Fig 1: High-throughput, siRNA-based screening to identify cellular factors regulating HIV-1 integrase stability. a Workflow for the siRNA-based screening. Cellular fluorescence, as surrogate of IN levels, was analyzed by automated, high-content fluorescent microscopy. b Results of screening. The graphs show the log10 values of the fold change of EGFP-positive cells over control in the two replicate screenings (R1 and R2). The dotted lines show 2x increase over Control (pool of results using 4 non-targeting siRNAs and mock-transfected cells). The 6 siRNAs in red are those that were in the top 10 in both screenings. The 4 siRNAs in green are those that were among the top 10 in one of screening while anyhow showing an effect ≥2 fold over control in the other screening. The effect of MG132 is shown in blue. c Confirmation of effective silencing of Pin1, TRIM33, FBOX28, RNF31, RNF125, RFPl3, and DTX by immunoblotting with the respective antibodies. Cells transfection of non-targeting siRNA1 (siNT1) was used as a control. HSC70 served as a loading control. d Representative immunoblot showing the levels of HIV-IN after knock-down of the top 10 E3 ligases from the screening. HeLa cells were transfected with siRNAs against the identified factors or with a siRNA against Pin1, followed by transfection of Flag-IN and EGFP. Forty-eight hour later the levels of IN were assessed by anti-Flag immunoblotting. Tubulin served as a loading control; β-catenin was used to confirm effect of the MG132 treatment. The bottom panel shows a western blotting for EGFP in a representative experiment to show lack of effect of any of the tested siRNAs on EGFP levels. e Quantification of the levels of HIV-1 IN after knock-down of the top 10 E3 ligases identified by the screening. Experiments were performed as in panel d. IN levels are expressed after normalization for tubulin and as fold over siNT1. Data are mean ± SEM; n = 3 independent experiments; *P < 0.05; **P < 0.01; one-way ANOVA. f Representative high-content microscopy images showing of EGFP-IN-expressing cells after depletion of four cellular ubiquitin-conjugation factors (TRIM33, FBXO28, siDTX2, and siUBE2J2) or Pin1