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. 2020 Jul 16;11:3571. doi: 10.1038/s41467-020-17391-2

Fig. 1. Identification of B. henselae gene essential in promoting vascular endothelial cell proliferation.

Fig. 1

a B. henselae (Bh) promotes vascular endothelial cell proliferation. Human umbilical vein endothelial cells (HUVECs) were co-cultured with or without Bh at multiplicity of infection (MOI) of 30 for 3 days. Fluorescence images stained with CellMask (red) and Hoechst 33342 (blue) are shown. Scale bar = 100 µm (upper). The numbers of cells from the images were counted, and the values were normalized to the uninfected cells as 100% (lower). b Screening process for Bh transposants lacking cell proliferative ability. c Cell proliferative ability of Bh-WT and two transposants isolated from the screening. HUVECs were co-cultured with Bh cells (WT, 623-125, or 804-29) at MOI of 3, 30, or 300 for 3 days, and the relative cell numbers are shown. d Diagram of bafA (AYT27_RS02860) in two transposants, showing the locations of transposon insertion in 623-125 (at base pair position 646,986 in the Bh genome; NCBI accession no. NC_005956.1) and 804-29 (at position 645,770). e Ectopic expression of bafA restores Bh-induced cell proliferation. HUVECs were co-cultured with WT or 623-125 transposants complemented with bafA expression plasmid pBAF. The transposants without vector and with empty vector (pBBR) were used as negative controls. After 3-day incubation, relative cell numbers were counted. f Schematic representation of indirect co-culture of Bh and HUVECs. g Bh cells with bafA promote cell proliferation without direct contact. HUVECs were co-cultured indirectly with or without indicated Bh strains. After 3-day incubation, cell numbers were calculated. Bars are colored based on the strain: white for no infection, gray for WT, red for 623-125 and blue for 804-29 (a, c, e, g). Data are mean ± s.d. (n = 8 biological replicates). Statistical significance was determined using a two-tailed unpaired Student’s t test (a), one-way ANOVA with Dunnett’s multiple comparisons test (c), or with Tukey’s multiple comparisons test (e, g). **P < 0.01; ***P < 0.001; NS not significant. All experiments were performed at least three times, and one of repeats is shown for each. Source data are provided as a Source Data file.