Fig 2: Effect of chronic exposure to ozone on 78-kDa glucose-regulated protein (GRP78) expression in rat hippocampus. (A) Shows the densitometry analysis of the protein levels for GRP78 from rat hippocampus following exposure to ozone for 7, 15, 30, 60 and 90 days. There is a significant increase at 60 and 90 days of treatment. (B) The results show an increase in the protein expression following 60 and 90-day exposure to ozone (*p < 0.05) compared with the control groups. GAPDH staining was used as an internal control. (C) Effects of ozone treatments on GRP78 immunoreactivity in the dentate gyrus of rat hippocampus. Light photomicrographs show immunoreactivity in GRP78 of cells from hippocampus of rats exposed to ozone. The control group showed low immunoreactivity. We observed an increase of immunoreactivity for GRP78 at 60 and 90 days of exposure to ozone. Arrows show GRP78 immunoreactivity 100×. The square indicates the area that is shown as 100× magnification.

Fig 3: S. flexneri deploys OspC3 to suppress neutrophil caspase-11-dependent pyroptosis and bacterial clearance(A–C) Pam3CSK4-primed neutrophils were infected with wild-type S. flexneri or S. flexneri ?ospC3 (MOI 100) for 4 hr. Cytotoxicity was determined by LDH release assay (A), IL-1ß was determined by ELISA (B) and gasdermin D, caspase-1, and caspase-11 cleavage were detected by western blot (C). GAPDH was used as a loading control. The asterisk indicates a non-specific band.(D and E) Indicated mice were infected i.p. with 2.5 × 107 CFU of both wild-type S. flexneri and S. flexneri ?ospC3 encoding ampicillin or kanamycin resistance, respectively. Bacterial loads from co-infected mice were determined 24 hr after infection and used to determine competitive index in the spleen (D) and liver (E).(F) Indicated mice were infected i.p. with 7.5 × 107 CFU of S. flexneri wild type or ?ospC3 and survival was monitored.Data are representative of three experiments. Bar represents mean ± SEM in (A-B and D-E). Dashed line in (D and E) indicates the x axis. For cytotoxicity and IL-1ß levels (A, B), statistical significance was assessed two-way ANOVA followed by Bonferroni's multiple comparison test with a statistical threshold of p = 0.05. For competitive index (D, E), statistical significance was determined by one-way ANOVA followed by Tukey's multiple comparison test with a statistical threshold of p = 0.05. Survival curves were compared using log rank (Mantel-Cox) to determine the p value (F). Groups that do not share a letter are significantly different from each other, p = 0.05.

Fig 4: ATF3 negatively regulates Atg5 gene. (a) Neuro2a cells were treated with either a control non-targeting siRNA or the Atf3-specific siRNA for 48 h and then infected with JEV (MOI 5). Relative expression of Atg5 at different times pi was studied by qRT PCR, and the mean is plotted (left panel). Cell lysates were prepared 24 h pi, and Western blotted for ATG5 and ATF3. Shown below is the ratio of ATG5/GAPDH. (b) MEF cells were treated with either a control non-targeting siRNA or the Atf3-specific siRNA for 48 h and then infected with JEV (MOI 5) (left panel) or transfected with 1 µg/ml poly(IC) (right panel). Relative expression of Atg5, 24 h post-treatment, was studied by qRT PCR, and the mean is plotted. Transcript level in control siRNA-treated cells is shown by the black bar while that in the Atf3 siRNA-treated cells is shown by the gray bar. The Gapdh levels were used for normalization. The experiments were done in triplicates on three different occasions. The statistical significance of the difference between the data was established by Student’s t-test; *p < 0.05; **p < 0.01; ***p < 0.001.

Fig 5: CSR and SHM are impaired in ROD1exon3-/- mice. a The absence of ROD1 protein in ROD1exon3-/- B cells. Diagram of gRNAs and the major AUG start codon in the ROD1 loci (upper panel). ROD1 and AID expression were examined by Western blotting. ß-actin served as the loading control. b RT-qPCR examination of post-switch transcripts containing Iµ and each of the CH exons. The RNA levels were normalized to GAPDH (n = 3). c Flow cytometric analysis of IgG1 and IgG3 with ex vivo cultured splenic B cells activated either by LPS or LPS plus IL4. Splenic B cells were isolated from wild-type and ROD1exon3-/- mice. Exogenous ROD1 was transduced into ROD1exon3-/- B cells before stimulation to rescue CSR defects (n = 3). d Quantification of IgG1 and IgG3 CSR efficiency by normalizing to WT levels as shown in (c). *P < 0.05, **P < 0.01 (two-tailed Student’s t test). e Flow cytometric analysis of Peyer’s patch germinal center B cells in wild-type (WT) and ROD1 knockout (KO) mice. Percentage of GL-7+/Fas+ germinal center B cells amongst all B220+ cells is indicated. f SHM analysis of Peyer’s patch germinal center B cells at AID hypermutation substrates located at the JH4 intron. The mean values were determined by Sanger sequencing from 73 (WT) and 88 (-/-) sequenced clones