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AML12 cells (ATCC, Manassas, VA, USA) were cultured in DMEM: F12 medium (Gibco, Carlsbad, CA, USA) supplemented with FBS (Gibco), ITTS, and dexamethasone. AML12 cells were exposed to hypoxia/reoxygenation (H/R) as described previously [29 (link)]. In brief, AML12 cells were cultured at 1% O₂ for 12 h, and then at 21% O₂ conditions for the indicated time points. Plasmids or small interfering RNAs (siRNAs) were transfected to AML12 cells using lipofectamine 3000 (Invitrogen, Carlsbad, CA, USA) for 48 h before H/R. The specific siRNA sequences (GenePharma, Suzhou, China) were listed in Table 1. After transfection, AML12 cells were incubated with 100 μM MitoTEMPO (Sigma, St Louis, Missouri, USA) or 10 μM Mdivi-1 (Selleck, Shanghai, China) for 3 h, followed by H/R. In addition, 20 μM MG132 (Selleck, USA) was administered for 3 h or 100 μM cycloheximide (CHX, Sigma) was applied for 0, 1, 2, and 4 h to AML12 cells after USP15 siRNA transfection.
HEK293 cells were grown in DMEM (Gibco) medium containing FBS (Gibco), penicillin and streptomycin (Beyotime, Hangzhou, China). Relevant plasmids including Myc-p66Shc, Flag-USP15 WT, Flag-USP15 C269A, and HA-Ub (GenePharma) were transfected into HEK293 cells using lipofectamine 3000 for 24–72 h, followed by a Co-IP assay.

The full-length ORF of SPOP (1125 bp, NM_001007226.1) was amplified from cDNA of FHC cells. The primers were as follows: F: 5′-AGAGAATTCATGTCAAGGGAAATCTTTGC-3′, R: 5′-AGAGGATCCTTAGGATTGCTTCAGGCGTT-3′. To construct the lentivirus production containing SPOP, the ORF of SPOP was subcloned into the pLenti-CMV-GFP vector (Addgene, Cambridge, MA, USA).
The synthesized DNA fragments encoding the short-hairpin RNA (shRNA) used for the knockdown of endogenous SPOP were inserted into the pGPU6/GFP/Neo vector (GenePharma, Shanghai, China). The sequences of the shRNAs were as follows: shSPOP, 5′-AACGCCTGAAGCAATCCTACTCGAGTAGGATTGCTTCAGGCGTT-3′, NC, 5′-TTCTCCGAACGTGTCACGTCTCGAGACGTGACACGTTCGGAGAA-3′. All plasmids were verified by sequencing.
Serial deletion fragments of SPOP promoter region (−1320, −1167, −655, −350, −221, −189, −10/+472) were amplified by PCR and cloned into a pGL3-basic luciferase vector (Promega). A NheI site was added to the 5′-primer, and a XhoI site was added to the 3′-primer. The constructs were verified by sequencing of both DNA strands. The sequences of mutated sites were synthesized by Genescript (Nanjing, China).
FLAG-Gli2, HA-Ub and pcDNA3.1(-)A-RXRA were purchased from GenePharma.