Journal: Pharmaceutics

Article Title: Modulating NPC1L1 to Potentiate PARP Inhibitor-Induced Ferroptosis and Immune Response in Triple-Negative Breast Cancer

doi: 10.3390/pharmaceutics17050554

Figure Lengend Snippet: PARP inhibitors regulate the PARP1-RELA-NPC1L1 signaling axis. ( A ) Three TNBC cell lines were treated with four clinical PARPis (40 μM) for 48 h. The protein expression levels of NPC1L1 in these cells were analyzed by immunoblotting, with each blot containing three samples from the respective cell lines. ( B ) The mRNA levels of NPC1L1 were analyzed after transfection with siCtrl negative control or siPARP1 for 48 h in three TNBC cell lines. ( C ) JASPAR predicted the binding sites of the transcription factor RELA in the CDS region with the PARP1 promoter region, as well as the binding site of the CDS region of RELA with the NPC1L1 promoter region. ( D ) MDA-MB-231 and MDA-MB-453 cells were transfected with siCtrl negative control or siPARP, and 48 h later, the protein expression of NPC1L1, PARP1, and RELA was analyzed by immunoblotting. ( E ) Four experimental groups—control, si-PARP1, si-PARP1 + PARP1-OE, and PARP1-OE—were established to analyze the expression levels of PARP1, RELA, and NPC1L1 in cells. ( F ) MDA-MB-231 and MDA-MB-453 cells were transfected with siCtrl negative control or shRELA; the protein expression of NPC1L1, PARP1, and RELA were analyzed by immunoblotting. ( G ) The interaction between RELA and NPC1L1 was confirmed using a dual-luciferase reporter assay in 293T cells, providing a robust validation of the regulatory targeting. Statistical significance between groups is indicated (* p < 0.05; ** p < 0.01; *** p < 0.001, ns: not significant).

Article Snippet: The following antibodies were used: NPC1L1 (rabbit monoclonal, 1:1000; Abcam, Cat# ab284598, Lot No: GR342448-1; Cambridge, UK), RELA (rabbit monoclonal, 1:1000; Cell Signaling Technology, Cat# 8242, Lot No: 14; Danvers, MA, USA), GAPDH (mouse monoclonal, 1:5000; Proteintech, Cat# 60004-1-Ig, Lot No: 00066073; Rosemont, IL, USA), Anti-PARP (46D11) Rabbit mAb (1:1000; Cell Signaling Technology, Cat# 9532, Lot No: 10; Danvers, MA, USA), HRP-conjugated anti-β-actin (1:1000; Abcam, Cat# ab49900, Lot No: GR3457139-1; Cambridge, UK), Anti-PI3 Kinase p85 alpha (1:1000; Abcam, Cat# ab191606, Lot No: 1011480-6), Anti-mTOR (1:1000; Abcam, Cat# ab134903, Lot No: 1000631-7), Anti-AKT1 + AKT2 + AKT3 (1:1000; Abcam, Cat# ab179463, Lot No: 1010423-9), Anti-(phospho S472 + S473 + S474) AKT1 + AKT2 + AKT3 (1:1000; Abcam, Cat# ab192623, Lot No: 10081813-8), Anti-mouse IgG HRP-linked (1:5000; Cell Signaling Technology, Cat# 7076, Lot No: 36), and Anti-rabbit IgG HRP-linked (1:5000; Cell Signaling Technology, Cat# 7074, Lot No: 31).

Techniques: Expressing, Western Blot, Transfection, Negative Control, Binding Assay, Control, Luciferase, Reporter Assay, Biomarker Discovery