Fig 2: Screening proteins that interact with sperm calcineurin. (A) Mouse proteins with PxIxIT, PxIxIN, or PxIxID that are expressed strongly in the testis were searched. There are eight genes that are included in both categories. (B) Testis-enriched genes that possess PxIxIT motif. Cklf, Spata33, and Tex43 were analyzed further. (C) RT-PCR of Cklf, Spata33, and Tex43 using RNAs obtained from mouse various tissues. Actb was the control. Br: brain, Th: thymus, Lu: lung, He: heart, Li: liver, Sp: spleen, Ki: kidney, Te: testis, Ov: ovary. (D) In silico data analysis of Ppp3cc, Ppp3r2, Cklf, Spata33, and Tex43 expression level. A1-A2: A1 and A2 differentiating spermatogonia, A3-A4: A3, A4, In, and B differentiating spermatogonia, Pre: preleptotene spermatocytes, Lep: leptotene/zygotene spermatocytes, Pac: pachytene spermatocytes, Dip: diplotene/secondary spermatocytes, Early: early round spermatids, Mid: mid round spermatids, Late: late round spermatids, Sertoli: Sertoli cells, Peritub: peritubular myoid cells, and Leydig: Leydig cells.

Fig 3: SPATA33 interacts with sperm calcineurin via PQIIIT sequence. (A) Spata33-PA (PQIIIT) or mutated Spata33-PA (AQIIIT, PQAIIT, or PQIIAT) was coexpressed with Ppp3cc-FLAG/Ppp3r2 in HEK293T cells, and immunoprecipitation (IP) with FLAG M2 antibody was performed. GAPDH was the control. (B) PPP3CC and PPP3R2 decreased in the spermatozoa from Spata33-11/-11 mice. BASIGIN was the control. (C) SPATA33 decreased in the spermatozoa from Ppp3cc-/- mice. BASIGIN was the control. The asterisk indicates a nonspecific band.

Fig 4: MiR-146a targets Tgif1, Camk2d and Ppp3r2. (a) Total RNA of mouse articular chondrocytes transfected with miR-146a inhibitor or control (NC) inhibitor was used for microarray analysis. Twelve genes were validated based on the seed region of miR-146a in 3'-UTR of candidate gene that is evolutionarily conserved in mammals. The gradient colors and numbers indicate fold change of gene expression in mouse chondrocytes transfected with the miR-146a inhibitor compared with control, as determined by microarray. Fold 146a/NC, fold change of gene expression in mouse chondrocytes transfected with miR-146a inhibitor compared with control inhibitor. (b) qPCR analysis of mRNA expression of candidate target genes in mouse articular chondrocytes transfected with miR-146a mimic or control mimic plasmid (n=3 for each treatment). (c and d) Luciferase activity analysis in HEK293T cells that were co-transfected with the indicated 3'-UTR reporter (c) or with the indicated WT or mutant 3'-UTR reporter (d) along with miR-146a mimic or control mimic plasmid (n=3 for each treatment). The results were normalized by internal firefly luciferase readout. (e-g) qPCR analysis of mRNA expression of Tgif1 (e), Camk2d (f) and Ppp3r2 (g) in articular cartilage of mice (WT, n=6; miR-146a KO, n=8) collected at 4 weeks after DMM surgery. (h and i) Western blot analysis of endogenous Sox9 and Col2a1 protein expression in mouse chondrocytes that were transfected with indicated expression vector constructs (h) or with gene-specific siRNAs as well as a scrambled control siRNA (siRNA-NC). For quantification, protein expression was normalized by the protein amount in the first lane. (i) Three independent experiments were performed with similar results in b-d. Relative mRNA expression was normalized to the expression of GAPDH. Data are mean±S.D. *P<0.05, **P<0.01 versus control group (Student's t-test) in b, c and d and Mann–Whitney test in e-g. NS, not significant between the indicated groups

Fig 5: Camk2d and Ppp3r2 are direct targets of miR-146a in vivo. (a-e) Immunohistochemical staining and quantitative analysis of the percentage of Camk2d, Ppp3r2, Nfatc1 and Nfatc2-positive chondrocytes in knee joint cartilage of 3- and 12-month-old WT or miR-146a KO mice. n=8 per group. Scale bars, 100 µm. (f-h) Immunohistochemical staining and quantitative analysis of the percentage of Camk2d and Ppp3r2-positive chondrocytes in knee joint cartilage harvested from the indicated groups. n=8–9 per group. Scale bars, 100 µm. Data are the mean±S.D. *P<0.05, **P<0.01 (Mann–Whitney test). NS, not significant between the indicated groups