\begin{figure}
\centering
\includegraphics{figure_2_1.pdf}
\includegraphics{figure_2_2.pdf}
\includegraphics{figure_2_3.pdf}
\includegraphics{figure_2_4.pdf}
\caption{p53 mediates IFN-$\gamma$–induced Bmf suppression. (A) Bmf expression levels in p53–sufficient and p53–deficient cells evaluated by qRT-PCR. Bmf mRNA levels relative to p53-sufficient A549 and AALEB cells and the p53-deficient SAOS-2 and Calu-6 cells. (B) Western blot analysis of p53 in Calu-6 cells infected with adenoviral expression vector for p53 and the relative Bmf mRNA levels quantified by qRT-PCR in these cell lines when exposed to 10 mJ UV radiation compared with the respective nontreated controls. (C) p53 protein levels in MAECs 6 h after exposure to 10 mJ UV radiation and Bmf mRNA levels quantified by qRT-PCR in MAECs exposed to 10 mJ UV and nontreated (NT) controls. (D) Bmf mRNA levels in AALEB cells 6, 12, and 24 h after exposure to 10 mJ UV radiation compared with nontreated controls. Data presented are means $\pm$ SEM for three independent experiments. *, P $<$ 0.05.}
\label{figure_2}
\end{figure}

\begin{figure}
\centering
\includegraphics{figure_3_1.pdf}
\includegraphics{figure_3_2.pdf}
\includegraphics{figure_3_3.pdf}
\includegraphics{figure_3_4.pdf}
\caption{p53 suppresses IFN-$\gamma$– and HDACi-induced Bmf expression. (A) Bmf mRNA in IFN-$\gamma$–treated and nontreated (NT) p53$^{-/-}$ MAECs. (B) Bmf protein levels in protein lysates prepared from p53$^{-/-}$ MAECs treated with nothing or IFN-$\gamma$ for 48 h. (C) Bmf mRNA levels in AALEB cells treated with 5 mM sodium butyrate, 300 nM TSA, and 5 $\mu$M MS-275 for 18 h compared with nontreated controls. (D) Bmf mRNA expression in p53$^{-/-}$ and p53$^{+/+}$ MAECs treated with 300 nM TSA for 18 h. Error bars indicate $\pm$ SEM (n = 3 independent experiments). *, P $<$ 0.05: statistically significant difference from controls.}
\label{figure_3}
\end{figure}

to activate p53 \cite{Zhang_Xiong_2001}, was necessary for reducing Bmf levels, whereas the same treatment significantly increased Bmf mRNA levels in cells infected with adenoviral GFP as a control (Fig. \ref{fig:figure_2}C). Identical results were obtained when p53 was expressed in Calu-6 cells using a lentiviral expression system after treatment with UV radiation (unpublished data). Exposure of MAECs to 10 mJ UV radiation increased p53 levels (Fig. \ref{fig:figure_2}C) and reduced Bmf mRNA levels to 50\% of nontreated controls in both MAECs (Fig. \ref{fig:figure_2}C) and AALEB cells (Fig. \ref{fig:figure_2}D) 6 h after treatment.

These findings suggested that suppression of Bmf expression by IFN-$\gamma$ may be mediated by p53. Therefore, we investigated the effect of IFN-$\gamma$ on primary MAECs from p53$^{-/-}$ and p53$^{+/+}$ mice and found that Bmf mRNA levels were significantly up-regulated by IFN-$\gamma$ in p53$^{-/-}$ MAECs (Fig. \ref{fig:figure_3}A), and Bmfcug and Bmfs proteins were detected in p53$^{-/-}$ MAECs treated with IFN-$\gamma$ (Fig. \ref{fig:figure_3}B). Similarly, suppression of p53 levels in AALEB cells using shRNA resulted in IFN-$\gamma$ failing to suppress Bmf mRNA levels (unpublished data). Having established that p53 mediates IFN-$\gamma$–induced suppression of Bmf expression, we investigated the possibility that p53 may be affecting Bmf mRNA stability. In general, 3$'$UTRs have conserved sequences that regulate mRNA stability \cite{Stoecklin_Anderson_2007}, and Bmf mRNA has an unusually long 4-kb 3$'$UTR. AALEB cells treated with and without IFN-$\gamma$ were harvested at 0, 0.5, 1, 2, and 4 h after blocking transcription with DRB (5,6-dichloro-1-$\beta$-D-ribobenzimidazole). On average, the half-life of Bmf mRNA in nontreated p53-sufficient A549 and AALEB cells was 1.94 and 2.43 h and in the p53-deficient Calu-6 and SAOS-2 cells, was 2.36 and 1.73 h, respectively. Bmf mRNA half-life in IFN-$\gamma$–treated p53-sufficient and p53-deficient cells was 1.5 and 1.8 h, respectively. The similarity of the mRNA half-lives among cells with and without p53 and IFN-$\gamma$ treatment suggested that p53 does not affect Bmf mRNA stability.

Inhibition of HDACs (HDACis) with sodium butyrate, TSA, or MS-275 significantly increased Bmf mRNA in AALEB cells (Fig. \ref{fig:figure_3}C) and Bmfcug and Bmfs protein levels in MAECs (Fig. 1). Although Bmf mRNA levels were increased by TSA in both p53$^{-/-}$ and p53$^{+/+}$ MAECs, Bmf mRNA expression was twofold higher in p53$^{-/-}$ compared with p53$^{+/+}$ MAECs (Fig. \ref{fig:figure_3}D), suggesting that the inhibitory effect of p53 was still present in cells treated with HDACis.

A previous study has established that overexpression of HDAC1 suppresses Bmf expression by inhibiting the promoter activity \cite{Zhang_2006a}. Therefore, we next evaluated whether IFN-$\gamma$ affects the interaction of p53 with the Bmf promoter. The Bmf promoter region at $-97$ is the region where histone hyperacetylation occurs in cancer cell lines in response to HDACi treatment to increase Bmf transcription \cite{Zhang_2006a}. We found by chromatin immunoprecipitation (ChIP) that region $-97$, but not $-560$, of the Bmf promoter interacted with HDAC1 in p53$^{-/-}$ but not in p53$^{+/+}$ HCT116 cells, and IFN-$\gamma$ had no effect on this interaction (Fig. 4A). In AALEB cells, p53 interaction with the Bmf promoter was absent in nonaffected controls but was present at region $-97$ and not $-560$ when cells were treated with IFN-$\gamma$ (Fig. 4B). These findings suggest that HDAC1 requires the absence of p53 for its interaction with the Bmf promoter and that IFN-$\gamma$ enhances the interaction of p53 with the Bmf promoter to suppress transcription.

Because the nuclear or cytosolic localization defines p53 function \cite{Lee_Gu_2010}, we assessed p53 localization in IFN-$\gamma$–treated cells and found that compared with nontreated controls, IFN-$\gamma$ increased nuclear p53 levels, whereas cytosolic p53 levels remained low and unchanged (Fig. 4C). Immunofluorescence also demonstrated that the percentage of cells with nuclear p53 was significantly increased by IFN-$\gamma$ treatment (Fig. 4D).