression and HMAs treatment. Relative expression of CD11b and CD15 in ABAT overexpressed THP-1 (G) and HL-60 (H) cells treated with HMAs. Data were shown as mean ± SEM from three biological samples. *P < 0.05, **P < 0.01, ***P < 0.001. Student’s t-test (two groups) or one-way ANOVA analysis followed by Dunnett’s test (multiple groups). ns. not significant.
Fig 5: Functional analysis of THP-1 and HL-60 cells with ABAT gene knockdown and HMAs treatment.A mRNA and protein expression of ABAT gene in THP-1 and HL-60 cells after ABAT interference. B GABAT enzyme activity in both cell lines with ABAT knockdown. C Evaluation of relative cell number in ABAT silenced cells treated with HMAs. Representative figures (D) and percentage (E) of apoptosis in ABAT silenced cells treated with HMAs. F Cell cycle analysis after ABAT interference and HMAs treatment. Relative expression of CD11b and CD15 in ABAT silenced THP-1 (G) and HL-60 (H) cells with HMAs treatment. Data were represented as mean ± SEM from three biological samples. *P < 0.05, **P < 0.01. Student’s t-test (two groups) or one-way ANOVA analysis followed by Dunnett’s test (multiple groups). ns. not significant.
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