Figure 2.

Downregulation of SMPD3 in epithelial cells following Cryptosporidium parvum infection is associated with delivery of Cdg7_FLc_1000 into the infected host cells. A, Downregulation of SMPD3 was further analyzed by real-time polymerase chain reaction (PCR) analysis in INT and HCT-8 cells following C. parvum infection or transfection of Full-Cdg7_FLc_1000 for various periods. *P<.05, by t-test, compared with control; **P<.01, by t-test, compared with control. B, Content of SMPD3 in HCT-8 cells following C. parvum infection for 24, 36, and 48 hours, as assessed by Western blot. Representative gel images are shown, and densitometric levels of SMPD3 signal were quantified. GAPDH was blotted for control. *P<.05, by t-test, compared with control. C, Inhibition of delivery of Cdg7_FLc_1000 into infected cells through pretreatment of host cells with a small interfering RNA (siRNA) to Cdg7_FLc_1000, followed by exposure of cells to C. parvum infection. INT and HCT-8 cells were treated with a siRNA to Cdg7_FLc_1000 for 12 hours and then exposed to C. parvum infection for additional 24 hours. Contents of Cdg7_FLc_1000 in the infected cells were quantified by quantitative real-time PCR analysis. A nonspecific scrambled siRNA was used as the control. D, Inhibition of Cdg7_FLc_1000 in host cells by the siRNA treatment attenuated the downregulation of SMPD3 following C. parvum infection. INT and HCT-8 cells were treated with a siRNA to Cdg7_FLc_1000 for 12 hours and then exposed to C. parvum infection for additional 24 hours. Expression levels of SMPD3 in the infected cells were quantified by real-time PCR. Data represent 3 independent experiments. *P<.01, by analysis of variance (ANOVA), compared with noninfected cells treated with the control siRNA; ^#P<.01, by ANOVA, compared with infected cells treated with the control siRNA.