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 Figs. 4 and 5. Source data are available for this figure: SourceData FS2.
Figure S2.

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 Figs. 4 and 5. Source data are available for this figure: SourceData FS2.

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