Fig 2: Drug inhibition of Nox4 suppresses RANKL-induced autophagy and osteoclastogenesis. (A) RAW264.7 cells were seeded overnight and incubated with or without RANKL (100 ng/ml) for the indicated times. The levels of Nox family proteins (Nox1, Nox2, Nox3, and Nox4) were assessed by western blot. (B–D) RAW264.7 cells were seeded overnight and treated with the Nox inhibitor DPI (B), Nox2 inhibitor GSK2795039 (C), Nox1 inhibitor ML171 and Nox4 inhibitor 5-O-methyl quercetin (D) in the presence of RANKL (100 ng/ml) for 3 days. The protein levels of LC3-I and LC3-II were tested by western blot, and the ratio of LC3-II/LC3-I was quantified by Image J. (E) RAW264.7 cells were seeded overnight and incubated with ML171 (2 and 5 μM) and 5-O-methyl quercetin (5 and 10 μM) in the presence of RANKL (100 ng/ml) for 3 days. The mRNA expression levels of TRAP, Cath K and MMP-9 were detected by qRT-PCR. (F) RAW264.7 cells were treated as described in (E) for the indicated times. Then, TRAP staining and bone resorption assays were performed to evaluate the formation of TRAP-positive multinucleated (≥3) osteoclasts and bone resorption pits respectively. All the data derived from at least three independent replicates and were presented as mean ± SD. *p < 0.05 versus corresponding control group; # p < 0.05 vs. corresponding RANKL group; ns, no significance, versus corresponding RANKL group.

Fig 3: MnTE-2-PyP decreased DNA binding of p50 subunit of NF-κB through NOX2 mediated oxidation of nuclear Trx. Human prostate fibroblast cells were treated with 20 mM glucose (HG) in the presence or absence of 3 Gy of X-rays (RAD) and 30 μM MnTE-2-PyP (T2E). A. Nuclear NFκB levels. NFκB p65 levels were significantly lower in RAD and RAD + HG groups as compared to control. T2E treatment restored NFκB p65 levels. NFκB p50 levels were significantly higher in RAD + HG group, which were suppressed by T2E. B. Nuclear thioredoxin (TRX) protein expression. T2E treatment significantly increased nuclear TRX levels in radiated and non-radiated conditions. C. Oxidized nuclear TRX protein levels. T2E treatment significantly increased oxidized nuclear TRX levels. The fully oxidized upper band of TRX is represented on a redox western blot. D. NFκB EMSA. DNA binding of NFκB p65-p50 heterodimer and p50-p50 homodimer. p65-p50 DNA binding was not altered. The DNA binding of p50-p50 homodimer was significantly less in T2E treatment after radiation. n ≥ 3. (*) denotes a significant difference as compared to control, ($) denotes a significant difference as compared to HG, (#) denotes a significant difference as compared to RAD and (@) denotes a significant difference as compared to RAD + HG group.

Fig 4: ROS production was involved in IS-induced reductions of Ito,f-related proteins.(A and D) Measurements of ROS productions based on DHE fluorescence in rat hearts. (A) Representative images of DHE immunofluorescence. Scale bar: 200 μm. (D) Relative ROS fluorescence intensities in sham, CKD, and CKD plus BB536 groups (n = 5 per group), and in vehicle and IS treatment groups (n = 6 per group). (B and E) Measurements of ROS productions based on flow cytometry in NRVMs treated with different concentrations of IS. (B) Representative flow cytometric histograms in NRVMs. (E) Relative ROS fluorescence intensities in NRVMs (n = 5 per group). (C and F) NAC reversed ROS production induced by IS in NRVMs. (C) Representative flow cytometric histograms in 4 groups. (F) Relative ROS fluorescence intensities detected by flow cytometry in 4 groups (n = 5 per group). (G and H) Representative immunoblots of NOX2 proteins in sham, CKD, and CKD plus BB536 groups (n = 5 per group) (G), and in vehicle and IS treatment groups (n = 6 per group) (H). (I) Average immunoblots data of NOX2 proteins in the hearts of rats. (J and M) Representative immunoblots (J) and average data (M) of NOX2 proteins in NRVMs treated with different concentrations of IS (n = 3 per group). (K and N) Representative immunoblots (K) and average data (N) of NOX2 proteins in control, DPI, IS, and IS plus DPI groups (n = 3 per group). (L and O) Representative immunoblots (L) and average data (O) of NOX2 proteins in control, APO, IS, and IS plus APO groups (n = 3 per group). (P and Q) Representative immunoblots (P) and average data (Q) of Kv4.2, Kv4.3, and KChIP2 proteins in control, NAC, IS, and IS plus NAC groups (n = 3 per group). (R) Relative mRNA expressions for Kv4.2, Kv4.3, and KChIP2 in control, NAC, IS, and IS plus NAC groups (n = 3 per group). NRVMs in IS and IS plus NAC groups were treated with 10 μM IS. Data are presented as mean ± SEM. Statistical analysis was performed using 2-tailed Student’s t test (D and I) and 1-way ANOVA followed by Bonferroni post hoc test (D–F, I, M–O, Q, and R). *P < 0.05, **P < 0.01.

Fig 5: Mechanisms of oxidative stress induced by NACT and docetaxel in the vasculature.(A) Protein levels of the NADPH oxidase homologs NOX1, NOX2, NOX4, and NOX5 in arteries from patients with or without prior NACT, with the densitometric analysis normalized to β-ACTIN (right) (n = 8/group). Data are expressed as the mean ± SEM. **P < 0.01 versus no NACT; 2-tailed, unpaired Student’s t test. (B) Heatmap depicting mRNA expression of selected key genes involved in the regulation of oxidative stress in human vasculature in arteries from patients with (n = 13) or without (n = 16) prior NACT. Expression was normalized to ACTB mRNA. *P < 0.05 versus no NACT; 2-tailed, unpaired Student’s t test. (C) Effects of a 24-hour organ culture with docetaxel (100 nM) or vehicle on the expression of NOX2 (also known as CYBB) and NOX4 mRNA in NACT-naive arteries (n = 5/group). Data are expressed as the mean ± SEM. *P < 0.05 versus vehicle; 2-tailed, paired Student’s t test. (D) Effects of 24 hours of docetaxel exposure (100 nM) on NOX2 (CYBB) and NOX4 mRNA in HASMCs and HDMECs (n = 4/group). Data are expressed as the mean ± SEM. **P < 0.01 versus vehicle; *P < 0.05 versus vehicle; 2-tailed, unpaired Student’s t test. (E) Immunofluorescence detection (red) of NOX2 and NOX4 expression in arteries from patients with or without NACT. Costaining (green) with CD31 and α-SMA was done to identify NOX2 and NOX4 expression in endothelial cells and SMCs (representative of 5/group). Scale bars: 200 μm.