Fig. 3

Validating promising genes with HIV-eGFP and HIV-HSA viruses by siRNAs. Kinetics of HIV-eGFP A and HIV-HSA B infection. Seven-day-old MDMs were infected with 150 ng p24 of HIV-eGFP or HIV-HSA viruses and harvested at various days after infection for flow cytometry analysis to quantify eGFP or HSA expressing cells (N = 3). C Apoptosis of HIV-eGFP-infected MDMs induced by siRNAs of 28 promising genes. Seven-day-old MDMs were infected with HIV-eGFP for 7 days and then transfected with siRNAs of 28 promising genes for 72 h. Cells were trypsinized, harvested, stained with Annexin-V antibody labelled with BV711 and apoptosis was quantified by flow cytometry. The fold change (HIV-eGFP-infected/mock-infected) of all Annexin-V+ apoptotic cells induced by each siRNA was calculated. D Apoptosis of HIV-HSA-infected MDMs induced by siRNAs of 12 candidate genes. Seven-day-old MDMs were infected with HIV-HSA for 7 days and then transfected with siRNAs of all 12 candidate genes for 72 h. Cells were trypsinized, harvested, stained with anti-mouse CD24 antibody (HSA-FITC) and Annexin-V-BV711 antibody and the apoptosis was analyzed with flow cytometer. Fold change (HIV-HSA-infected/mock-infected) of all Annexin-V+ apoptotic cells induced by each siRNA was calculated. The p-values of C and D were calculated using student’s t-test (n = 4). E siRNA transfection silenced the expression of all four identified genes in primary MDMs. Seven-day-old MDMs were infected with HIV-HSA for 7 days and then transfected with siRNAs specific for Cdk2, Cox7a2, Cstf2t or Znf484 genes for 72 h. Cell lysates were subjected to Western blot analysis by probing with antibodies specific for CDK2, COX7A2, CSTF2T or ZNF484. The results shown are representative of three independent experiments. The full-length blots for silencing of all the four genes were shown in Supplementary Fig. 3, 4, 5, 6 and 7