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. 2020 Sep 1;9(9):2010. doi: 10.3390/cells9092010

Figure 4.

Figure 4

Cul3 reduces HIV-1 mRNA expression. (a,b) To measure the amounts of reverse transcription intermediates in infected cells, HEK293T cells were transfected with two independent siRNAs targeting Cul3 (CUL3_5 and CUL3_9) or a non-targeting siRNA as control (Scr). 48 h post transfection, cells were infected with a VSVg-pseudotyped HIV-1 NL4-3 luciferase reporter virus. 24 h post infection, cells were harvested and DNA was isolated and analyzed by qRT-PCR, using specific primers to amplify HIV-1 early or late RT (n = 1–3 +/− SD). (c) To analyze HIV-1 mRNA levels, total RNA was isolated 24 h post infection, reversely transcribed into cDNA and analyzed by qRT-PCR (n = 6 +/− SD). (d) To analyze virus particle release, HIV-1 p55 and p24 levels in the supernatant of HEK293T cultures were measured by western blot. (e) HEK293T cells were transfected with two independent siRNAs targeting Cul3 (Cul3_5 and Cul3_9) or a non-targeting (NT) siRNA as control. 48 h post transfection cells were harvested and cell cycle transition was analyzed by FACS using propidium iodine staining (n = 2). * p < 0.05; ** p < 0.01; n.s.: not significant.