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. 2021 Feb 15;12:619362. doi: 10.3389/fimmu.2021.619362

Figure 3.

Figure 3

Apoptosis of BoMac cells induced by MbovP280 in mycoplasmas cells or as the recombinant protein. Cells (5 ×105 per well) were treated with MbovP280 (A) or MbovP280Δ210−269 (B) at concentrations of 0.25, 0.5, or 1 μM for 24 h. The cells were then stained with annexin V and propidium iodide (PI) and detected with flow cytometry. (C) Growth curves of strains HB0801, the Mbov_0280 mutant T9.297, and its complement CT9.297. Growth of M. bovis at each time point was determined with a plating assay. (D) Visualization of MbovP280 expression in T9.297 and CT9.297 strains with a western blotting assay. Wild-type strain HB0801 was used as the positive control. (E) The cleaved caspase-3 of BoMac cells treated with rMbovP280. The cell lysates of BoMac treated with 0.5 μM rMbovP280 or rMbovP280Δ210−269 were resolved with SDS-PAGE, transferred onto membrane, and then immunodetected with an antibody directed against cleaved caspase-3. β-actin was used as the internal control. The ratio of the amount of cleaved caspase-3 to the amount of β-actin was calculated and normalized to the NC. (F) The cleaved caspase-3 of BoMac cells infected with M. bovis. The cell lysates of BoMac infected with HB0801, T9.297, or CT9.297 (MOI = 1,000) were resolved with SDS-PAGE, transferred onto PVDF membrane, and then immunodetected with the antibody directed against cleaved caspase-3. β-actin was used as the internal control. The ratio of the amount of cleaved caspase-3 to the amount of β-actin was calculated and normalized to the NC. (G) Single peak around the protospacer adjacent motif (PAM) in the sequence of BoMac cryab− clonal cell line. BLAST analysis of sequences around the PAM sequence of WT BoMac (BoMac WT) and BoMac cryab− cell lines. (H) Expression of CRYAB in BoMac WT and BoMac cryab− cells. Whole-cell lysates of BoMac WT and BoMac cryab− cells were resolved with SDS-PAGE, transferred onto membrane, and then immunodetected with the antibody directed against CRYAB. β-actin was used as the internal control. (I) Early apoptosis of BoMac induced by HB0801, T9.297, or CT9.297 was compared. BoMac (5 ×105 cells per well) were treated with 5 ×108 CFU of HB0801, T9.297, or CT9.297, or with PBS for 24 h. The cells only stained with annexin V are defined as undergoing early apoptosis. (J) Early apoptosis of BoMac-cryab− cell line induced by M. bovis. BoMac (5 ×105 cells per well) were infected with HB0801 (5 ×108 CFU), T9.297 (5 ×108 CFU), or CT9.297 (5 ×108 CFU), or treated with PBS (NC) for 24 h. *p <0.05, **p <0.01, ***p <0.001 indicate statistically significant differences; ns indicates no difference.