Sinegative

BCE and BAE cells were plated onto six‐well culture plates at a density of 1.5 × 10⁵ cells/well and cultured overnight. Two milliliter of antibiotics‐free culture medium was used to replace the old medium. siRNAs, including siNegative (Ambion, CAT# AM4611), siSTAT3‐915 (Invitrogen, CAT# 361146C04), siSTAT3‐1492 (Invitrogen, CAT# 361146C05), and siSTAT3‐454 (Invitrogen, CAT# 384235A10), were mixed with Lipofectamine RNAiMAX reagent (ThermoFisher Scientific, CAT# 13778150) in Opti‐MEM™ I Reduced Serum Medium (Gibco, CAT# 31985062) according to manufacturer’s instructions. Briefly, a mix containing 25 pmol of siRNA, 7.5 μl of RNAiMAX reagent, and 125 μl of Opti‐MEM medium was used to transfect cells in each well, which makes a final siRNA concentration of 12.5 nM. A mix of RNAiMAX and Opti‐MEM was used as no siRNA control. Cells were incubated with siRNAs for 8 h and then cultured in fresh medium. Twenty‐four hours after transfection with siRNAs, cells were used for EC proliferation assays and RNA/protein extraction.

BCECs were plated in 6-well culture plates at a density of 1.5 × 10⁵ cells/well and cultured overnight. Two milliliters of antibiotics-free culture medium was used to replace the old medium. siRNAs, including siNegative (Ambion, AM4611), siRNA against JNK1#2 (Invitrogen, NM_001192974.2_siRNA_266), JNK1#4 (Invitrogen, NM_001192974.2_siRNA_485), siRNA against JNK2#2 (Invitrogen, XM_005208371.4_siRNA_1240), and JNK2#4 (Invitrogen, XM_005208371.4_siRNA_696), were mixed with Lipofectamine RNAiMAX reagent (ThermoFisher Scientific, 13778150) in Opti-MEM™ I Reduced Serum Medium (Gibco, 31985062) according to manufacturer’s instructions. Briefly, a mix containing 25 pmol of siRNA, 7.5 μl of RNAiMAX reagent, and 125 μl of Opti-MEM medium was used to transfect cells in each well, to a final siRNA concentration of 12.5 nM. A mix of RNAiMAX and Opti-MEM was used as no siRNA control. Cells were incubated with siRNAs. Eight hours later, the siRNA-containing medium was replaced with fresh medium. Twenty-four and/or 48 h after transfection with siRNAs, cells were used for proliferation assays and protein extraction.

Small interfering RNA (siRNA) targeting Nrf2 (si-Nrf2), and negative scramble control siRNA (si-Negative) were purchased from Thermo Fisher Scientific (USA). C8-D1A astrocytes were cultured in complete medium in 6-well plates for 48 h, and then transfected with either Nrf2 siRNA or Nrf2-negative siRNA using Lipofectamine RNAi MAX (Invitrogen, Waltham, MA, USA). After 24 h, the transfected cells were exposed to OGD/R for 4 h before Western blotting and further analysis.