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Fig. 2. ER stress induces CAP2 expression via promoter binding of ATF2. (A) SNU423 and Huh7 cells are treated with/without thapsigargin (Thap; 50 nM and 100 nM, respectively), tunicamycin (Tuni; 50 ng/ml), or DTT (1 mM) for 24 h. Con, control. Expression of CAP2 mRNA is measured by qRT-PCR and normalized to β-actin mRNA. Data represent mean ± SEM of 4 independent experiments. (B) SNU423 and Huh7 cells are treated with thapsigargin (50 nM or 100 nM, respectively) for indicated times. Cell lysates are examined by western blotting with the indicated antibodies. CAP2 is indicated by asterisks (*). (C) Schematic representation of the CAP2 luciferase reporters. ChIP primer sets are shown. (D and E) Huh7 cells are transiently co-transfected with pTurbo-GFP and deleted constructs (D) or ATF2 binding site mutated construct (ATF2-Mut) (E) of the CAP2 promoter. After treatment with 100 nM thapsigargin for 24 h, total RNAs are extracted. Luciferase expression is monitored by qRT-PCR; Luciferase expression levels are normalized by that of GFP mRNA (right). Data represent mean ± SEM of the three independent experiments. (F) Cross-linked chromatin preparation from control and thapsigargin (100 nM, 24 h) treated cells are immunoprecipitated with anti-ATF2, anti-Histone H3 (H3), or normal rabbit IgG (IgG) antibodies. The ATF2 binding sites on the immunoprecipitated DNA are determined by RT-PCR using their corresponding primers. Amplificons of the input chromatin (input) prior to immunoprecipitation were served as controls for each chromatin extraction and PCR amplification. Chromatin immunoprecipitation using a Histone H3 antibody is served as a positive control (P.C) and a non-specific antibody (normal rabbit IgG) is served as a negative control (N.C). (G and H) Huh7 cells are transiently transfected with siNegative control (siNeg) or siATF2. After 24 h, thapsigargin (100 nM) is treated for 24 h and the CAP2 expression levels are measured by qRT-PCR (G) and western blotting (H). CAP2 is indicated by asterisk (*). The data represent mean ± SEM of three independent experiments. *P < 0.05, **P < 0.01, and ***P < 0.001.
Mol. Cells 2021;44:569~579
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