Global Epigenetic Changes Induced by SWI2/SNF2 Inhibitors Characterize Neomycin-Resistant Mammalian Cells
Figure 6
Expression of endogenous SG2NA is influenced by ADAADi production.
The transcript as well as protein expression was monitored in the untransfected cells as well as in cells stably transfected with pcDNA 3.1 myc/his (−) vector at passage 13. (A). Endogenous SG2NA transcript was analyzed by quantitative RT-PCR in untransfected and transfected Neuro 2Acells. (B). Endogenous SG2NA protein analyzed by western blot using antibody against SG2NA. (C). sg2na promoter occupancy by RNAPII, Brg1, H3K9Ac, and H3K9Me2 was analysed in untransfected and transfected Neuro 2A cells using ChIP. Fold enrichment was calculated with respect to the mock ChIP done using IgG antibodies. (D). SG2NA transcript level in transfected cells at passage 4. (E). Expression of exogenous SG2NA expressed using pcDNA 3.1 myc/his (−) vector is also influenced by ADAADi production. Overexpression of three variants of SG2NA in Neuro2A cells were monitored using anti-myc antibody. Transfected cells (passage 4) were grown as indicated for 12 hours before analysis. Two clones of 87 kDa (87.1 and 87.2), one clone each of 78 kDa (78.1) and of 52 kDa (52.2) were analyzed for protein expression. The cells transfected with vector alone were used as control. Protein expression was observed only when cells were grown in the absence of both antibiotics. (F). Western blot analysis of expression of 87- and 78-kDa proteins in clones 87.1 and 78.1 in stably transfected cells grown in the presence of antibiotics using anti-myc antibody. Lane 1: vector alone transfected cells; Lane 2: 78.1 clone; Lane 3: 87.1 clone. N.S., indicating non-specific band, was used as loading control. (G). The expression of 78- and 87- kDa protein in 78.1, 78.2, 87.1 and 87.2 clones was monitored in stably transfected cells grown in the absence of antibiotics. Lane 1: vector transfected cells; Lane 2: 78.1 clone; Lane 3: 78.2 clone; M: marker; Lane 4: 87.1 clone; Lane 5: 87.2 clone. N.S., indicating non-specific band, was used as loading control. (H). Semi-quantitative RT-PCR analysis done using insert-specific forward primer and vector-specific reverse primer confirms that 87 kDa transcript expression is observed only when the cells are grown the absence of antibiotics. (I). The expression of 87 kDa, 78 kDa, and 52 kDa was not observed in stably transfected cells even after removal of antibiotics when the cells were freeze-thawed at passage 9. (J). Semi-quantitative RT-PCR analysis of APH transcript. Lane1: Untransfected Neuro2A cells; Lane 2: Stably transfected Neuro2A cells; Lane 3: control reaction using purified pcDNA 3.1 myc/his (−) vector.