Figure 3(A) Representative confocal fluorescence micrographs of HeLa cells transfected for 24 h to produce either mCherry or mCherry-BspF (grayscale panels), incubated on ice with AlexaFluor™488-Cholera Toxin subunit B (CTxB; green) and shifted to 37˚C for 20 min to allow for CTxB retrograde transport to the Golgi apparatus (stained using an anti-GM130 antibody; purple). CTxB accumulation within Golgi structures appears white in overlays. Scale bars: 10 and 2 µm (insets). (B) Quantification of CTxB transport to the Golgi apparatus in HeLa cells producing either mCherry, mCherry-BspF or HA-BspF over a 30 min time course, expressed as percentages of cells in which CTxB colocalized with the GM130 Golgi marker, as in (A). Data are means ± SD from n=3 independent experiments, in which 100 cells were analyzed per experiment. Asterisks indicate statistically significant differences compared to mCherry-producing cells as determined by a two-way ANOVA with Tukey's multiple comparisons test (P<0.05). ( (C) Representative Western blot analysis of Arf6, Rab8a and Rab6a/a' depletions in BMMs following siRNA-mediated knockdowns, compared to non-targeting siRNA (siNT) treatments. β-actin was used as loading control. (D) Quantification of CTxB transport to the Golgi apparatus in BMMs following siRNA-mediated depletion of either Arf6 (siArf6), Rab8a (siRab8a) or Rab6a/a' (siRab6a/a') after AlexaFluor™488-CTxB binding on ice followed by 30 min incubation at 37˚C. Data are means ± SD from n=3 independent experiments, in which 100 cells were analyzed per experiment. Asterisks indicate a statistically significant difference compared to siNT control cells as determined by a one-way ANOVA with Tukey's multiple comparisons test (P<0.05). (E) Quantification of CTxB transport to the Golgi apparatus in BMMs that were either mock-infected or infected with wild type (2308), ΔbspF, complemented ∆bspF (ΔbspF::bspF), ∆bspB or complemented ∆bspB (ΔbspB::bspB) bacteria for 24 h, incubated for 30 min with AlexaFluor™488-CTxB on ice for binding followed by 30 min incubation at 37˚C. Data are means ± SD from n=3 independent experiments, in which 100 cells were analyzed per experiment. Asterisks indicate a statistically significant difference compared to mock-infected cells as determined by a one-way ANOVA with Tukey's multiple comparisons test (P<0.05). ( (F) rBCV biogenesis in BMMs treated with either non-targeting siNT, siRab6a/a' or siRab8a siRNAs and infected with wild type (2308) bacteria. Data are means ± SD of n=3 independent experiments, in which 100 BCVs were analyzed per experiment. Asterisks indicate statistically significant differences (P<0.05, two-way ANOVA followed by Dunnett's multiple comparisons test) compared to control (2308). (G-I) Brucella replication in BMMs treated with non-targeting siRNAs (siNT), or siRNAs against Rab6a/a' (siRab6a/a') (G), Rab8a (siRab8a) (H) or Arf6 (siArf6) (I) and infected with either wild type (2308), ΔbspF, or complemented ∆bspF (ΔbspF::bspF) bacteria, measured as number of bacteria per cell at 24 h pi. Data are means ± SD of n=3 independent experiments in which at least 100 cells were analyzed per experiment. Grey dots represent individual cells analyzed; black dots indicate means of individual experiments. Asterisks indicate statistically significant differences (P<0.05, one-way ANOVA followed by Dunnett's multiple comparisons test) between test and control conditions.