PLCB1 and PRKCA were direct transcript targets of KLF16. (IMAGE)

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PLCB1 and PRKCA were direct transcript targets of KLF16.

Caption

(A) The flow chart for down-regulated KLF16 in gastric cancer (GC) MKN-74 cells. (B) KEGG pathway analysis identified significantly enriched genes, including PLCB1 and PRKCA, in the phosphatidylinositol (PI) signaling pathway. (C) After KLF16 overexpression (C1) or siRNA knockdown (C2) in AGS and MKN-74 cells, KLF16, PLCB1, and PRKCA expression were assessed with quantitative PCR. ∗∗∗P < 0.01 and ∗∗∗∗P < 0.001 for Vector vs. KLF16 or Scr-siRNA vs. KLF16-siRNAp. (D1/2) AGS and MKN-74 cells were transfected with either a KLF16 plasmid (D1) or KLF16-siRNAp (D2), and Western blotting was used to measure the expression of KLF16, PLCB1, and PRKCA. (E) Informatics analysis predicted the binding motif for the transcription factor KLF16. (F1/2) Schematic illustration of the PLCB1 (F1) and PRKCA (F2) promoters with potential GC-box sites. TSS, transcription start site. WT, wild-type; MT, mutated. (G1/2) DNA probes were validated through nucleic acid electrophoresis (G1), and biotin-labeled DNA pulldown experiments were used to confirm the binding relationship between KLF16 and PLCB1 promoter or PRKCA promoter (G2). (H1/2) A luciferase reporter assay evaluated the impact of KLF16 on PLCB1 (H1) and PRKCA (H2) promoter sites in GC cells co-transfected for 48 h, with results shown as the ratio of promoter to control vector luciferase activity. RLU, relative luciferase units; P1, promoter site 1; P2, promoter site 2. ∗P > 0.05, ∗∗P < 0.05, and ∗∗∗∗P < 0.001. (I1/2) Chromatin immunoprecipitation-PCR confirmed the direct binding of KLF16 to the PLCB1-p1 (I1) or PRKCA-p2 (I2) in GC cells.

Credit

Linjie Hong, Siyang Peng, Yidong Chen, Yanci Xie, Yuting Lei, Jieke Wu, Xiaodong Huang, Xiangyang Wei, Ping Yang, Jieming Zhang, Qiong Yang, Weimei Tang, Xiaosheng Wu, Ying Peng, Zhizhao Lin, AiminLi, Side Liu, Jiaying Li, Weiyu Dai, Jide Wang

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