Fig 2: CAF1 is a key partner of MIWI in piRNA-induced target repression. (A) Co-IP assays of the interaction between Myc-CAF1 and Flag-MIWI in GC-2spd (ts) cells. Anti-Flag IP (top) and anti-Myc IP pellets (middle) were respectively immunoblotted by anti-Myc and anti-Flag antibodies. Bottom, cell lysates immunoblotted by anti-Flag or anti-Myc antibodies, with ß-actin serving as a loading control. (B) GST pull-down assays of the interaction between CAF1 and MIWI. The GST-fused CAF1 was generated in bacteria and purified. [35S]-Met-MIWI protein prepared by in vitro transcription/translation was incubated with GST (lane 2) or GST-CAF1 (lane 3), respectively. Input represents 10% of [35S]-Met-MIWI (lane 1). Samples were analyzed by autoradiography (top) or visualized by Coomassie blue staining (bottom). (C) Co-IP assays of the interaction between endogenous CAF1 and MIWI in adult mouse testes. Anti-CAF1 IP pellets from RNase A untreated (lane 2) or treated testis lysate (lane 3) immunoblotted by anti-MIWI (top) and anti-CAF1 (bottom) antibodies, respectively. Immunoblot of MIWI or CAF1 in testis lysate (lane 1) or IgG IP pellet (lane 4) served as positive and negative controls, respectively. (D and E) The effect of Caf1 knockdown (D) or ectopic Myc-CAF1 (E) on piR_010111-induced Grk4 reporter repression in GC-2spd (ts) cells. Left, dual-luciferase reporter assays. Middle, PAT assays. Right, western blot assays of CAF1 proteins, with ß-actin serving as a loading control. (F) RIP combined with northern blot assays of pi-RISC complex assembly in GC-2spd (ts) cells. Cells were cotransfected with the indicated expression vectors and mouse test piRs plus Miwi siR (lanes 1-3) or test piR (lanes 4-12). Anti-Flag RIP (lanes 2, 5, 8, and 11) and anti-Myc RIP (lanes 3, 6, 9, and 12) were blotted with the probes of piR_010111, piR_013474, piR_027161, piR_035327, piR-1, piR-2, and piR-3. Total RNAs from respective transfected cells served as positive controls (lanes 1, 4, 7, and 10). The average values ± s.d. of three separate experiments were plotted. **P < 0.01, ***P < 0.001. Results shown are representative of three independent experiments.

Fig 3: pi-RISC is predominantly assembled in ES. (A) Co-IP assays of the interaction between CAF1 and MIWI in enriched-SC (lane 1), RS (lane 2), and ES (lane 3). Anti-MIWI IP (top) and anti-CAF1 IP pellets (middle) immunoblotted by anti-CAF1 and anti-MIWI antibodies, respectively. Bottom, respective cell lysates immunoblotted by anti-MIWI or anti-CAF1 antibodies, with ß-actin serving as a loading control. (B) Double-immunostaining of MIWI (red) and CAF1 (green) in SC (top), RS (middle), and ES (bottom). Nuclei were counterstained with DAPI (blue). Colocalization of MIWI and CAF1 was shown as yellow. Scale bar, 10 µm. Z-stacks (right) was used to project the 6.0-µm side view (x axis) of MIWI (red) CAF1 (green) in the lined region. (C) RIP assays of CAF1-associated RNAs in SC (lane 1), RS (lane 3), and ES (lane5), with IgG-IPed RNA as negative controls (lanes 2, 4, and 6), and immunoblot of IP pellets with anti-CAF1 as loading references (bottom). Results shown are representative of three independent experiments.

Fig 4: CAF1 knockout increases LPS-induced IL-8 and ICAM-1 protein levels.Notes: ELISA data indicated IL-8 levels in CAF1-knockout mice challenged with ns were comparable to those in wild-type mice (A). It revealed a great increase in IL-8 both in wild-type and CAF1-knockout mice following LPS stimulation, and IL-8 levels in CAF1-knockout mice were greatly higher than those in wild-type mice (A). Western immunoblot band density data showed that ICAM-1 levels in wild-type mice challenged with ns were comparable to those in CAF1-knockout mice (B). LPS stimulation greatly increased ICAM-1 expression in both wild-type and CAF1-/- mice compared to ns stimulation. ICAM-1 levels in CAF1-/- mice stimulated with LPS were greatly more than those in LPS-stimulated wild-type mice (B). ELISA and Western immunoblot band density data are expressed as mean ± SEM. n=3, *P<0.05 vs NS, #P<0.05 vs wild-type mice. An independent sample t-test was used.Abbreviations: WT, wild-type mice; KO, CAF1-knockout mice; ns, normal saline; LPS, lipopolysaccharide; ICAM-1, intercellular adhesion molecule-1; IL-8, interleukin-8; SEM, standard error of the mean; ELISA, enzyme-linked immunosorbent assay.

Fig 5: CAF1 knockout influences LPS-induced IL-8 and ICAM-1 mRNA expression. IL-8 and ICAM-1 mRNA levels in wild-type mice were comparable to those in CAF1-/- mice after NS stimulation and were greatly increased in both wild-type and CAF1-/- mice following LPS stimulation (A and B). IL-8 mRNA levels in CAF1-/- mice challenged with LPS were significantly higher than those in wild-type mice (A). ICAM-1 mRNA levels in CAF1-/- mice following LPS stimulation were significantly more than those in wild-type mice (B). The data are expressed as mean ± SEM. n=3, *P<0.05 vs NS, #P<0.05 vs wild-type mice. An independent sample t-test was used.(/P)(P)Abbreviations: WT, wild-type mice; KO, CAF1-knockout mice; ns, normal saline; LPS, lipopolysaccharide; ICAM-1, intercellular adhesion molecule-1; IL-8, interleukin-8; SEM, standard error of the mean.