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. 2021 Oct 19;13(12):e14351. doi: 10.15252/emmm.202114351

Figure 5. YAP/TAZ‐ATF4 collaboratively regulate SLC7A11 expression.

Figure 5

  1. A total of 264 common genes were identified downregulated by both deficiency for YAP/TAZ and deficiency for ATF4, among which were known ATF4 targets, such as SLC7A11 and CHAC1. HLE cells were transfected with siCtrl, siYAP/TAZ, or siATF4 and cultured with 6 μM Sorafenib for 18 h and RNA was subjected to next‐generation RNA sequencing. Venn diagram analysis was conducted using VENNY 2.1.0.
  2. Quantitative RT‐PCR analysis verified that depletion of YAP/TAZ or ATF4 declined the expression levels of SLC7A11 and CHAC1. HLE cells were transfected with siCtrl, siY/T, or siATF4 and cultured with 6 μM Sorafenib for 18 h. Quantitative RT‐PCR was conducted to determine SLC7A11 and CHAC1 mRNA levels. Data are shown as mean ± standard deviation (SD). Statistical significance was calculated using two‐way ANOVA. Results represent 3 independent experiments.
  3. Binding of ATF4 to DNA fragments containing the AARE binding motif in the promoters of SLC7A11 and ATF3. ChIP was performed on HLE cell lysate with antibodies against ATF4 and rabbit IgG as control. DNA fragments were amplified using the primers specific for AARE binding motif in the SLC7A11 promoter region. The non‐coding region NC10 served as negative control. Data are shown as mean ± standard deviation (SD). Statistical significance was calculated using one‐way ANOVA. Results represent three independent experiments.
  4. Schematic representation of the ATF4 binding motif AARE located at – 95 bp of the SLC7A11 promoter region. YAP/TAZ were predicted to interact with ATF4 binding to the AARE binding motif in the promoter of SLC7A11.
  5. Binding of YAP and TAZ to DNA fragments containing the AARE binding motif in the SLC7A11 promoter. ChIP was performed on HLE cell lysate with antibodies against YAP and TAZ and rabbit IgG as control. DNA fragments were amplified using the primers specific for AARE binding motif in the SLC7A11 promoter region. The non‐coding region NC10 served as negative control, and the bona fide TEAD target genes CYR61 and ANKRD1 served as positive controls. Data are shown as mean ± standard deviation (SD). Statistical significance was calculated using one‐way ANOVA. Results represent three independent experiments.
  6. Schematic representation of the ChIP‐ReChIP strategy to test whether YAP/TAZ bind to the AARE binding motif within the SLC7A11 promoter via binding to ATF4.
  7. YAP/TAZ bind to DNA fragments containing the AARE binding motif within the SLC7A11 promoter via ATF4. HLE cells were cultured with 6 μM Sorafenib for 18 h before harvest. In a 1st round ChIP ATF4 was immunoprecipitated with antibody against ATF4, rabbit IgG was used as control. DNA‐protein immunocomplexes were eluted and in a 2nd round ChIP antibody against YAP/TAZ was used to precipitate DNA fragments which were then amplified and analyzed by quantitative PCR for the AARE motif in the SLC7A11 promoter. NC10 served as negative PCR control. Data are shown as mean ± standard deviation (SD). Statistical significance was calculated using one‐way ANOVA. Results represent three independent experiments.

Source data are available online for this figure.