Fig 1.
NKAPL expression and localization analyses.
(A) RT-PCR analysis of systemic tissue. The targeted amplicon was 471 bp. (B) Immunoblotting analysis of systemic tissue protein. (C) RT-PCR analysis of testis by age. RT+ or RT- represents the presence or absence of reverse transcription, respectively. M: DNA size marker. (D) Immunoblotting of testis by age. (E) Immunohistochemistry of adult testis with Nkapl antibody. Black scale bar = 50 μm. (F) Immunofluorescence of adult testis with Nkapl, TRA98, E-cadherin, and c-kit antibodies. White arrows indicate E-cadherin-positive undifferentiated spermatogonia, and white arrowheads represent c-kit-positive differentiating spermatogonia. White scale bar = 50 μm.
Fig 2.
Nuclear localization signal (NLS) analyses of NKAPL in vitro.
(A) Immunofluorescence with GFP antibody after transfection of EGFP-tagged vectors into 293T cells. Upper row shows images of GFP expression, and lower row shows those merged with DAPI staining to visualize nuclei. White scale bars = 50 μm. (B) The upper bar represents the open reading frame (ORF) of the Nkapl gene, and the lower bars illustrate the fragmented sequences of the Nkapl gene, which were inserted into EGFP-tagged vectors. Numbers over the bars indicate the fragmented sites on the ORF. Black regions indicate the sequence including the putative NLS signal amino acids predicted by cNLS Mapper prediction program. (C) Immunofluorescence of GFP antibody after the transfection of EGFP-tagged fragmented Nkapl. Left columns show the immunofluorescent images with GFP antibody, and the right columns show those merged with DAPI staining.
Fig 3.
NKAPL interacts with other Notch signaling co-repressors and suppresses downstream transcription of all Notch family receptors.
(A-C) interactions between Nkapl and CIR (Corepressor interacting with RBPJ), HDAC3 or CSL (Cbf1/Rbp-jk) by immunoprecipitation (IP) and immunoblotting (WB) with whole-cell extract to confirm expression of co-transfected genes. Nkap was used as positive control. (A) Interaction of NKAP and NKAPL with CIR (Corepressor interacting with RBPJ). (B) Interaction of NKAP and NKAPL with HDAC3. (C) Interaction of NKAP and NKAPL with CSL (Cbf1/Rbp-jk). (D)The Luciferase assay of transcription levels with pEluc-CSL and pcDNA-Notch 1 to 4 co-transfected NIH3T3 cells. Mock represents co-transfection with no insertion of pcDNA vector. (E) The luciferase assay after co-transfection with pEluc-CSL and pcDNA-Notch1 to 4 and pCAGGS-Nkap or Nkapl vectors. Mock represents co-transfection with no insertion of pCAGGS vectors. Error bars show standard deviation (SD) from the means.
Fig 4.
NKAPL suppresses the transcription of downstream target genes through Notch signaling and plays critical roles in spermatogonial stem cell (SSC) maintenance and differentiation.
(A) Immunoblotting for proteins extracted from testis or GS cells. Black arrowheads represent the specific reaction for Nkapl antibody. Asterisks represent non-specific reactions. (B) Immunoblotting of GS and GS-Nkapl cells to confirm the expression of the lentiviral-induced Nkapl gene. The reaction with FLAG antibody demonstrates its expression. Black arrows represent the specific reactions by each of the antibodies. (C) The transcriptional changes of Notch1 to 3 between GS and GS-Nkapl cells by qRT-PCR. (D) The transcriptional changes of Hes1 and Hes5. (E) The transcriptional changes of SSC markers. (F) The transcriptional changes of differentiation-associated factors. Error bars indicate standard deviation from the means. *P<0.05.
Fig 5.
NKAPL deletion caused complete maturation arrest at meiosis through elevation of the Notch signaling pathway.
(A) Nkapl mRNA transcription in testis of genetically modified mice by Northern blotting. (B) NKAPL expression in testis of genetically modified mice by immunoblotting. (C) Macroscopic appearances of testes in Nkapl+/- and Nkapl-/- mice. (D) Histology of testis and epididymis stained with hematoxylin and eosin. (E) Immunohistochemistry of Nkapl antibody for testes of Nkapl+/- and Nkapl-/- mice. (F) Transcriptional changes of Hes1 and Hes5 between testes of Nkapl+/- and Nkapl-/- mice by qRT-PCR. (G) Transcriptional changes of Notch1 to 4. Error bars indicate standard deviation from the means. *P<0.05.
Fig 6.
Nkapl deletion caused significant apoptosis at the level of pachytene spermatocytes due to aberrant changes of meiosis-related genes.
(A) Immunohistochemistry of cleaved caspase-3 antibody for testes of Nkapl+/- and Nkapl-/- mice to detect apoptotic cells. (B) The average counts of apoptotic cells per seminiferous tubule. One hundred seminiferous tubules were randomly selected, and cleaved caspase-3-positive germ cells were counted. Error bars are standard deviation (SD) from the means. (C) Immunohistochemistry of MCA in testes of Nkapl+/- and Nkapl-/- mice. (D) Transcriptional changes of meiosis-related genes between testes of Nkapl+/- and Nkapl-/- mice by qRT-PCR. Error bars indicate SD from the means. *P<0.05.
Fig 7.
Nkapl deletion caused significant changes in spermatogonial stem cell (SSC) maintenance markers and increases in differentiation-related factors.
(A) Transcriptional changes of SSC maintenance markers between testes of adult Nkapl+/- and Nkapl-/- mice by qRT-PCR. (B) Transcriptional changes of differentiation-related factors. Error bars indicate standard deviation from the means. *P<0.05.