Figure 3 Effect of targeted silencing of the c-Abl gene on the mRNA and protein levels of PSMB8 and its alternatively spliced isoforms. (A) JHU-011 cells and FaDu cells were divided into 6 groups (described previously). In the 3 groups with IFN-γ (final concentration of 150 U/μl), the transcription of PSMB8 in the IFN-γ+/Nor group and the IFN-γ+/Arg group was significantly upregulated but was inhibited in the IFN-γ+/c-Abl-shRNA group due to the targeted silencing of the c-Abl gene (** P<0.01). (B) PSMB8 protein expression levels were detected in JHU-011 cells by Western blotting, and the ImageJ software was used to determine the gray value and drew a histogram. Lanes 1–6 represent IFN-γ−/RNAi−/Arg−, IFN-γ−/RNAi−/Arg+, IFN-γ−/RNAi+/Arg−, IFN-γ+/RNAi−/Arg−, IFN-γ+/RNAi−/Arg+, and IFN-γ+/RNAi+/Arg−, respectively. The results show that the PSMB8 protein expression level was inhibited in the IFN-γ+/RNAi+/Arg− group (** P<0.01). (C) The PSMB8 protein expression level was inhibited in the IFN-γ+/RNAi+/Arg- group of FaDu cells (** P <0.01). (D) FaDu cells were divided into 6 groups. RT-PCR was performed to determine the ratio between E1 and E2 in each group. The transcription level of isoform E2 was significantly upregulated by IFN-γ (** P<0.01). The targeted silencing of c-Abl gene (IFN-γ+/c-Abl-shRNA) downregulated the transcription of E2 (** P<0.01). (E) JHU-011 cells were divided into 6 groups. RT-PCR was performed to determine the ratio between E1 and E2 in each group, and the results were consistent with those of FaDu cells (** P<0.01). Each group contained 3 independent replicates. Quantitative data are expressed as the mean±SD.