Control sirna

A total of 2.5 × 10⁵ AF5 cells/well were seeded into 24-well plates the day before transfection with 200 ng of gene-specific dsRNAs or control dsRNA (dsLacZ) per well and transfected using 1 μL of Dharmafect2 reagent (GE Dharmacon). For siRNA knockdowns in knockout cells, 20 nM either Piwi4-specific siRNAs or control siRNA (Horizon Discovery) was transfected using 2 μL Dharmafect2 reagent (GE Dharmacon), as previously described (18 (link)). The following day, ASALV infection (MOI of 0.5) was performed. At 48 hpi, total RNA was isolated from cells using TRIzol (Ambion). cDNA of 1.5 μg RNA was produced using Moloney murine leukemia virus (M-MLV) reverse transcriptase (Promega) and oligo(dT)₁₅ primers (Thermo Fisher Scientific) according to the manufacturers’ protocols. SYBR green qRT-PCR for mRNA targets was performed using gene-specific primers (Table S1) and ribosomal protein S7 RNA as the housekeeping gene transcript. Results were analyzed using the 2^−ΔΔCT method with LacZ dsRNA samples as the control. All qPCRs were performed in technical triplicates.

Rat CaMKIIδ-specific siRNA and control siRNA (Horizon Discovery) were resuspended at 20 µM in distilled water. One day after plating, primary cardiomyocytes were transfected were transfected with DharmaFECT® Duo (Thermo Scientific) using 100 nM of siRNA per well.

For co-culture experiments, 100,000 P3 BM MSCs were plated in 12-well tissue-culture treated plates in αMEM/10% FCS. After 24 h, cells were washed with PBS and 1000 E16.5 or P0 FL LT-HSCs (LSK CD150⁺CD48⁻) were added to the cultures. Cultures were maintained in polyvinyl alcohol (PVA, Sigma) supplemented with HEPES, Insulin-Transferrin-Selenium-X, mTPO and mSCF^{81 (link)}. After 8–9 days, the number of CD45⁺ cells, LSK cells, and LT-HSCs (c-Kit⁺Sca1⁺EPCR⁺CD150⁺)⁸² were analyzed by flow cytometry to determine the relative expansion of each cell type. Here, 200 c-Kit⁺Sca1⁺EPCR⁺ CD150⁺ cells were also isolated from cultures via FACS and plated into methylcellulose (M3434) to interrogate CFU potential.
For knockdown of IGF1 and IGF2 in P0 BM MSCs, 100,000 P0 MSCs/well were plated in 12-well plates in 10% FCS αMEM media. After 24 h, the media was replaced with 1 mL fresh αMEM media. Fifty nanomolar IGF1, IGF2, IGF1 and IGF2, or control siRNA (Horizon Discovery) suspended in 1 mL of Darmafect transfection reagent (Horizon Discovery) was then titrated to each well. After 24 h, media was removed and 1000 P0 FL LT-HSCs added in PVA-based media. HSCs and MSCs were co-cultured for another 8 days and then analyzed as described for CD45+ cells, HSPCs and phenotypic HSCs. IGF1 and IGF2 knockdown efficiency was confirmed with ELISA one, four and seven days post-siRNA treatment.

Rictor and raptor small interfering RNA (siRNA) and control siRNA were purchased from Dharmacon. Transfection of ECs with siRNA (100 nM) was performed using Hiperfect (Qiagen) according to the manufacturer's instructions. In brief, subconfluent HUVECs were grown on coverslip in serum free medium with siRNAs. After 24 h of transfection, the cells were washed one time and cultured in medium with 10% FBS. Western blot was performed to confirm the efficiency of siRNA knockdown.

Human monocytic THP-1 cells (BCRC 60430, the Bioresource Collection and Research Center, Taiwan) were grown in RPMI 1640 (Gibco, Thermo Fisher Scientific Inc.) with glutamine (Invitrogen, Carlsbad, CA, USA) medium supplemented with 10% fetal bovine serum in an incubator containing 5% CO₂ at 37°C. THP-1 cells were differentiated into macrophage-like cells by incubation with 10 nM PMA (phorbol 12-myristate 13-acetate) (Sigma-Aldrich, St. Louis, MO, USA) for 48 hours. To examine the effect of CLEC5A in NLRP3-inflammasome expression by using an in vitro cell-based assay, the cells were transiently transfected with CLEC5A siRNA (cat# D-001810-10-05, Dharmacon, Lafayette, CO, USA) or control siRNA (cat# D-001810-10-05, Dharmacon, Lafayette, CO, USA) using the DharmaFECT Transfection Reagent (Qiagen, Valencia, CA, USA) for 48 hours. To confirm the efficacy of transfection, cells were harvested for subsequent detection of CLEC5A expression by Western blotting. For inducing the activation of NLRP3-inflammasome, the siCLEC5A knockdown THP-1 cells were treated with plasma from active AOSD patients or healthy controls for 6 hours. NLRP3-inflammasome expression levels from cell lysates were then analyzed by Western blotting.