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. 2017 Jul 17;8(38):64050–64065. doi: 10.18632/oncotarget.19300

Figure 6. Sp110 promotes Ncl and Rps3a degradation.

Figure 6

(A) Overexpression of Sp110 in RAW264.7 cells decreased the protein levels of Ncl and Bcl2. The protein levels of Sp110, Ncl and Bcl2 in Uninfected and H37Ra infected (MOI 5:1, 24 h) RAW-Control or RAW-Sp110 cells were determined by immunoblotting. (B) and (C) RAW-Control and RAW-Sp110 cells were infected with H37Ra (MOI 5:1, 24 h), and uninfected cells were used as controls. Expression of Bcl2 and Ncl was monitored by qPCR. (D) Sp110 promotes Ncl protein degradation. RAW-Control and RAW-Sp110 cells were treated with protein synthesis Cycloheximide (10 μg/ml) or proteasome inhibitor MG132 (10 μM) for different periods of time. Expression changes of Ncl were determined by immunoblotting. The intensity of Ncl bands was quantified with ImageJ and normalized to the intensity of Actin. (E) Sp110 inhibits Bcl2 expression via downregulating Ncl. Expression of Ncl and Bcl2 in RAW-Control, empty vector transfected RAW-Sp110, and pCMV-Myc-Ncl transfected RAW-Sp110 cells was monitored by immunoblotting. (F) and (G) Knockdown of Ncl leads to the downregulation of both mRNA and protein levels of the Bcl2. RAW264.7 cells were transfected with siRNAs targeting Ncl or negative control siRNA for 48 h, and then the mRNA and protein expression of Ncl and Bcl2 was determined by qPCR and immunoblotting respectively. (H) Sp110 decreases the stability of the reporter gene that contains the ARE sequence of Bcl2 mRNA. RAW264.7 cells were cotransfected with psiCHECK2-Bcl2 ARE and pCDNA3.1-FLAG-Sp110 or pCMV-Myc-Ncl for 48 h followed by luciferase assays. (I) and (J) Expression of Rps3a in RAW-Control and RAW-Sp110 cells was determined by immunoblotting and qPCR respectively. (K) Sp110 promotes Rps3a protein degradation. RAW-Control and RAW-Sp110 cells were treated with protein synthesis Cycloheximide (10 μg/ml) for different periods of time. Expression changes of Rps3a were determined by immunoblotting. (L) Overexpression of Sp110 enhances PARP activity. Data are present as means ±SD of three independent experiments, **p< 0.01, and *** p< 0.001.