Fig 1: mEMC10 maintains the homeostasis of cytoplasm sodium via positively regulating ATP1B3 in germ cells. (a) The protein levels of EMC10 and ATP1B3 in GC-1 and GC-2 cells after the transfection of siRNA targeting mouse Emc10 gene or control siRNA for 48 h. (b) Atp1b3 mRNA levels in GC-1 and GC-2 cells transfected with the indicated siRNAs for 48 h. (c) Cytoplasm sodium was determined using CoroNa Green, a sodium-sensitive fluorescent dye, in Emc10 siRNA or control siRNA-transfected GC-1 and GC-2 cells. (d) mEMC10 and ATP1B3 protein expression, (e) Atp1b3 gene expression, and (f) cytoplasm sodium in GC-1 and GC-2 cells transfected with a plasmid-encoding mouse membrane-bound EMC10 or control plasmid for 48 h. The levels of (g) ATP1B3 protein, (h) Atp1b3 mRNA, and (i) cytoplasm sodium in GC-1 and GC-2 cells transfected with different plasmids and siRNAs as indicated for 48 h. siCtr, control siRNA; siEMC10, EMC10 siRNA; pCtr, control plasmid; pmEMC10, mEMC10 plasmid. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001.
Fig 2: ATP1B3 expression and localization in male germ cells of mouse. (a) Immunohistochemistry analysis of ATP1B3 in the testis and epididymis tissues of mouse. Scale bar = 50 µm. (b) EMC10 and ATP1B3 protein expression measured in the testis, spermatozoa, GC-1, and GC-2 cells by Western blotting. (c) Immunofluorescence analysis of ATP1B3 localization (red) in spermatozoa from mouse epididymis (100×). Scale bar = 10 µm. Te, testis; Cap, caput epididymis; Cor, corpus epididymis; Cau, cauda epididymis; PNA-FITC (green), sperm acrosome; DAPI (blue), sperm nucleus.
Fig 3: The protein levels of EMC10 and ATP1B3 in human spermatozoa. (a–e) EMC10 and ATP1B3 protein expression were determined by Western blotting in 61 human sperm samples. (f) Based on the Western blotting results shown in (a–e), gray values of EMC10 and ATP1B3 were evaluated using ImageJ software version1.8.0 (National Institutes of Health, Bethesda, MD, USA). Correlation analysis of the levels of EMC10 and ATP1B3 protein in these samples were performed using Spearman’s analysis.
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