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. 2019 May 22;10:1106. doi: 10.3389/fimmu.2019.01106

Figure 4.

Figure 4

Expression of the MHC II gene activated by IRF1 and CIITA. (A,B) IRF1 and CIITA induce MHC II expression. GCO cells were transfected with 1.0 μg pcDNA3.1, pcIRF1, or pcCIITA respectively. Cells were harvested at 24 h after transfection and fold changes of the gene expression were measured through qRT-PCR. (C) Activation of grass carp CIITA promoter by IRF1. GCO cells seeded in 24-well plate were cotransfected with (0.25 μg pcDNA3.1 or pcIRF1) and CIITA promoter at the ratio of 1:1. pRL-TK was used as an internal control. Promoter activities were monitored at 24 h after transfection. Asterisks (*) indicate significant differences from control (p < 0.05). (D) The binding of IRF1 with CIITA promoter. 293T cells seeded in 10-cm dishes were cotransfected with 5 μg Myc-IRF1 and 5 μg CIITA-pro. Empty vector PCMV-Myc (5 μg) was transfected in parallel as control. After 24 h, cell lysates were immunoprecipitated with anti-Myc-Agarose beads. Then the DNA binding to CIITA promoter was checked by semiquantitative PCR. The input was used as a control in semiquantitative PCR to quantify the DNA concentration. (E) Schematic representation of mutated CIITA promoter-driving luciferase constructs. The base substitution mutations of each ISRE site are shown. (F) The binding of IRF1 with ISRE motifs of CIITA promoter. The cells seeded in 10-cm dishes were cotransfected with 5 μg Myc-IRF1 and (5 μg CIITA-pro, Mut 1-pro or Mut 2-pro). Cell samples were harvested at 24 h after transfection and cell lysates were immunoprecipitated with anti-Myc-Agarose beads. The input was used as a control in semiquantitative PCR to quantify the DNA concentration. (G) The function of ISRE motifs on activation of CIITA. GCO cells were cotransfected with (pcDNA3.1, IFN-γ, or IRF1) and (CIITA-pro, Mut 1-pro, or Mut 2-pro). Luciferase activities were monitored at 24 h after transfection. (H) Schematic representation of wild type IRF1 and its two mutants. (I) Effect of the two structural domains of IRF1 on the CIITA promoter. GCO cells were cotransfected with (IRF1, IRF1-ΔN or IRF1-ΔC) and CIITA promoter, luciferase activities were monitored at 24 h after transfection.