Fig. 4.

Biological consequences of MCPIP-1 downregulation. a Mobilization of intracellular free calcium levels in Fura-2-loaded cells was monitored by spectrofluorometry after the treatment of neutrophils with culture media collected from noninfected and E. coli-infected HeLa cells (shNC - NC, shRNA MCPIP-1 silenced); the arrow indicates the time of media addition. Representative ratios of fluorescence intensities (340/380 nm) from three independent experiments are shown. b MCPIP-1 was silenced in HeLa cells using specific siRNA or shRNA sequences; scrambled nonselective sequences were used as NCs. Forty-eight hours later, cells were infected with E. coli for 8 and 24 h. The level of iNOS mRNA, determined by RT-PCR, was calculated across the samples; the value in the NC sample was defined as 1. c Four hours after E. coli infection of MCPIP-1-silenced HeLa cells (MOI = 5), cell lysates were collected and plated onto LB agar for the counting of intracellular bacteria (CFU). Data represent mean values from three independent experiments ± SD. * p < 0.05; ** p < 0.01.