HIV-1 infection modifies migration modes of macrophages in 2D and 3D environments via Nef. (A-E) hMDMs were infected or not with HIV-1 or HIV-1Δnef and seeded (A) in Ibidi culture inserts or (B-C) on thick layers of fibrillar collagen I (D-E) or of Matrigel polymerized in transwell chambers. (A) The percentage of cells closing the gap after 24 hours was measured and reported as 100% for controls. Mean ± standard deviation (SD), n = 4. *P ≤ .05; **P ≤ .01; and ***P ≤ .001. (B,D) The percentage of migrating cells after 72 hours was measured and reported as 100% for controls. Mean ± standard error of the mean (SEM), n = 7 in B and n = 11 in D. (C,E) 3D positions of macrophages and MGCs for representative experiments using TopCat Software. (F-G) hMDMs were transduced with GFP (control), mCherry-LifeAct, or Nef_SF2-GFP lentivirus and layered (F) on fibrillar collagen I or (G) on Matrigel. The percentage of migrating cells after 72 hours was measured. Mean ± SEM, n = 4.