Fig 1: miR-155 is critical for DC-mediated T cell activation. Naive T cells isolated from spleens of healthy WT mice were co-cultured with tumor cell lysate and ECM pulsed WT or miR-155-/- BMDCs (T cells:DCs = 10:1). Twenty-four hours after co-culture, T cell activation was determined by examining CD25 and CD69 expression on CD3+ cells by flow cytometry. T cell only group served as control. (A) Representative scatter plots (left) and percentage of CD3+ CD25+ cells (right) are shown (n = 3). (B) Representative scatter plots (left) and percentage of CD3+ CD69+ cells (right) are shown (n = 3). (C) Five days after co-culture, T cell proliferation was measured by [3H] thymidine incorporation assay (n = 4). (D) Co-culture media was collected on day 3 and IFN? concentrations were measured by ELISA; BMDC and T cell only group were used as controls (n = 4). (E) and (F) mRNA levels of IL-12 p35 (E) and IL-12 p40 (F) were determined by RT-PCR in WT and miR-155-/- BMDCs with or without treatment (n = 3). (G) Forty-eight hours post-treatment, IL-12 p70 concentrations in BMDC culture media were determined by ELISA; BMDCs without tumor lysate and ECM treatment were used as controls (n = 4). (H) and (I) IL-12 p70 (H) and IFN? (I) concentrations in sera of WT and miR-155-/- tumor-bearing mice at the end point (Day 25) were determined by ELISA (n = 6). (J) and (K) mRNA (J) and protein levels (K) of SOCS1 in BMDCs treated with tumor lysate and ECM were determined by RT-PCR and western blot, respectively. LPS stimulated cells were used as positive control. (A)–(D) one-way ANOVA followed by the Tukey multiple comparison test. #p < 0.05 versus T cell only group. (E)–(G) two-way ANOVA following by the Tukey multiple comparison test. #p < 0.05 versus control group. (H)–(J) Student's t test. *p < 0.05; **p < 0.01; ***p < 0.001 versus WT group.
Fig 2: HLJ1 deletion alleviates IL-12-dependent septic death.Dnajb4+/+ and Dnajb4-/- mice were intraperitoneally injected with 20 mg/kg lipopolysaccharide (LPS). (A) After 4 or 8 hr, the RNA from n = 6–8 total livers were isolated and gene expression levels were quantified via qRT-PCR. IL-12b, p = 0.029. (B) Serum levels of IL-12p70 in LPS-treated Dnajb4+/+ and Dnajb4-/- mice were quantified via ELISA 4 hr (n = 11–14) and 8 hr (n = 4–7) after LPS administration. 4 hr IL-12p70, p < 0.001; 8 hr IL-12p70, p = 0.033. (C) Dnajb4+/+ and Dnajb4-/- mice were intraperitoneally injected with anti-IL-12 neutralizing antibodies 1 hr prior to the injection of 20 mg/kg LPS. After the administration of LPS and anti-IL-12 antibodies, the serum was collected at the indicated time points and analyzed for IL-12 and IFN-? levels. (D) Kaplan–Meier analysis of overall survival of Dnajb4+/+ and Dnajb4-/- mice injected with 100 µg anti-IL-12 neutralizing antibodies 1 hr before the 20 mg/kg LPS challenge (n = 9–11 per group). Dnajb4+/+ versus Dnajb4-/- mice, p = 0.014; Dnajb4+/+ versus Dnajb4+/+ + anti-IL-12, p = 0.007. Data presented are means ± standard deviation (SD). Statistical analysis was performed by using the two-tailed, unpaired Student’s t-test. Log-rank Mantel-Cox test was used to compare survival curve. *p < 0.05, **p < 0.01, ***p < 0.001, ns, not significant. Figure 7—source data 1.Data for graphs depicted in Figure 7A–D.
Fig 3: HLJ1 deletion leads to the accumulation of homodimeric IL-12p35 and reduced levels of heterodimeric IL-12p70.(A) Bone marrow-derived macrophages (BMDMs) isolated from n = 6–7 Dnajb4+/+ and Dnajb4-/- mice were treated with 10 ng/ml lipopolysaccharide (LPS) and 20 ng/ml IFN-?. Supernatant was collected at the indicated time points, and IL-12p70 was quantified via ELISA. 12 hr, p = 0.003; 24 hr, p = 0.003. (B) LPS/IFN-?-treated BMDMs from n = 4–5 mice were lysed at the indicated time points and intracellular IL-12p70 was quantified via ELISA. 5 hr, p = 0.006; 8 hr, p = 0.012. (C) IL-12a, IL-12b, IL-6, and IL-18 expression was determined via quantitative real-time PCR (qRT-PCR) in LPS/IFN-?-treated BMDMs isolated from n = 5 mice. (D) Intracellular IL-12p40 and HLJ1 expression levels were analyzed in LPS/IFN-?-treated BMDMs isolated from Dnajb4+/+ (+/+) and Dnajb4-/- (-/-) mice. Representative samples of n = 3–5 biological replicates are shown. GAPDH served as a loading control. In comparisons with the 0 hr group (right panel): 2 hr, p = 0.001; 4 hr, p < 0.001; 8 hr, p = <0.001; 16 hr, p = 0.02. (E) The influence of human HLJ1 knockdown on the redox state of human IL-12p35 was analyzed via non-reducing sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE). 293T cells were (co-)transfected with the indicated IL-12p35 subunits and shRNA targeting HLJ1. The percentage of high-molecular-weight (HMW) and low-molecular-weight (LMW) IL-12p35 species in the presence or absence of shHLJ1 was quantified (right panel, n = 4 biological repeats for shHLJ1- and control-transfected cultures; p = 0.001). Where indicated, samples were treated with ß-mercaptoethanol (ß-Me) after cell lysis to provide a standard for completely reduced protein. GAPDH served as a loading control. Data presented are means ± standard deviation (SD). Statistical analysis was performed by using the two-tailed, unpaired Student’s t-test. *p < 0.05, **p < 0.01, ***p < 0.001. Figure 9—source data 1.Data for graphs depicted in Figure 9A–E. Figure 9—source data 2.Original and labeled blots images of Figure 9D, E.
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