Fig 2: The levels of S-glutathionylation and Nrf2 in growth-arrested versus rescued pulp microtissues at early and advanced stages of development. (A,C,E,G) Fluorescence stock photos and (B,D,F,H) phase-contrast images of growth-arrested spherical microtissues after (A,B) 2 d and (E,F) 7 d versus growth-resumed pulp microtissues treated with NAC after (C,D) 2 d and (G,H) 7 d. White arrows point to the interactions between the spherical structures. (I) Bar graph measuring the fluorescence intensity of the GSH marker (S-glutathionylated proteins). *** p = 0.001. (J,L) Fluorescence and (K,M) phase-contrast images of (J,K) growth-arrested spheroids versus (L,M) growth-resumed pulp microtissues treated with NAC. White arrows point to the interactions between the spherical structures. (N) Bar graph measuring the fluorescence intensity of Nrf2 marker. * p = 0.05.

Fig 3: Immunoblot analysis of kidney lysates.(A) Blot membranes were incubated with the antibodies against Nrf2, p53 and 8-oxoguanine glycosylase a (OGG1a). The mean integrated optical density is related to actin for Nrf2 (B), p53 (C) and mature 8-oxoguanine glycosylase a (D) levels. Results are given as a mean ± S.E.M. KD4, KD6, KD8 – groups of Eker rats (Tsc2+/-) treated with HFKD for four, six or eight mo., respectively; ST – Eker rats fed with a standard diet; LE ST – wild-type Long Evans rats treated with a standard diet; LE KD – wild-type Long Evans rats treated with a ketogenic diet similarly to the KD6 group. *P < 0.05, **p < 0.01, ***p < 0.001 as compared to ST unless otherwise stated (horizontal line) for ANOVA with a Fisher post hoc test.

Fig 4: Activation of Nrf2 drives antioxidant responses to HIV-1-induced oxidative stress ATotal-to-oxidized glutathione ratio (GSH/GSSG) in mock-infected or HIV-1-infected CD4+ T cells (7 dpi, n = 4 biological replicates; mean ± SEM).B–DRepresentative picture (B) and quantification (C, D) of ROS content in HIV-1-infected or mock-infected primary CD4+ T cells at 7 dpi (productive infection) and 14 dpi (latent infection). ROS content was measured by immunofluorescence; HIV-1 DNA+ cells were identified by FISH (D). The black dash in (C) indicates the median. Box plots in (D) depict median and 25–75 percentile, while whiskers extend from min to max. n = number of cells. Scale bar = 2 µm.ESubcellular localization of Nrf2 in CD4+ T cells infected with HIV-1 or mock infected (3 dpi) as analyzed by biochemical fractionation and Western blot, whole cell extract (WCE), cytoplasm (Cyt), nucleoplasm (Nuc).FNuclear localization (left) and content (right) of Nrf2 in HIV-1 RNA+ and HIV-1 RNA- cells (7 dpi) as measured by combining immunofluorescence and HIV-1 RNA FISH. Black dash indicates median, n = number of cells. Scale bar = 2 µm.G, HTime course of the mRNA (G) and protein (H) levels of main downstream antioxidant targets of Nrf2 during the transition from productive (3–9 dpi) to latent (14 dpi) infection, as measured by qPCR (G) and Western blot (H). Data in (G) are depicted as mean ± SEM of 3 biological replicates. GCLC and HMOX-1 in (H) were probed upon membrane stripping.IRelative mRNA expression of Nrf2 downstream antioxidant targets in J-Lat 9.2 cells left untreated (latent) or treated for 24 h with 10 µM TPA (reactivated). Data are depicted as mean ± SEM of 3 biological replicates.Data information: Values shown in (C, D and F) were calculated as nuclear corrected total cell fluorescence (as in McCloy et al, 2014) and analyzed by Kruskal–Wallis multiple comparison test followed by Dunn's post-test (C) or by two-tailed Wilcoxon signed-rank test (D, F). For (G, I), raw data were first normalized using 18S as housekeeping control and then expressed as log2 fold mRNA expression in infected vs. mock-infected cells (G), or TPA-reactivated vs. latent cells (I) which were calculated as in Livak and Schmittgen (2001). Data were analyzed by two-way ANOVA followed by Tukey's post-test for multiple comparisons. *P < 0.5; **P < 0.01; ***P < 0.001; ****P < 0.0001. Trx = thioredoxin; NQO1 = NAD(P)H [quinone] dehydrogenase 1; HMOX-1 = heme oxygenase 1; G6PD = glucose-6-phosphate dehydrogenase; GCLC = glutamate-cysteine ligase; TrxR1 = thioredoxin reductase 1.Source data are available online for this figure.

Fig 5: Overexpression of KLF15 regulates NF-?B and Nrf2 signaling pathways in Eahy926 cells. (A) Reverse transcription-quantitative polymerase chain reaction analysis was performed to investigate the mRNA expression of Nrf2 and p65. (B) Western blotting was performed to investigate the protein expression levels of Nrf2 and p-p65, which was normalized to ß-actin expression. *P<0.05, **P<0.01 and ***P<0.001 vs. respective NC; ^P<0.05, ^^P<0.01 and ^^^P<0.001 vs. respective TNF-a + NC. NC, negative control pTA2 vector; KLF15, Krüppel-like factor 15; TNF-a, tumor necrosis factor-a; Nrf2, nuclear factor erythroid 2-related factor 2; p65, transcription factor p65; p-, phosphorylated.