Nef stabilizes podosomes and increases the size of the F-actin core. (A) Confocal images of Nef_NL4-3-expressing hMDMs on glass. Inset, 2× zoom; arrow, large podosomes where Nef_NL4-3 localizes. (B) Normalized fluorescence intensity profiles of F-actin and Nef_NL4-3 along the pink dotted line in A. (C) Confocal images of hMDMs expressing Nef_SF2-GFP inside Matrigel. Inset, 3.5× zoom; arrowheads, colocalization of Nef_SF2 with F-actin at 3D podosomes. (D) FRAP analyses in hMDMs cotransfected with mRFP-β-actin, and Nef_NL4-3-GFP or GFP (control). Mean ± SEM, representative of 6 independent experiments (donors) (>20 podosomes per condition for each donor). (E-F) Time-lapse analysis of podosomes. hMDMs were cotransfected with LifeAct-mCherry (F-actin) and Nef_NL4-3-GFP or GFP. (E) Images from supplemental Videos 2 and 3. (F) Quantification of podosome life span. n = 4 (>100 podosomes per condition). Red dotted lines: median. (G) Automatic quantification of F-actin intensity, fluorescence area, and F-actin density inside podosomes in hMDMs expressing the indicated Nef mutants. Nef from different strains were used: 2 HIV-1 (NL4-3 and SF2), 2 HIV-2 (rod and nep from Nepal84), and 1 SIV. Mean ± SEM, n = 3 (>1000 podosomes from ≥10 cells per donor). Scale bars, 10 μm.