Fig 2: Cell surface binding and TFCP2 rescue experiments.A, TFCP2C21 cells show a significant decrease in binding of a subset of protein ligands by flow cytometry (n ≥ 4, t test, ∗∗p < 0.01, ∗∗∗p < 0.001). B, Transfection of an HA-tagged human TFCP2 cDNA cassette restored TFCP2 expression in TFCP2C21 cells. C, Reintroduction of TFCP2 restored fibroblast growth factor 1 (FGF1) binding to wildtype levels (n = 3, t test, ∗∗p < 0.01). TFCP2, transcription factor cellular promoter 2.

Fig 3: The functions of YAP, TFCP2 and PCBP2 in CCT3 knockdown-suppressed transformative phenotypes.a Representative WB images of YAP/TFCP2 and their targets in control cells, Bel-7402 and SMMC-7721 cells with CCT3 knocked down in the presence or absence of YAP, TFCP2 or PCBP2 overexpression. b YAP boosted TFCP2 binding to PCBP2, as measured by co-IP in SMMC-7721 cells. c–e The cell proliferation capacity (c), caspase 3/7 activity (d) and colony formation capacity (e) were evaluated in Bel-7402 and SMMC-7721 cells with CCT3 knockdown in the presence or absence of YAP, TFCP2 or PCBP2 overexpression. f CCT3 knockdown-reduced in vivo tumour growth could be reversed by YAP and TFCP2, which were blocked by increasing PCBP2. n = 5/group. The data were analysed from three independent experiments (except Fig. 6f) using a one-way ANOVA test. **p < 0.01 indicates statistical significance

Fig 4: CCT3 blocked the interaction between PCBP2 and YAP/TFCP2.a PCBP2 directly interacted with YAP but not with TFCP2, as evaluated by PLA in Bel-7402 and SMMC-7721 cells. Scale bar, 50 μm. b YAP, TFCP2 bond with PCBP2, as measured by co-IP in Bel-7402 cells. c Schematic presentation of the hypothesis that CCT3 might prevent YAP/TFCP2 from interaction with PCBP2. d Interaction between exogenous PCBP2 and YAP, but not between PCBP2 and TFCP2, was identified by co-IP in Bel-7402 cells. e The WB2 motif was essential for the interaction between PCBP2 and YAP, as measured by co-IP in Bel-7402 cells. d, deletion. f The PCBP2-YAP interaction replied on the two WW domains in YAP, as measured by co-IP in Bel-7402 cells. d, deletion. g The WB2 motif was critical for ubiquitination of YAP and TFCP2. h The WB2 motif of PCBP2 was essential for CCT3-mediated suppression of ubiquitination of YAP and TFCP2, as measured in Bel-7402 cells with or without expressing increasing concentrations of CCT3. i The two WW domains of YAP were prerequisite for suppression of TFCP2 by PCBP2, as measured in control cells, Bel-7402 cells with YAP knocked down with or without simultaneous expressing exogenous YAP-WT/YAP-d2WW. j The two WW domains of YAP were essential for CCT3-induced disassociation of PCBP2 from YAP/TFCP2, as measured by co-IP in Bel-7402 cells. To clearly identify the interaction between PCBP2, TFCP2 and YAP, we artificially adjusted the concentrations of PCBP2 to the same level in the immune precipitates that were immunoprecipitated by the anti-PCBP2 antibodies. Images of WB are representative of three independent experiments

Fig 5: 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.