Allstars negative sirna af 488

Small interfering RNAs (siRNAs) targeting SKP2 were purchased from Qiagen. For transient expression, the cell lines were transfected using HiPerFect Transfection Reagent (Qiagen), as outlined in the HiPerFect Transfection Reagent Handbook (Fifth edition, Qiagen). AllStars Negative siRNA AF 488 (Qiagen) was used both as a negative control and to determine the transfection efficiency. Two independent experiments were performed. SKP2 siRNA data: the target sequence is 5′-AAGTGATAGTCATGCTAAA-3′, the sense strand is 5′-GUGAUAGUGUCAUGCUAAATT-3′ and the antisense strand is 5′-UUUAGCAUGACACUAUCACTT-3′.

Control (AllStars Negative siRNA AF488, catalog no. 1027284), YAP siRNAs (catalog no. GS22601), and TAZ siRNA (catalog no. SI01442511 and SI01442532) were obtained from Qiagen, USA. HDAC3 silencer pre-designed siRNA (catalog no. AM16708) and silencer negative control siRNA (catalog no. AM4620) were purchased from Invitrogen, USA. The siRNA was transfected in BMDMs using Lipofectamine RNAiMAX transfection reagent (Thermo Fisher, Singapore, catalog no. 13778150). Briefly, after a transient transfection, BMDMs were incubated for 72 hours and treated with either pro-inflammatory (LPS 100 ng/ml + IFNγ 10 ng/ml) or reparative stimuli (IL4 10 ng/ml + IL13 10 ng/ml) diluted in culture medium (DMEM containing 10% FBS and 1% penicillin/streptomycin). Cells were collected for total RNA isolation and qRT-PCR analysis using Favorgen RNA extraction kit (Favorgen Biotech, Taiwan, catalog no. FATRK 001–2). Cell lysates were prepared using RIPA buffer (Thermo Fisher, catalog no. 89901) for western blot analysis.