V binding buffer at a density of 1 106 cells/mL. Then, one hundred mixture was transferred into a brand new tube. Five microliter Annexin V-FITC and five PI have been added into the mixture followed by incubation for 15 min at space temperature in the dark. Another 300 Annexin V binding buffer was added into the mixture for further flow cytometry within 1 h employing FACSVerse flow cytometer (BD Biosciences). 4.7. RNA Extraction and RT-qPCR Total RNA was extracted from mouse tissues and spermatogonia using TRIzol reagent (Invitrogen). The concentration of total RNA was measured with a Nano-100 Nav1.2 Storage & Stability spectrophotometer (Allsheng, Hangzhou, China) as well as a 260/280 ratio of 2.0 was accepted as pure RNA. Then, applying PrimeScriptTM RT reagent kit with gDNA eraser (TaKaRa, Cat. # RR047A), 1 RNA was reverse transcribed based on the manufacturer’s directions. For RT-qPCR, a final volume of 20 reaction technique contained 10 SYBRPremix Ex TaqTM (TaKaRa, Cat. # RR420A), 1 cDNA, 0.4 forward primer (work concentration 0.two ), 0.four reverse primer (operate concentration 0.two ), 0.four ROX reference dye II, and 7.eight sterile distilled H2 O was prepared. RT-qPCR was carried out on an ABI 7500 Rapidly Real-Time PCR system (Applied Biosystem, Waltham, MA, USA). Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was employed as an internal reference gene. Relative mRNA expression was detected by normalizing gene expression against GAPDH expression making use of 2-ddct system [40]. The primer sequences applied for RT-qPCR are shown in Table S1. Benefits are presented as fold adjustments relative to the manage groups. 4.8. Western Blotting Mouse tissues and spermatogonia have been lysed working with protein extraction reagent containing protease and phosphatase inhibitor (Thermo Scientific, Waltham, MA, USA). The protein extracts were denatured, electrophoresed on ten SDS-PAGE gel, then transferred (blotted) to nitrocellulose membranes. The PPARβ/δ manufacturer membranes were blocked with 5 nonfat milk for 1 h at space temperature then incubated with principal antibodies overnight at 4 C. The key antibodies utilized were PRMT7 (D1K6R) Rabbit mAb (1:1000, #14762), Cyclin A2 (BF683) Mouse mAb (CCNA2, 1:1000, #4656), Cyclin B1 (D5C10) XPRabbit mAb (CCNB1, 1:1000, #12231), cdc2 (E1Z6R) Rabbit mAb (CDK1, 1:1000, #28439), CDK2 (78B2) Rabbit mAb (1:1000, #2546), P53 (1C12) Mouse mAb (1:500, #2524), BCL2 (D17C4) Rabbit mAb (1:500, #3498), Phospho-Akt (Ser473) Antibody (P-AKT, 1:1000, #9271), -ActinInt. J. Mol. Sci. 2021, 22,12 of(13E5) Rabbit mAb (1:1000, #4970), GAPDH (14C10) Rabbit mAb (1:1000, #2118) from Cell Signaling Technologies, AKT Mouse mAb (1:2000, #60203-2-Ig) and BAX Mouse mAb (1:2000, #60267-1-Ig) from Proteintech. Later, the membranes have been incubated with secondary antibody (Anti-rabbit IgG, HRP-linked antibody, 1:3000, #7074 or Anti-mouse IgG, HRP-linked antibody, 1:3000, #7076) for 1 h at space temperature. The blots have been created employing Pierce ECL Western Blotting Substrate according to the manufacturer’s directions (Pierce) plus the protein bands have been visualized on a Tanon-5200 Chemiluminescent Imaging System (Shanghai, China). 4.9. Library Preparation for RNA Sequencing Total RNA was extracted from spermatogonia transfected with Prmt7 siRNA or NC siRNA making use of TRIzol reagent (Invitrogen). A total volume of 1 RNA per sample was employed as input material for the RNA library. Sequencing libraries for RNA were generated using NEBNextUltraTM RNA Library Prep Set for Illumina(New England Biolabs, Ipswich, MA, USA) following
