pcdna3 flag sp1 addgene  (Addgene inc)

Journal: Cell Death & Disease

Article Title: Orphan nuclear receptor 4A1 (NR4A1) and NR4A2 are endogenous regulators of CD71 and their ligands induce ferroptosis in breast cancer

doi: 10.1038/s41419-025-08143-5

Figure Lengend Snippet: MDA-MB-231 cells were transfected with oligonucleotides targeting NR4A1 ( A ), NR4A2 ( B ), Sp1 ( C ), Sp4 ( D ) and Sp3 ( E ), and after 72 h, whole cell lysates were analyzed by Western blots and quantified as outlined in the “Methods”. F Effects of Sp1 overexpression on CD71 were obtained by transfection of the p CVM-Sp1 expression plasmid and subsequent western blot analysis of whole cell lysates and quantified as outlined in the “Methods”. Cells were treated with 10 µM DIM-3,5-CI 2 ( G ) and DIM-3-CI-5-CF 3 ( H ) for up to 24 h, and effects on CD71, Sp1 and Sp4 (separate blot) were determined by Western blots of whole cell lysates and quantified as outlined in the “Methods”.

Article Snippet: The plasmid containing Sp1 coding sequence (Addgene plasmid # 12097) and empty vector as negative control (Addgene plasmid # 20783) using LipofectamineTM 3000 reagent (Invitrogen, # L3000008) according to the manufacturer’s instructions.

Techniques: Transfection, Western Blot, Over Expression, Expressing, Plasmid Preparation

Journal: Cell Death & Disease

Article Title: Orphan nuclear receptor 4A1 (NR4A1) and NR4A2 are endogenous regulators of CD71 and their ligands induce ferroptosis in breast cancer

doi: 10.1038/s41419-025-08143-5

Figure Lengend Snippet: Interactions of NR4A1 ( A ), NR4A2 ( B ), Sp1 ( C ) and Sp4 ( D ) with the CD71 gene promoter containing the Sp binding region was determined after treatment with DIM-3,5 compounds in a ChIP assay (in triplicate) as outlined in the “Methods section”. E Summary of the CD71 gene promoter, Sp binding sites (−1576 to −1566) and the primers used for detecting protein interactions in this region of the promoter, along with the overall mechanism of ferroptosis induction. Results are expressed as means ± SD for replicate (3) determinations for each treatment group ( A – D ) and significant ( p < 0.05) induction is indicated (*).

Article Snippet: The plasmid containing Sp1 coding sequence (Addgene plasmid # 12097) and empty vector as negative control (Addgene plasmid # 20783) using LipofectamineTM 3000 reagent (Invitrogen, # L3000008) according to the manufacturer’s instructions.

Techniques: Binding Assay