Fig 2: Erastin inhibits YAP and TFCP2 interaction. (A) A Co-IP experiment was performed using anti-YAP antibodies in control cells and PC9 or H1299 cells with or without erastin (10 μM, 24 h) treatment. The YAP level in each co-IP samples was adjusted to the same protein content. The interaction between YAP and TFCP2, LATS, SMAD2, TRIB2, and CREB were measured using the indicated antibodies by IB. (B) A Co-IP experiment was performed using anti-Myc antibodies in control cells and PC9 or H1299 cells with the indicated vectors transfected before erastin (5 μM, 4 h) treatment. The indicated proteins in co-IP samples or WCL were measured by IB. (C) Schematic representation for interactions between the PSY motif of TFCP2 and the WW domains of YAP. (D–F) Co-IP experiments were performed using anti-Myc (D,F) and anti-HA (E) antibodies in PC9 cells with the indicated vectors transfected before erastin (5 μM, 4 h) treatment. The TFCP2-Myc (D,F) and YAP-HA (E) level in each co-IP samples was adjusted to the same protein content. The indicated proteins in co-IP samples or WCL were measured by IB. (G) PLA experiments were performed using anti-Myc and anti-HA antibodies in PC9 cells with indicated vectors transfected before erastin treatment (5 μM, 4 h). Scale bar, 50 μm. Images of IB are representative ones of 3 independent experiments.

Fig 3: YAP and TFCP2 suppression upregulates labile iron through inhibiting FTL. (A) Labile iron was measured in control cells and PC9 or H1299 cells with YAP and TFCP2 individually or simultaneously knocked out. (B) Schematic representation of pathways and regulators that affect ferroptosis via labile iron levels. (C) The indicated mRNA levels measured in control cells and PC9 cells with YAP overexpression or knockdown. (D) YAP, FTL, HSPB1, and CISD1 expressions were measured using the indicated antibodies in control cells and PC9 or H1299 cells with YAP overexpression or knockdown. (E) YAP and FTL expressions measured using the indicated antibodies in control cells and PC9 or H1299 cells with YAP knockout. (F) FTL promoter luciferase (Luc) activity measured in control cells and PC9 cells with the indicated genes overexpression or knockdown. (G) TFCP2 and FTL expressions measured using the indicated antibodies in control cells and PC9 cells with TFCP2 overexpression, knockdown or knockout. (H) FTL promoter Luc activity measured in control cells and PC9 cells with YAP and TFCP2 individually or simultaneously knocked out. The data are shown as the mean ± SD from three biological replicates (including IB). *P < 0.05, **P < 0.01 indicates statistical significance. Data in (A,C,F,H) were analyzed using a one-way ANOVA test.

Fig 4: PCBP2 was involved in the regulation of YAP and TFCP2.a PCBP2 was identified by MS and UniProt software to be a ubiquitin-associated protein with YAP and TFCP2. b PCBP2 did not act as an RNA-binding protein to YAP and TFCP2, as measured by RIP in Bel-7402 cells. The α-globin mRNA was examined in parallel as a positive control. c PCBP2 negatively regulated the expression of YAP and TFCP2, as measured by WB in Bel-7402 cells. d PCBP2 boosted ubiquitination of YAP and TFCP2, as measured by co-IP in Bel-7402 cells. e CCT3 regulated ubiquitination of YAP and TFCP2 in a PCBP2-dependent manner, as measured by co-IP in Bel-7402 cells with or without CCT3 overexpression. f, g CCT3 regulated the expression and activity of YAP and TFCP2 via PCBP2, as measured by WB (f) and a pUAS-LUC/TEAD-Gal4 reporter system (g), respectively, in Bel-7402 cells. Images of RIP and WB are representative of threee independent experiments. **p < 0.01 indicate statistical significance. The data were analysed by a one-way ANOVA test from three independent experiments

Fig 5: CCT3 interacted with YAP and TFCP2 and was critical for liver cancer.a MS and bioinformatics revealed that CCT3 was associated with both YAP and TFCP2 in Bel-7402 cells, and might located on the membrane or in the cytoplasm. b Endogenous CCT3 interacted with endogenous YAP and TFCP2, as measured by co-IP in Bel-7402 cells. c Exogenous CCT3-HA interacted with YAP-FLAG and TFCP2-MYC, as measured by reciprocal co-IP in Bel-7402 cells. d The sub-cellular localisation of CCT2 and CCT3 in Bel-7402 and SMMC-7721 cells. Scale bar, 20 μm. e CCT3 was highly upregulated in liver cancer compared to adjacent liver, as evaluated in five paired adjacent (A) and tumourous (T) liver tissue. The representative IHC images of CCT3 are also shown on the right side. Scale bar, 200 μm. f The TCGA liver cancer cohort (50 normal liver, 371 liver cancer samples) identified CCT3 was upregulated in liver cancer. g Overall survival plots of liver cancer patients (n = 365, p < 0.001) in TCGA stratified by CCT3 expression. Images of IF, IHC and WB are representative of three independent experiments