Fig 2: Suppression of GSH triggers NF-κB activation both in in vitro and in vivo. EOC cells were either untreated (control) or pretreated with 500 μM NAC or 200 μM BSO for 18 h, prior to 22 mM EtOH exposure for 6 h. Western blot analysis was performed to determine the expression and activation of NF-κB p105 (a) phosphorylated p65 at ser536 and total p65 protein (b). β-actin served as a loading control. In similar experiments, nucleus and cytosol fractions were separated to determine expression levels of p50 (c). Lamin B and β-actin served as nuclear and cytosolic loading control, respectively. Representative immunoblot of p105 in fetal brain samples taken 24 h after dams were pretreated 1 h with either NAC (4 mg/dose) or BSO (1.5 g/kg) followed by ethanol exposure (2.5 g/kg, s.c) (d)

Fig 3: Deletion of NFKB1 does not affect uninjured tendon gliding function or biomechanical properties. Western blotting for NFKB1 proteins p105/p50 demonstrates partial and complete depletion in NFKB1Het and NFKB1KO uninjured tendon, respectively, compared to wildtypes littermates (A). Full length blots are presented in Supplementary Fig. 2. Measurement of metatarsophalangeal (MTP) joint flexion angle (B), gliding resistance (C), stiffness (D), and maximum load at failure (E) of uninjured wildtype, NFKB1Het, and NFKB1KO tendons showed no significant differences.

Fig 4: Deletion of NFKB1 increases activation of canonical NF-κB signaling post-repair. NFKB1 proteins p105 and p50 are involved in both canonical NF-κB and MAPK signaling cascades (A). Western blots to assess canonical NF-κB (p-p65) and MAPK (p-ERK1/2) activation in uninjured (B), 3-(C) and 14-days (D) post-repair in NFKB1WT, NFKB1Het, and NFKB1KO. Blots were then stripped and re-probed for total p65, total ERK1/2, and GAPDH (B). Full length blots are presented in Supplementary Figs 3–5.

Fig 5: p50‐KOIMC slows high‐risk neuroblastoma tumor growth. (A) Lineage‐negative (Lin−) murine marrow cells were nucleofected with sg3 (targeting exon 14 of Nfkb1) complexed with Cas9, or with no ribonucleoproteins (RNPs). Western blotting for NF‐κB p50 and β‐actin was performed 18 days later using total cellular protein from 2E5 cells. (B) 2E6 Lin− murine marrow cells were nucleofected with Cas9:p50 sg3 RNP or a nontargeting (NT) RNP and then cultured in IMDM with serum‐free BIT, SCF, TPO, FL, SR‐1, and UM171. The fold change in viable cell numbers, with data shown representative of three independent experiments. (C) WT mice inoculated with Nb cells on day 0 received 5FU on day 28, followed by 1E7 nontargeting NT‐IMC or Nfkb1 gene‐edited p50KO‐IMC on days 33, 36, and 40. The P‐value shown compares the entire growth curves using an exponential model. P was 0.031 comparing tumor volumes on day 44.