YAP/TAZ and ATF4 drive resistance to Sorafenib in hepatocellular carcinoma by preventing ferroptosis (2021)
Figure 4

Figure 4. YAP/TAZ stabilize ATF4 protein and direct it into the nucleus.

(a) YAP/TAZ deficiency repressed Sorafenib-induced expression of ATF4, whereas ATF4 depletion had no effect on YAP/TAZ protein levels. HLE cells were transfected with siCtrl or siATF4 or siY/T and treated with 6μM Sorafenib or not for 18 hours. ATF4 and YAP/TAZ protein levels were analyzed by immunoblotting. GAPDH served as loading control. Results represent three independent experiments.

(b) YAP/TAZ deficiency induced a reduction of ATF4 protein level and nuclear localization. HLE cells were transfected with siCtrl or siY/T, and ATF4 and YAP/TAZ were visualized by immunofluorescence microscopy. DAPI visualized nuclei. Scale bars, 50μm. Results represent three independent experiments.

(c) Cellular fractionation revealed that YAP/TAZ deficiency reduced ATF4 nuclear localization. HLE cells were transfected with siCtrl or siY/T, and cell lysates were separated into cytoplasmic and nuclear fractions and analyzed by immunoblotting for ATF4 and YAP/TAZ. GAPDH served as loading control of total lysate and cytoplasmic fractions, Lamin A/C served as loading control for nuclear proteins. Results represent three independent experiments.

(d) Cellular fractionation revealed that high cell density, and with it, lower YAP/TAZ activity reduced the levels of nuclear ATF4. HLE cells were seeded at different cell numbers, and cell lysates of high cell density cells (HD) and low cell density (LD) were fractionated into cytoplasmic and nuclear fractions and analyzed for ATF4 and YAP/TAZ by immunoblotting. GAPDH served as loading control for total lysate and cytoplasmic proteins, Lamin A/C served as loading control for nuclear proteins. Results represent three independent experiments.

(e) Increasing cell density declined YAP/TAZ stability and led to a reduction of ATF4. HLE cells were seeded to reach low (L), medium (M) and high (H) cell densities and transfected with siCtrl or siY/T. Protein levels of ATF4 and YAP/TAZ were determined by immunoblotting blotting. GAPDH served as loading control. Results represent three independent experiments.

(f) YAP/TAZ physically interacted with ATF4. HLE cells were cultured with 6μM Sorafenib for 18 hours and ATF4 was immunoprecipitated and precipitated YAP, TAZ and ATF4 were visualized by immunoblotting. Unrelated IgG was used as control for immunoprecipitation. Input represents cell lysates directly analyzed by immunoblotting. Results represent three independent experiments.

(g) YAP/TAZ directed the nuclear localization of ATF4. HLE cell were transfected with plasmid constructs coding for wild-type TAZ (1C-TAZ-WT), constitutive-active TAZ (1C-TAZ- 4SA) or constitutive-active TAZ lacking a nuclear localization signal (NLS) (1C-TAZ-mNLS) and analyzed for TAZ and ATF4 expression and localization by immunofluorescence microscopy. DAPI visualized nuclei. Scale bars, 66.2μm. Results represent three independent experiments.

(h) Representative images of immuno-histochemical staining of ATF4 and YAP proteins in HCC samples from patients. Scale bars, 50μm.

(i) Quantification of the immunohistochemical staining in (j) showed a positive correlation of ATF4 and YAP expression (N=18). Statistical significance was calculated using Pearson correlation analysis.