c hek293t cells  (ATCC)

Journal: The Journal of Experimental Medicine

Article Title: PROTAC-mediated NR4A1 degradation as a novel strategy for cancer immunotherapy

doi: 10.1084/jem.20231519

Figure Lengend Snippet: NR-V04 induces NR4A1 degradation. (A) NR-V04 effectively promoted the degradation of NR4A1 in two more human melanoma cell lines in 16 h, including WM164 and M229, while simultaneously stabilizing VHL expression; two experiments. (B) NR-V04 effectively promoted the degradation of NR4A1 in two mouse melanoma cell lines in 16 h, including SM1 and SW1; two experiments. (C) NR-V04 did not induce the degradation of NR4A2 and NR4A3; two experiments. (D) NR-V04 induces a transient elevation of NR4A1 mRNA in short time. CHL-1 cells were treated with 500 nM of celastrol or NR-V04 at the indicated time points. RNA was prepared for reverse transcription and qPCR. ACTB was used as control; N = 4, one experiment. (E) Ternary complex formation was observed in CHL-1 cells with 16-h treatments of 500 nM NR-V04, rather than DMSO or 500 nM celastrol, as detected by PLA (63× magnification); two experiments. Bar represents 10 μm for both images. (F) NR4A1 degradation by 500 or 1,000 nM of NR-V04 for 16 h in the WT or VHL KO HEK293T cells; two experiments. A two-sided unpaired t test was performed, with P values indicated (P = 0.0104 between control and 2 h of celastrol treatment; P = 0.023 and 0.008, respectively, between control and 2 or 4 h of NR-V04 treatment). Supplementary to and . Source data are available for this figure: .

Article Snippet: HEK293T cells were transfected with 3 µg Flag-NR4A1 (HG17699-CF; SinoBiological) for 36 h in Opti-MEM (31985062; Thermo Fisher Scientific) medium with 10 µl GeneTran III reagent (GT2211; Biomiga).

Techniques: Expressing

Journal: The Journal of Experimental Medicine

Article Title: PROTAC-mediated NR4A1 degradation as a novel strategy for cancer immunotherapy

doi: 10.1084/jem.20231519

Figure Lengend Snippet: NR-V04 induces a ternary complex formation and mediates NR4A1 degradation through the ubiquitin-proteasome system. (A) PLA showing ternary complex formation induced by NR-V04. CHL-1 (left panels) and A375 (right panels) cells were pretreated with 0.5 μM MG132 for 10 min and then treated with DMSO, 500 nM celastrol, or 500 nM NR-V04 for 16 h. Representative images were shown for PLA assay (20× magnification); two experiments. Bar represents 50 μm for all images. (B) Co-IP experiment showing complex formation between NR4A1 and VHL by NR-V04 treatment. Co-IP was performed in NR4A1-Flag overexpressed HEK293T cells that were pretreated with 0.5 μM MG132 for 10 min, followed by 16-h treatment with DMSO or 500 nM NR-V04. NR4A1 was pulled down using an anti-Flag antibody conjugated to magnetic beads; two experiments. (C) NR-V04 induces NR4A1 degradation via VHL E3 ligase- and proteasome-dependent manner. CHL-1 cells were pretreated with 0.5 μM MG132 or 10 µM VHL 032 for 10 min, followed by 16-h treatment with DMSO or 500 nM NR-V04, three experiments. Source data are available for this figure: .

Article Snippet: HEK293T cells were transfected with 3 µg Flag-NR4A1 (HG17699-CF; SinoBiological) for 36 h in Opti-MEM (31985062; Thermo Fisher Scientific) medium with 10 µl GeneTran III reagent (GT2211; Biomiga).

Techniques: Co-Immunoprecipitation Assay, Magnetic Beads