The code files import the data files and exactly reproduce Figure 10

The code files import the data files and exactly reproduce Figure 10. different spermatogenic arrest states, as described in Spiess et al. (Hum Reprod transcripts are expressed predominantly in spermatocytes and spermatids in mouse, and are reduced in men with impaired spermatogenesis. DOI: http://dx.doi.org/10.7554/eLife.08916.001 and GnT1IP/genes in male Sertoli and germ cells, and show that transcripts of human GnT1IP/are markedly reduced in testis biopsies of men with impaired spermatogenesis. Results GnT1IP-L inhibits MGAT1 via its luminal domain To investigate whether the TM or luminal domain of GnT1IP-L is important for inhibition of MGAT1 in CHO cells, different mutant and chimeric expression plasmids were constructed (Figure 1 and Table 1). Constructs were transfected into CHO cells and stable populations selected for hygromycin resistance were examined for resistance to the toxicity of leukoagglutinin (L-PHA), and/or binding of the lectin agglutinin (GNA). Resistance to L-PHA, accompanied by increased expression of cell surface oligomannose N-glycans detected by GNA, are hallmarks of inhibition of MGAT1 activity in CHO cells (Chen and Stanley, 2003; Huang and Stanley, 2010). The subcellular localization of each construct was investigated by transient transfection of HeLa cells and analysis of immunofluorescence using antibodies to Myc or HA, Golgi -mannosidase II (MAN2A1), or GM130, or ER protein disulfide isomerase (PDI). In initial experiments, five Phe residues in the GnT1IP-L TM domain were all replaced with either Leu (similar hydrophobicity index to Phe) or Ala (hydrophobicity reduced 50% compared to Phe or Leu). Transfectants expressing GnT1IP-L(F/L) or GnT1IP-L(F/A) (Table 1) at similar levels based on western analysis, had an increased ability to bind GNA, and exhibited resistance to the toxicity of L-PHA (Figure 2B and data not shown). Thus, replacement of five Phe residues with Ala in the TM domain of GnT1IP-L did not markedly reduce its MGAT1 inhibitory activity. Open in a separate window Figure 1. Expression constructs.Mouse GnT1IP-L (417 aa) contains an N-terminal cytoplasmic domain of 48 aa, a transmembrane (TM) domain of 21 aa (shaded), and a luminal domain of 348 amino acids. The location of the Myc tag (red) is shown for each construct. Chimeric constructs contained the cytoplasmic and TM domain of MGAT1 (green) linked to the luminal domain of GnT1IP-L (blue), or the cytoplasmic and TM domain of GnT1IP-L linked to the luminal domain of MGAT1, or N-terminal aa 1C47 of human Invariant chain p33 (Iv; beige) linked to aa 45 to 417 of ML604440 GnT1IP-L. Predicted TM domains are shown in darker colors. Numbers on top of each chimera are aa from ML604440 the N-terminal domain and underneath are aa from the luminal domain. DOI: http://dx.doi.org/10.7554/eLife.08916.003 Table 1. Primers for expression constructs DOI: http://dx.doi.org/10.7554/eLife.08916.004 GnT1IP-L-Myc?For: 1301: (Kozak) CAGATCKozak, HA-GnT1IP-L) GGAACTMyc-KDEL) Kozak) GGACCGgene is very highly expressed in mouse testes compared to all other tissues (see BioGPS microarray data [Wu et al., 2009, 2013]). In mouse germ cells, expression of GnT1IP/based on microarray and RT-PCR data is very low in ML604440 spermatogonia, highest in spermatocytes and intermediate in spermatids (Chalmel et al., 2007; Huang and Stanley, 2010). This expression pattern ZNF538 in mouse germ cells is complementary to that is high in spermatogonia, and greatly reduced in spermatocytes (Chalmel et al., 2007). Very similar results are evident from an analysis of mouse RNA-Seq data that we interrogated for GnT1IP/and transcripts (Gene Expression Omnibus Dataset “type”:”entrez-geo”,”attrs”:”text”:”GSE43717″,”term_id”:”43717″GSE43717; [Soumillon et al., 2013a, 2013b]). Mapping the relative expression values of GnT1IP/(ENSMUSG00000035057) and (Figure 9, blue) is exclusively expressed in post-meiotic germ cells (Figure 9source data 1). In contrast, (Figure 9, red) is expressed at lower levels in all germ cell types, as well as somatic Sertoli cells. These results, as well as the observation that ML604440 antibodies to rat GnT1IP (GL54D) detect signals in spermatocytes and spermatids but not spermatogonia (Au et al., 2015), suggest post-meiotic transcriptional activation of the GnT1IP/gene. Interestingly,.