Fig. 3: Delivery of CSpV1-dsRNAs into the cytoplasmic region of intestinal epithelial cells following C. parvum infection.

a Detection of CSpV1-dsRNAs in the cytoplasm of infected cells. IEC4.1 cells were exposed to C. parvum for 24 h and cytoplasmic fractions were isolated. CSpV1-dsRdRp, CspV1-dsCA, and several parasite RNAs (e.g., Cgd7_4540) in the cytoplasmic fractions were measured by RT-qPCR. Data are presented as the fold change to control normalized to host Gapdh. Cytoplasmic extract from cells collected from cell suspension mixed with the same amount of parasites was used as control (Ctrl). Data are from three biological replicates and presented as mean values ± SD. p values were determined by two-tailed unpaired Student’s t-test. b In situ hybridization of CSpV1-dsRdRp in C. parvum-infected cell. IEC4.1 cells were exposed to C. parvum for 24 h and CSpV1-dsRdRp was detected in the cytoplasm (arrows) using DIG-labeled CSpV1-dsRdRp probes. Representative images from three independent experiments are shown. Blue: DAPI (DNA), green: CSpV1-dsRdRp (arrows), C. parvum: arrowheads. Bars = 1 µm. c Immunofluorescent staining of CSpV1-dsRNAs using J-2 antibody in IEC4.1 cells following C. parvum infection (24 h) or in cells transfected with CSpV1-dsRdRp, CSPv1-dsCA, or a non-specific double-stranded siRNA (control RNA) for 24 h. Representative images from three independent experiments are shown. Blue: DAPI (DNA), red: C. parvum, green: CSpV1-dsRNAs (positive for J2 antibody, arrows). Bars = 1 µm. d–f Dot blots using J2 antibody to detect CSpV1-dsRNAs. Detection of in vitro-transcribed CSpV1-dsCA and CSpV1-dsRdRp (template plasmid DNA for in vitro transcription as control) (d); detection of CSpV1-dsRNAs in different doses of C. parvum sporozoite lysate (e); detection of CSpV1-dsRNAs in untreated IEC4.1 cells and cells infected by inactivated C. parvum or infective C. parvum or transfected with CSpV1-dsRNAs (f). Representative dot blots from three independent experiments are shown. g RIP assay for CSpV1-dsRNAs from IEC4.1 cells following C. parvum infection or CSpV1-dsCA or CSpV1-dsRdRp transfection. Cells were exposed to C. parvum or transfected with CSpV1-dsCA or CSpV1-dsRdRp for 24 h. CSpV1-dsRNAs were pulled down using J2 antibody. Isotype IgG was used as negative control. Data are from three biological replicates and presented as mean values ± SD. p values were determined by two-tailed unpaired Student’s t-test. Source data are provided as a Source Data file.