2.7.4. Silencing RNA (siRNA)-mediated gene knockdown [40]

All siRNAs were purchased from Beijing Yibaike Co. Ltd., Beijing, China. To knock down TF and NOX2, the siRNA sequences were chosen as follows:

si-hTransferrin-1:5′-GUGUCUGGCUGUCCCUGAUAATT-3′,

si-hTransferrin-2:5′-CUUGAUCUGGGAGCUUCUCAATT-3′,

si-hNOX2-1:5′-GCUGUGCCUCAUAUUAAUUTT-3′,

si-hNOX2-2:5′- CCAUGGAGCUGAACGAAUUTT-3′.

The negative control was 5′-UUCUCCGAACGUGUCACGUdTdT3′.

Briefly, 143B cells were seeded in six-well plates at a density of 2 × 10⁵ cells per well and transfected with siRNA using Lipofectamine-3000 reagent (Invitrogen, Carlsbad, CA, USA) according to the manufacturer's protocol. The cells were then incubated for two days, and the knockdown efficacy was validated using Western blot.