Scramble control sirna

To test whether varlitinib alters LPS-stimulated neuroinflammatory responses by modulating NLRP3 inflammasome activation, BV2 microglial cells were transfected with nlrp3 siRNA or scramble (control) siRNA (Dharmacon, Rafayett, CO, USA). First, siRNA at a final concentration of 30 nM in Opti-MEM (Thermo Scientific, Waltham, MA, USA) was incubated with 1 µL of Lipofectamine^® RNAiMAX (Thermo Scientific) for 40 min. Second, BV2 microglial cells in a 24-well cell culture plate (2 x 10⁵ cells/well) were transfected with the siRNA mixture for 24 h. Finally, the transfected cells were treated with 200 nM LPS or PBS for 30 min and 5 μM varlitinib or 1% DMSO for 23.5 h, and real-time PCR was performed using previously described primers (7 (link)).

In order to analyze the role of leptin, the effect of leptin knockdown on HaCaT cells, and to see if adalimumab exerts its effect via the leptin pathway, leptin siRNA was used. The keratinocyte cell line was exposed to 8 µg/mL adalimumab and/or 1 µg/mL LPS or without LPS and adalimumab was transfected with leptin siRNA (sequence 5′-CCAAAUAUCCAACGACCUG-3′) or scramble control siRNA (Dharmacon, Lafayette, CO, USA) using Lipofectamine2000 reagent (Thermo Fisher Scientific, Lipofectamine™ 2000 Transfection Reagent; Catalog Number 11668027) in accordance with the protocol. The culture was harvested 48 h after transfection. To evaluate whether the anti-TNF drug exerts its effect via the leptin pathway in the keratinocytes treated with LPS and adalimumab, siRNA was compared to the control culture, and the levels of JAK-2 (JAK2 ELISA Kit ab253224) and STAT3 (STAT3 ELISA KIT ab126427) were assessed by the ELISA assay.

The A549 and 95D cells were transfected with leptin siRNA or scramble control siRNA (Dharmacon, Lafayette, CO, USA) using Lipofectamine 2000 reagent as suggested by the manufacturer (Invitrogen). In addition, the A549 cells were transfected with Notch-1 siRNA and siRNA control (Santa Cruz Biotechnology, Santa Cruz, CA, USA) using Lipofectamine 2000 reagent (Invitrogen). All the cells were harvested 48 h after transfection.

Small short interfering RNA (siRNA) was used to transiently silence KDM5A, KDM5B, and KDM5C in Schwann cells during cAMP experiments. To decrease the expression of KDM5 isoforms, Accell siRNA duplexes targeting KDM5A (Cat. no. A-095654-24-0010; Dharmacon), KDM5B (Cat. no. A-082318-16-0010; Dharmacon), KDM5C (Cat. no. A-095923-14-0010; Dharmacon), and non-targeting Scramble control siRNA (Cat. no. D-001910-01-05; Dharmacon) were used. siRNAs combined and delivered in DMEM containing 2.5% FBS for a final concentration of 1 μM according to the manufacturer’s instructions. After two 72-h transfections, the cells were treated with cAMP and collected after 8 h to assess H3K4me3 via IF.

To evaluate the role of the leptin, the effect of leptin knockdown on Ishikawa cells, and to test whether cisplatin exerts its effect via leptin path, leptin siRNA was used.
The Ishikawa endometrial cancer cell line (exposed to 5 µM cisplatin as an average inhibitory concentration or without the drug) was transfected with leptin siRNA (sequence 5′-CCAAAUAUCCAACGACCUG-3′) or scramble control siRNA (Dharmacon, Lafayette, CO, USA) using Lipofectamine2000 reagent (Thermo Fisher Scientific, Lipofectamine™ 2000 Transfection Reagent; Catalog Number 11668027) according to the instruction in the manufacturer’s protocol. The culture was harvested 48 h after transfection.