Fig. 3

IFITMs block SAMHD1 degradation after treatment with VLPs-Vpx pseudotyped with VSV-G. a Knockout efficiency as measured by protein levels. THP1 were cells transduced with a lentivector coding for Cas9 and sgRNAs targeting either IFITM1 (IFITM1-KO, lanes 2 and 5) or IFITM2 and IFITM3 (IFITM2/3-KO, lanes 3 and 6) or encoding a non-targeting sgRNA (NTC, lanes 1 and 4). Cells were selected in puromycin for 2 weeks. Cells were treated for 24 h with 0 (lanes 1 through 3) or 1000 (lanes 4 through 6) U/mL IFNα and protein expression was measured by Western Blot. For IFNα treated cells, three times less lysate was loaded, in order to be able to visualize levels of IFITMs in the absence or presence of IFNα stimulation in the same exposure. Tubulin was used as a loading control. b Genomic DNA was extracted from THP1 NTC, IFITM1-KO or IFITM2/3-KO cells. The ifitm2 and ifitm3 loci were amplified using specific PCR primers with a 3’ mismatch. For each cell type, we verified that the PCR was specific by Sanger sequencing. The sequences at the two loci were compared to the reference sequence using TIDE analysis and the percentage of editing was quantified. c Cells were treated for 24 h with 0 or 1000 U/mL IFNα and incubated with the indicated amount of VLPs-Vpx pseudotyped with VSV-G. SAMHD1 degradation was measured by flow cytometry 16 h after treatment. One dose response experiment is shown. d Combined data from three independent experiments, using a viral dose within linear range. Each symbol represents an experiment. *p < 0.05 (t test). e Overexpression of IFITM3 in THP1 cells was measured by Western Blot. Cells were lysed in NP40-DOC buffer and probed for IFITM3. Tubulin was used as a loading control. The band indicated with an asterisk corresponds to the HA tagged version of IFITM3. f SAMHD1 degradation assays. Cells were treated for 24 h with 0 or 1000 U/mL IFNα, then incubated with the indicated amount of VLPs-Vpx pseudotyped with VSV-G for 16 h and SAMHD1 degradation was measured by flow cytometry. Combined data from three independent experiments using a viral dose within linear range. *p < 0.05 (t test)