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. 2016 Apr 15;6:24324. doi: 10.1038/srep24324

Figure 4. CLOCK enhances PFKFB3 expression.

Figure 4

The promoter -reporter assay and ChIP assay were performed in SCC9 cells. (A) Schemes of a 6.1 kb PFKFB3 promoter and a 3.2 kb irrelevant DNA fragment. The strict binding site for BMAL1/CLOCK is about 431 bp upstream of the starter codon of PFKFB3 DNA. A genuine E-box validated by the ChIP assay (F,G) was underlined. (B) Verification of reporter constructs. Un-digested plasmids were used for DNA gel electrophoresis. pGL3-6.1K, luciferase expression driven by a 6.1 kb fragment of PFKFB3 promoter; pGL3-3.2K, luciferase expression driven by a 3.2 kb irrelevant DNA fragment, and pGL3, a basic reporter construct. (C) Effects of endogenous clocks on PFKFB3 promoter activity. SCC9 cells were synchronized and transfected with pGL3-6.1K, pGL3-3.2K, or pGL3 at 7 hr and/or 19 hr post synchronization (zeitgeber time, ZT7 and/or ZT19). (D) CLOCK stimulation of PFKFB3 promoter transcription activity. SCC9 cells were co-transfected with pGL3-6.1K and BMAL1-, CLOCK-, or green fluorescent protein (GFP)-expressing plasmid. Some cells were co-transfected with pGL3-6.1K, BMAL1-expressing plasmid, and CLOCK-expressing plasmid. (E) Effects of mutant CLOCK on PFKFB3 promoter transcription activity. SCC9 cells were co-transfected with pGL3-6.1K and wild-type CLOCK- or mutant CLOCK-expressing plasmid. For (C–E), cells were harvested for analysis of luciferase activity at 24 hr post transfection. Luciferase activity was normalized to protein concentrations, and expressed as arbitrary units. (F) ChIP assay in SCC9 cells. The PCR products of the DNA prepared from chromatin immunoprecipitated with IgG and/or antibodies against BMAL1 or CLOCK. (G) The resultant DNA of chromatin immunocomplex was subjected to quantitative real-time PCR. For bar graphs, data are means ± SE. n = 5–6. ††P < 0.01 and †††P < 0.001 pGL3-6.1K vs. pGL3-3.2K or pGL3 for the same Zeitgeber time (in C); *P < 0.05; **P < 0.01; and ***P < 0.001 ZT19 vs. ZT7 for the same condition (pGL3-6.1K in (C)), CLOCK vs. GFP (in D) under the same condition (pGL3-6.1K in (E)), or BMAL1 or CLOCK vs. IgG (in G); ‡‡P < 0.01 mCLOCK vs. CLOCK under the same condition (in (E)).