Fig 1: Comparison of gene and protein expression between iDC and 27DC.(A). A microarray analysis was performed on iDC and 27DC from 4 different donors, and a total of 51 genes (S1 Table) were significantly changed by > 3-fold in 27DC. The Venn diagram analysis was conducted using reported genes associated with HIV replication by siRNA/shRNA screening (siRNAs/shRNAs), CRISPR libraries screening (CRISPR), and the reported Interferon-stimulated genes (ISGs). Of 51, 19 genes overlapped with the reported genes. The gene names with the red font were targeted for further analysis. The genes with black font encode receptors or transcriptional factors. (B) The qRT-PCR was conducted to define the expression level of each gene of interest using total cellular RNA from iDC and 27DC from three independent donors. Data show mean ± SEM from three independent donors’ cells. (C) The total cellular protein lysate was collected from iDC and 27DC, and Western blotting was performed using anti-ANKRD22, anti-STAB1, anti-SERPING1, and anti-GAPDH antibodies. The band intensity of each protein was normalized by the band intensity of GAPDH (Image J); the values are indicated below images.
Fig 2: 27DC produce CCL3 and SERPING1 in culture supernatants.(A) The concentrations of secreted CCL3, CCL4 and CCL5 in culture supernatants on day 7 were measured by ELISA. A total of eight different donor culture supernatants were subjected to the assay. (B) Real-time RT-PCR was conducted to compare the expression level of CCL3, CCL4 and CCL5 gene. Data show mean ± SEM from three independent donors’ cells. (C) The total cellular protein lysate was collected from three independent donors’ iDC and 27DC, Western blotting was performed using anti-CCL3, anti-CCL4, anti-CCL5, and anti-β-actin antibodies. As a positive control for WB, 10 ng and 1 ng recombinant CCL5 (R&D system) was used. The band intensity of each protein was normalized by the band intensity of β-actin (Image J); the values are indicated below images. (D) The concentrations of secreted SERPING1 in culture supernatants on day 7 were measured by ELISA. A total of eight different donor culture supernatants were subjected to the assay. Data indicates mean ± SEM (n = 8). **: p<0.01, ***: p<0.001, and ****: p<0.0001.
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