Human rna panel

To generate human testicular complementary DNA (cDNA), human testicular RNA was obtained from a human RNA panel (Clontech, Mountain View, CA, USA). Total testicular cDNA was generated from this RNA by Superscript^™ III Reverse Transcriptase (Invitrogen), which was stored at −20 °C until use, as described in our previous study [39 (link)]. Full-length SEPT14 transcripts were amplified and cloned into the pFLAG-CMV2 plasmid. The construct was confirmed by Sanger sequencing. Next, NTERA-2 cl.D1 (NT2D1) cells (ATCC, Manassas, VA, USA), a pluripotent human testicular embryonal carcinoma cell line, or HeLa cells were transfected with these plasmids using Lipofectamine reagent (Cat No.: 11668; Invitrogen, Carlsbad, CA, USA). The protocols used were published in our previous studies [38 (link),43 (link)]. Total cell lysates were then collected for co-immunoprecipitation (co-IP).

Human SEPT12 and NDC1 were amplified from a human RNA panel (Clontech, Mountain View, CA, USA) and cloned into pEGFP-N1 and pFLAG-CMV2 vectors, as described previously [33 (link)]. NT2D1 cells were transfected with the vectors through lipofection. Overexpression of NDC1 or co-transfection with SEPT12 was replicated three times and more than 100 cells were counted per assay. The cells lysates and the antibodies (Anti-GFP antibody: sc-9996, Santa Cruz Biotechnology Inc.; anti-FLAG antibody: F1804, Sigma-Aldrich, St. Louis, MO, USA; Anti-NDC1 antibody: sc-161929, Santa Cruz Biotechnology Inc.) were used in the Co-IP and IB assays, by employing the procedure described in our previous study [33 (link)].

Human SEPT7 and PKA were amplified from a human RNA panel (Clontech, Mountain View, CA, USA) and cloned into pFLAG-CMV-2, pEGFP-C3, and pCDNA3-HA vectors, as described previously [27 (link)]. All constructs were verified by DNA sequencing. For transient transfection, malignant human testis pluripotent embryonic carcinoma NT2/D1 cells, REP cells, and human embryonic kidney 293 T cells were incubated in Dulbecco’s minimal essential medium (DMEM), supplemented with 10% fetal bovine serum (FBS) and 1% antibiotics. Cells were transfected with plasmids using Lipofectamine 2000 (Thermo Fisher Scientific, Waltham, MA, USA) according to the manufacturer’s instructions. After 24 h, the cells were subjected to immunofluorescence staining or immunoblotting. Alternatively, cells were treated with 8-bromo-cAMP or Na₃VO₃ (Sigma-Aldrich, Darmstadt, Germany) as indicated for analysis of protein expression.