Fig 1: T3SS1 effector translocation occurred only in very few infected AML12 cells. AML12 cells were exposed for 30 min to ST or ST ?invA strains expressing SopD effector-TEM-1 ß-lactamase in pCX340 and DsRed in pGG2, then washed and infection was continued for 60 min. Subsequently, cells were loaded with CCF4-AM for 20 min and visually inspected (A–E) using an inverted confocal microscope equipped with a 100 × oil immersion objective (LEICA TCS SP8, Germany). Images of 1024 × 1024 pixels were acquired using LaserX software (LEICA). Green fluorescence indicates that CCF4-AM was loaded and the presence of blue cells due to CCF4-AM cleavage reveals SopD translocation. White arrowheads indicate Salmonella. (A) non-infected AML12 cells, (B) cells infected with the ST DsRed strain carrying the empty vector pCX340, (C) cells infected with the ST DsRed strain expressing SopD effector-TEM-1 ß-lactamase in pCX340, (D) cells infected with the ST ?invA DsRed strain carrying the empty vector pCX340 and (E) cells infected with the ST ?invA DsRed strain expressing SopD effector-TEM-1 ß-lactamase. The cells emitting blue fluorescence were considered as positive for effector translocation. (F) HeLa and AML12 cells were infected as described above with the ST strain expressing SopD effector-TEM-1 ß-lactamase and DsRed, CCF4-AM loaded and finally trypsinized. Cell resuspensions were acquired using cytometry (BD LSRFortessa X-20 cell analyzer) and analyzed by BD FACSDiva Software. Green squares represent cells infected without SopD translocation; Blue dots represent infected cells with SopD translocation. Scale bars 10 µm.