Fig 1.
The structure of BBF2H7 and its expression in tumors or cancer cell lines.
(A) Increased expression of Bbf2h7 in human cancers. Microarray datasets of tumors were accessed in the ONCOMINE Cancer Profiling Database (version 4.4.4.4, www.oncomine.org). Box plots showing increased expression of Bbf2h7 during tumorigenesis of various cancers were constructed from ONCOMINE. The y-axis represents the log2 median-centered intensity (normalized expression). The line within the box represents the median expression value for each group, and the upper and lower edges of the box indicate the 75th and 25th percentiles of the distribution, respectively. The lines (whiskers) from each box extend to the 90th and 10th percentiles of the distribution. The black dots outside the ends of the whiskers represent the largest and smallest data points. Box plots depicting the distribution of Bbf2h7 expression within each sample and a Student’s t-test giving a P value for the comparison of Bbf2h7 expression between normal and malignant tissue samples were obtained directly through ONCOMINE. Normal colon tissue samples included ascending colon, descending colon and rectum. (B) Predicted peptide features of human BBF2H7. Transcription activation, basic leucine zipper (bZIP) and transmembrane domains, as well as a site-1 protease (S1P) site are indicated. (C) Under ER stress conditions, BBF2H7 is transported from the ER to the Golgi apparatus and cleaved at the S1P site [9]. The cleaved BBF2H7 N-terminus acts as a transcription factor via binding to the cyclic AMP response element (CRE) of target genes [15]. In contrast, the cleaved BBF2H7 C-terminus is extracellularly secreted [16]. (D) Western blotting of the endogenous full-length BBF2H7 and the N- or C-terminus of BBF2H7 using cell lysates (left panel) or culture media (right panel) of several human cancer cell lines. BBF2H7 N-terminus, BBF2H7 C-terminus or IL-6 in the culture media was immunoprecipitated using anti-BBF2H7 N-terminus (top panel), anti-BBF2H7 C-terminus (middle panel), or anti-IL-6 antibodies (bottom panel), respectively. β-actin or IL-6 was used as loading controls. (E) RT-PCR analysis of Xbp1 mRNA expression in several human cancer cell lines. Xbp1-s and Xbp1-u indicate spliced and unspliced forms of Xbp1, respectively. Note that Xbp1-s was detected in all cancer cell lines, indicating that these cells are undergoing ER stress. (F) RT-PCR analysis of Xbp1 mRNA expression in U251MG cells treated with 2 mM 4-PBA, a chemical chaperon, for 3 h. (G) Quantification of Xbp1-s expression in F. Error bars represent the mean ± SD of five independent experiments. **P < 0.01. (H) Western blotting of BBF2H7 in U251MG cells treated with 1 μM thapsigargin (TG), an inhibitor of ER Ca2+-ATPase, for 6 h. (I) Immunohistochemistry of brain sections from glioblastoma patients using an anti-BBF2H7 C-terminus-specific antibody. Strong signals were detected both in the cytosol of the cancer cells (indicated with an arrowhead) and in their surrounding extracellular space (indicated with arrows). Left panel shows a low magnification, and right panel shows a higher magnification of the left panel. Scale bars indicate 50 μm (left panel) and 10 μm (right panel).
Fig 2.
Cancer cell proliferation in an Shh-dependent manner.
(A) RT-PCR analysis of Hh target genes in several cancer cell lines treated with 500 ng/ml recombinant Shh for 48 h. (B) Quantitative real-time PCR analyses of Hh target genes expression in A. Error bars represent the mean ± SD of five independent experiments. *P < 0.05, **P < 0.01. (C) The cancer cell lines were cultured for 5 days after treatment with 500 ng/ml recombinant Shh and analyzed by WST-8 assays at day 5. Error bars represent the mean ± SD of five independent experiments. *P < 0.05.
Fig 3.
Activation of Hh signaling by BBF2H7 C-terminus.
(A, B) Bbf2h7-/- MEFs were cultured for 5 days after transfection with BBF2H7 full-length (Full), N-terminus (N), C-terminus (C) or an empty vector (Mock), and analyzed by cell counting at day 5 (A) and WST-8 assay at the indicated time points (B). WT and KO indicate wild type MEFs and Bbf2h7-/- MEFs, respectively. Error bars represent the mean ± SD of five independent experiments. *P < 0.05, **P < 0.01. (C) RT-PCR analysis of Hh target genes in several cancer cell lines exposed to conditioned medium collected from HEK293T cells transfected with BBF2H7 C-terminus (C-Sup. +) or an empty vector (C-Sup.–) for 48 h. (D) Quantitative real-time PCR analyses of Hh target genes expression in C. Error bars represent the mean ± SD of five independent experiments. *P < 0.05, **P < 0.01. (E) RT-PCR analysis of Hh target genes in U251MG cells treated with C-Sup., C-Sup. depleted of BBF2H7 C-terminus by immunoprecipitation using an anti-BBF2H7 C-terminus antibody (C-Sup. + Ab.), or C-Sup. in the presence of 5 μM cyclopamine (C-Sup. + CPN). Mock indicates conditioned medium collected from HEK293T cells transfected with an empty vector. (F) Quantitative real-time PCR analyses of Hh target genes expression in E. Error bars represent the mean ± SD of five independent experiments. *P < 0.05, **P < 0.01. (G, H) U251MG cells were cultured for 5 days after conditioned medium treatment and analyzed by cell counting at day 5 (G) and WST-8 assays at the indicated days (H). Error bars represent the mean ± SD of five independent experiments. *P < 0.05, **P < 0.01.
Fig 4.
Suppressed proliferation of cancer cells by knockdown of Bbf2h7.
(A) Western blotting of BBF2H7 in U251MG cells knocked down by siRNAs. Scramble and siBBF2H7 indicate non-targeting siRNA and Bbf2h7-targeting siRNAs, respectively. The sequences of siBBF2H7-1 and siBBF2H7-2 have different nucleotide positions. (B) Quantification of full-length BBF2H7 expression in A. Error bars represent the mean ± SD of five independent experiments. ***P < 0.001. (C) Western blotting of full-length BBF2H7 or the C-terminus of BBF2H7 using cell lysates (left panel) or culture media (right panel) of U251MG cells knocked down by siRNAs. BBF2H7 C-terminus or IL-6 in the culture media was immunoprecipitated using anti-BBF2H7 C-terminus (upper panel) or anti-IL-6 antibodies (lower panel), respectively. β-actin or IL-6 was used as loading controls. (D–G) U251MG cells were knocked down by siRNAs, and then cell growth was monitored by cell counting and WST-8 assays. (D, E) U251MG cells were cultured for 5 days after transfection with each siRNA in the absence or presence of 2 μM PPN, and then analyzed by cell counting at day 5 (D) and WST-8 assays at the indicated days (E). Error bars represent the mean ± SD of five independent experiments. *P < 0.05, **P < 0.01, ***P < 0.001. (F) RT-PCR analysis of Hh target genes in U251MG cells at 5 days after transfection with each siRNA in the absence or presence of 2 μM PPN. (G) Quantitative real-time PCR analyses of Hh target genes expression in F. Error bars represent the mean ± SD of five independent experiments. *P < 0.05.
Fig 5.
Secreted BBF2H7 C-terminus is involved in the proliferation of cancer cells.
U251MG cells were knocked down by siRNAs, followed by treatment with conditioned medium collected from HEK293T cells transfected with an empty vector (Mock) or BBF2H7 C-terminus (C-Sup.), or by treatment with C-Sup. depleted of BBF2H7 C-terminus by immunoprecipitation using an anti-BBF2H7 C-terminus antibody (C-Sup. + Ab.), and then cell proliferation was monitored by cell counting and WST-8 assays. Scramble and siBBF2H7-1 indicate non-targeting siRNA and Bbf2h7-targeting siRNA, respectively. (A, B) U251MG cells were cultured for 5 days after conditioned medium treatment, and then analyzed by cell counting at day 5 (A) and WST-8 assays at the indicated days (B). Error bars represent the mean ± SD of five independent experiments. *P < 0.05, **P < 0.01, ***P < 0.001. (C) RT-PCR analysis of Hh target genes in U251MG cells at 5 days after conditioned medium treatment. (D) Quantitative real-time PCR analyses of Hh target genes expression in C. Error bars represent the mean ± SD of five independent experiments. *P < 0.05.