Plvx shrna2

The mRNA sequence of IDOL can be retrieved using RefSeq in the National Center for Biotechnology Information (NCBI) Entrez Gene database, and DNAMAN (version 6) (Lynnon Biosoft) was used to search for mRNA conserved sequences. The IDOL-targeting siRNAs were designed using DSIR version 7 (http://biodev.extra.cea.Fr/DSIR/DSIR.html)⁴⁵ and RNAstructure version 6.4 (http://rna.urmc.rochester.edu/RNAstructure.html).⁴⁶ Moreover, the homology analysis of the candidate siRNAs was conducted by using BLAST provided from NCBI GeneBank with the transcript reference sequence database (Transcript Reference Sequences) for excluding the possibility that siRNA non-specifically inhibits genes other than IDOL. All siRNAs (Table S2) were synthesized and purified by GenePharma (Shanghai, China) as well as dissolved to a concentration of 20 μM with sterilized and RNase-free water according to the manufacturer’s protocol. The selected IDOL-targeting siRNA and a scrambled siRNA (catalog #A06001NC-RL; Genepharm) were embedded in shRNA scaffolds as previously described,³⁰ (link) and the shRNA sequences used in this study are listed in Table S3. The lentiviral expression vectors pLVX-shIDOL and pLVX-shNC were constructed by subcloning the synthetic annealed shRNA duplexes at BamH I/EcoR I sites of pLVX-shRNA2 (catalog #632179; Clontech).

Sponge oligos containing 6 tandem antisense sequences of miR-20a-5p were synthesized and cloned into the shRNA lentiviral transfer vector pLVX-shRNA2 (Clontech, Palo Alto, CA, USA). The lentiviruses expressing miR-20a-5p sponge or the empty vector were packaged as previously described (21 (link)). The 3T3-L1 cells were infected with the lentiviruses at an MOI (multiplicity of infection) of 10. Then, the cells of 100% confluence were allowed to differentiate in presence of adipogenic medium.

ShRNAs for knockdown of TET1, TET2, TET3 and GFP-control were constructed into pLvx-shRNA2 (Clontech, Takara) digested with BamHI and EcoRI, and tranfected to HEK293T cells with packaging plasmids (pspax2, pMD2G) using Lipofectamine® 3000 (Thermo, USA). Supernatant was harvested at 24 h and 72 h after transfection and infected anti-CD3ε/CD28 mAb-stimulated naïve T cells. Transfected T cells were then treated with decitabine (10 μM) or vehicle for 96 h. Cells were harvested for real-time PCR to analyze the efficiency of knockdown. ShRNA sequences were detailed in Supplementary Table 2.

ShRNA sequences targeting human Trx2 and Prx3 as well as a non-targeting control were designed using BLOCK-iT RNAi Designer software from Thermo Fisher Scientific (S3 Table) and were cloned into the pLVX-shRNA2 (Clontech, Mountain View, CA). Following the manufacturer’s instructions for the Lenti-X system, Lenti-X 293T cells were transfected with pLVX-shRNA2 and HTX packaging mix to produce ≥2.5x10⁸ IFU/mL as determined by qPCR. Cells were seeded in 6-well dishes and transduced with 0.5mL viral media supplemented with 8 μg/mL polybrene for 6 hours. Transduction efficiency after 48 hours was ≥85% by flow cytometry for ZsGreen1.

ShRNA plasmids were used to interfere with the translation of TRAF6 and IRAK1. The TRAF6 and IRAK1 shRNA sequences were inserted into pLVX-shRNA2 (Clontech 632179, Mountain View, USA). The vector pLVX-CMV-IRES-PURO was employed to construct the TRAF6 and IRAK1 overexpression plasmids. (The primer and shRNA sequences are listed in Supplementary Table S4). The vector GV298 (U6-MCS-Ubiquitin-Cherry-IRES-puromycin) was employed to construct the has-miR-146a-5p overexpression plasmid. All plasmids were transformed into E. coli strain Stbl3. The lentivirus was then packaged in HEK293T cells with psPAX2 (Addgene #12260) and pMD2.G (Addgene #12259). After transfection, HEK293T cells were incubated for 48 h. The supernatant with virus particles was collected. After being filtered, the virus particles were added to infect U87MG and A172 cells for infection. Successfully infected cells were screened with puromycin. To validate the knockdown or overexpression of IRAK1 and TRAF6, Western blot assays were conducted.