Hck is necessary for HIV-1 effect on podosomes and 3D mesenchymal migration of macrophages. (A) Representation of the siRNA experimental design. (B) Dosage of p24 from day 15 supernatants of HIV-1_ADA–infected hMDMs. Mean ± SEM, n = 5. (C) (Upper) Western blot analysis of Hck depletion in infected hMDMs. (Lower) Quantification of Hck normalized to tubulin. Mean ± SD, n = 7. (D) The percentage of hMDMs migrating in Matrigel after 72 hours was measured and reported as 100% for controls. Mean ± SD, n = 5. (E) IF microscopy of HIV-1_ADA–infected macrophages on glass subjected to control siRNA or Hck siRNA. Arrowhead: podosome rosettes/belts; arrow: podosome clusters. Scale bar, 10 μm. (F) Quantification of the number of cells forming podosome rosettes/belts. Mean ± SD, n = 3. (G) Automatic quantification of F-actin intensity inside podosomes. Mean ± SEM, n = 3 (>1000 podosomes from ≥10 cells per donor). NI, not infected.