Fig 2: SH3BP5-AS1 recruits IGF2BP2 to positively regulate VDAC2. (A) In RIP assay, the enrichment of SH3BP5-AS1 in Anti-U2AF2, Anti-HNRNPA1, Anti-IGF2BP2 and Anti-SRSF1 was calculated by qPCR. (B) The possible binding between SH3BP5-AS1 and four RBPs was evaluated by RNA pulldown assay. (C-D) IGF2BP2 level pulled down by SH3BP5-AS1/VDAC2 sense was detected via WB. (E) WB was done to measure IGF2BP2 and VDAC2 protein levels in BC cells with overexpressed SH3BP5-AS1. (F) qPCR analysis was done to testify IGF2BP2 knockdown efficiency. (G) qPCR and WB analyses were applied to measure VDAC2 expression in IGF2BP2-depleted cells. (H) In RIP assay, the level of VDAC2 precipitated in Anti-IGF2BP2 was detected in T24 and J82 cells transfected with pcDNA3.1 or pcDNA3.1-SH3BP5-AS1. (I) VDAC2 mRNA and protein levels were examined via qPCR and WB in the cells with transfection of sh-IGF2BP2 or sh-IGF2BP2 + pcDNA3.1-SH3BP5-AS1. **p < 0.01, n.s.: no significance.

Fig 3: SH3BP5-AS1 facilitates BC cell ferroptosis. (A) Bioinformatics tool (starBase) was applied to predict the RBPs potentially binding to SH3BP5-AS1 under the indicated conditions (CLIP Data> = 5, pan-Cancer> = 10). (B) The ferroptosis pathway map obtained from KEGG was demonstrated. (C) The four candidate RBPs (U2AF2, HNRNPA1, SRSF1 and IGF2BP2) were predicted to potentially bind with key genes of ferroptosis on starBase. (D) The ferrous iron content in T24 and J82 cells with overexpressed SH3BP5-AS1 was measured with a microplate reader. (E) Flow cytometer was applied to detect lipid ROS in BC cells with SH3BP5-AS1 up-regulation. **p < 0.01.

Fig 4: SH3BP5-AS1 recruits IGF2BP2 to positively regulate VDAC2. (A) In RIP assay, the enrichment of SH3BP5-AS1 in Anti-U2AF2, Anti-HNRNPA1, Anti-IGF2BP2 and Anti-SRSF1 was calculated by qPCR. (B) The possible binding between SH3BP5-AS1 and four RBPs was evaluated by RNA pulldown assay. (C-D) IGF2BP2 level pulled down by SH3BP5-AS1/VDAC2 sense was detected via WB. (E) WB was done to measure IGF2BP2 and VDAC2 protein levels in BC cells with overexpressed SH3BP5-AS1. (F) qPCR analysis was done to testify IGF2BP2 knockdown efficiency. (G) qPCR and WB analyses were applied to measure VDAC2 expression in IGF2BP2-depleted cells. (H) In RIP assay, the level of VDAC2 precipitated in Anti-IGF2BP2 was detected in T24 and J82 cells transfected with pcDNA3.1 or pcDNA3.1-SH3BP5-AS1. (I) VDAC2 mRNA and protein levels were examined via qPCR and WB in the cells with transfection of sh-IGF2BP2 or sh-IGF2BP2 + pcDNA3.1-SH3BP5-AS1. **p < 0.01, n.s.: no significance.