Figure 4

Thioredoxin depletion in a neutrophil-like cell line increases ROS but does not intrinsically affect motility. (A) Western blot of thioredoxin confirming CRISPR/Cas9 mediated txn knockdown in three HL60 clonal lines (KD4, KD6 and KD7) compared to luciferase control, with beta-tubulin as a loading control. (B) Quantification of intracellular ROS production (Carboxy-H2DCFDA probe) over time in txn knockdown lines compared to control (n=3). (C) Quantification of extracellular H₂O₂ production (Amplex Red/HRP Probe) over time in txn knockdown lines compared to control (n=3). (D) 2D chemotactic index and (E) mean track speed for txn knockdown lines compared to control (N = 2 repeats, n = 5-12 devices). Samples were analyzed for statistical significance via generalized estimating equation (GEE) fit to the log fold change with confidence intervals set to 95% coverage (B, C) or t-test (D, E) (p < 0.001***).