Fig 2: MALAT1 and ID4 favor circ_0076611 expression by counteracting RNA-binding protein PTBP1.a Schematic representation of the genomic regions cloned in pCDNA3.1 vector to generate a circ_0076611 expression vector (indicated as CIRC) and a deletion mutant lacking an inverted repeated sequence required for circ_0076611 expression (indicated as CIRC#del). b Expression of circ_0076611 (log10), evaluated by RT-qPCR, in MDA-MB-468 cells transiently transfected with a pCDNA3.1 vector (EV) or with the circ_0076611 expression vectors indicated in a. c Ribonucleoprotein Immunoprecipitation assays (RIP) in MDA-MB-468 cells (after UV crosslinking) showing the enrichment of VEGFA precursor RNA (pre-VEGFA) in samples immunoprecipitated with control IgG or with antibodies directed to PTBP1 (left) or SRSF1 (right) proteins. Data are normalized to 28S rRNA and presented as folds of enrichment over IgG. d, e Expression of circ_0076611, evaluated by RT-qPCR, in MDA-MB-468 cells transfected with a pool of siRNAs directed to PTBP1 (left) or two different siRNAs directed to SRSF1 for 72 h (right) (d). WB showing the efficiency of PTBP1 and SRSF1 interference after 72 h of siRNA transfection is shown in e. f Scheme summarizing the regulatory roles of lncRNA MALAT1 and of proteins ID4, SRSF1 and PTBP1 in the control of circ_0076611 back-splicing. g, h RIP assay in the indicated cell lines (after UV crosslinking) showing the level of MALAT1 lncRNA, evaluated by RT-qPCR, in samples immunoprecipitated with control IgG or with an antibody directed to PTBP1 protein (g). A fraction (25%) of eluates recovered during RIP experiments has been used for protein analysis by WB to control PTBP1 IP and co-IP of ID4, mutant p53 and SRSF1 proteins (h). Enrichment of SRSF1 (i) and PTBP1 (j) proteins on pre-VEGFA mRNA evaluated by RIP assay in control MDA-MB-468 cells (CTR) and in ID4 knocked-out counterpart (ID4-KO) (after formaldehyde crosslinking) by using antibodies directed to SRSF1 or PTBP1 and IgG as negative control. Data are presented as folds of enrichment over IgG. Expression of circ_0076611 (k) and MALAT1 (l) and PTBP1 (l), evaluated by RT-qPCR, in MDA-MB-468 cells transfected with siRNAs directed to MALAT1, or to MALAT1 and PTBP1, for 72 h. Data are presented as mean plus standard deviation. p values have been calculated by paired, two-tailed Student’s t test. Results from at least three biological replicates are shown. Bars indicate standard deviation.

Fig 3: NCOA4 overexpression increases the sensitivity of liver cancer cells to SF-induced ferroptosis. (A) NCOA4 overexpression significantly reversed the si-PTBP1-mediated changes in the percentage of iron. (B) Changes in the percentage of MDA in Huh-7 and HepG2 cells treated with SF. (C) Changes in the percentage of GSH in Huh-7 and HepG2 cells treated with SF. *P<0.05, **P<0.01 and ***P<0.001 (Student's t-test). NCOA4, nuclear receptor coactivator 4; SF, sorafenib; si-, small interfering RNA; PTBP1, polypyrimidine tract-binding protein 1; MDA, malondialdehyde; GSH, glutathione; NC, negative control; oe-, overexpression.

Fig 4: Polypyrimidine tract binding protein 1 (PTBP1) contributed to transforming growth factor‐β1 (TGF‐β1)‐induced collagen accumulation and proliferation of cardiac fibroblasts (CFs). CFs were transfected with oe‐PTBP1 or si‐PTBP1. (A) Quantitative real‐time PCR analysis of PTBP1 level in CFs with various transfections. (B) The protein abundance of PTBP1, collagen I, and collagen III was detected by western blotting. (C) 5‐Ethynyl‐2′‐deoxyuridine (EdU) proliferation assay was utilized to determine proliferative ability of CFs (N = 3). DAPI, 4′,6‐diamidino‐2‐phenylindole; GAPDH, glyceraldehyde 3‐phosphate dehydrogenase. **P < 0.01, ***P < 0.001.

Fig 5: Polypyrimidine tract binding protein 1 (PTBP1)/nuclear receptor NR4A1 (Nur77)/fatty acid‐binding protein 5 (FABP5) axis modulated transforming growth factor‐β1 (TGF‐β1)‐induced collagen accumulation and proliferation of cardiac fibroblasts (CFs). (A) Western blotting analysis of Nur77 and FABP5 levels in heart tissues of mice (N = 5). CFs were transfected with si‐PTBP1 in combination with or without si‐Nur77 or si‐FABP5. (B) The mRNA levels of Nur77 and FABP5 were assessed by quantitative real‐time PCR. (C) The protein levels of Nur77, FABP5, collagen I, and collagen III were detected by western blotting. (D) The proliferative capacities of CFs from different groups were evaluated by 5‐ethynyl‐2′‐deoxyuridine (EdU) proliferation assay (N = 3). DAPI, 4′,6‐diamidino‐2‐phenylindole; GAPDH, glyceraldehyde 3‐phosphate dehydrogenase; MI, myocardial infarction. *P < 0.05, **P < 0.01, ***P < 0.001.