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. 2022 Aug 8;18(13):5136–5153. doi: 10.7150/ijbs.76261

Figure 5.

Figure 5

CircLIMA1-dependent sperm nuclear actin organization along the epididymis. qRT-PCR detection of circLIMA1 expression levels in caput and cauda SPZ (A) or epididymis (B) from WT and CB1-/- mice. (C) Immunofluorescence analysis of F-actin by phalloidin staining in caput and cauda SPZ from WT and CB1-/- mice. qRT-PCR detection of circLIMA1 expression levels (D) and Immunofluorescence analysis of F-actin by phalloidin staining (E) in caput SPZ from WT mice in vitro co-incubated with: WT Caput ELF (CTRL group), WT Caput ELF combined with Cytochalasin-D (C8273; Sigma-Aldrich, Milano, Italy) at the concentration of 10 μM (ELF WT+CYTOD); CB1-/- Caput ELF (ELF CB1-/-); CB1-/- Caput ELF combined with Cytochalasin-D 10 μM (ELF CB1-/- +CYTOD); (n=3 different samples for each experimental group from 8 different animals in triplicate). qRT-PCR data are normalized using Cyclophilin and RPS18, for SPZ and epididymis respectively, expressed as DeltaCq and reported as mean value ± S.E.M. Experimental groups with statistically significant differences (p<0.05; p<0.01) were indicated with different letters; the experimental groups without statistically significant differences were indicated with the same letter. White full arrowheads and asterisks represent F-actin localization (red) in sperm head and tail, respectively. Nuclei were labeled with DAPI (blue). Scale bar: 15 µm (F) Immunofluorescence analysis of QKI protein in caput and cauda SPZ from WT and CB1-/- mice. White full arrowheads and asterisks represent QKI localization (green) in sperm head and tail, respectively. Nuclei were labeled with DAPI (blue); F-actin was labeled with phalloidin staining (red). Scale bar: 15 µm (G-H) The enrichment levels of circLIMA1 in the products of RIP assay (QKI-IP compared with IgG-IP) in caput and cauda SRN from WT (G) and CB1-/- (H) mice detected by qRT-PCR. Data are reported as mean ± SEM from three independent experiments. **p<0.01. (J-K) Western blot analysis of RIP protein fraction immunoprecipitated with QKI Ab (QKI-IP) in caput and cauda SRN from WT and CB1-/- mice. QKI-IP was analyzed in comparison to control IgG-IP and Input protein extracts.