Research Paper Volume 12, Issue 3 pp 2507—2529

PRMT5-TRIM21 interaction regulates the senescence of osteosarcoma cells by targeting the TXNIP/p21 axis

Figure 4. TXNIP is essential for the induction of cellular senescence by PRMT5 depletion. (A) The protein expression of TXNIP was determined by WB with or without PRMT5 knockdown in U2 OS cells. (B) siRNA targeting TXNIP was transfected into SC, shP5#1 or shP5#3 Saos-2 cells, and the expressions of PRMT5, TXNIP, and p21 were measured by WB; β-actin was used as the internal control. (C) Plasmids encoding HA-PRMT5 were transfected into SC or shP5#3 U2 OS cells, and the expression of PRMT5 and TXNIP was determined by WB. (D) Two independent siRNAs targeting TXNIP (siTXNIP#1 and siTXNIP#2) were transfected into SC or shP5#3 U2 OS cells for 3 days, the percentage of senescent cells was quantified. ****, p< 0.0001. (E) siRNAs targeting TXNIP were transfected into SC, shP5#1 or shP5#3 U2 OS cells, and the expressions of PRMT5, TXNIP, γ-H2A.X, and p21 were measured by WB; β-actin was used as the internal control. (F) DSBs were quantified by Open Comet software. ***, p< 0.001; ****, p< 0.0001. (G, H) siRNA targeting TXNIP was transfected into SC and shP5#1 U2 OS cells, and cellular senescence was visualized using a SA-β-gal staining kit. Scale bar = 10 μm. the percentage of senescent cells was quantified. ****, p< 0.0001. (I) U2 OS cells were treated with CDDP for different durations, the expression of TXNIP was measured by WB. (J) 10 μM CDDP was added to SC, shP5#1, and shP5#3 cells for 12 h, the expressions of PRMT5 and TXNIP were determined by WB. (K) U2 OS cells were transfected with plasmids encoding HA-PRMT5, followed by treated with CDDP for 12 h, the expressions of PRMT5 and TXNIP were determined by WB.