Fig. 5: Influence of persistent GALNTL5 expression on GC-2spd(ts) cells. | Cell Death Discovery

Fig. 5: Influence of persistent GALNTL5 expression on GC-2spd(ts) cells.

From: GALNTL5, which is restricted to mouse spermatids, impairs endoplasmic reticulum (ER) function through direct interaction with ER chaperone proteins

Fig. 5

A Continuous culture of GALNTL5-Flag/GC-2spd cells with or without cumate solution for 3 days. The N-glycosylated products of GALNTL5-Flag were detected for 3 days in lanes indicating chemical induction (+). Signal intensities of calnexin and of histone H3 nuclear protein as a control were almost identical at 3 days, with or without chemical induction. With accumulation of GALNTL5 in GALNTL5-Flag/GC-2spd cells, the levels of three ER proteins (BiP, UBE2J1, and CREB3L4), Golgi proteins, (GM130 and GOPC), and cytoplasmic proteins (HSC70, β-actin, and GAPDH) were hardly detected by western blotting after 3 days. B Continuous culture of GALNTL5Quad-Flag/GC-2spd cells with or without cumate solution for 3 days. In GALNTL5Quad-Flag/GC-2spd cells, signals of calnexin or of histone H3 as a control were also detected uniformly for 3 days, with or without chemical induction. The levels of the three ER marker proteins, Golgi markers, and cytoplasmic markers were slightly decreased after induction for 3 days. Histograms representing signal intensities detected with anti-calnexin antibody (C), anti-BiP antibody (D), anti-GOPC antibody (E), or anti-GAPDH antibody (F), in continuous culture of GALNTL5-Flag/GC-2spd cells and GALNTL5Quad-Flag/GC-2spd cells with or without cumate solution for 3 days. All values are means ± SEM (error bars, n = 3). Two-tailed Student’s t-test with or without cumate treatment. *p < 0.05, ***p < 0.001. G siRNA knockdown of calnexin or BiP in GC-2spd(ts) cells. Knockdown with calnexin-specific siRNA decreased the amount of BiP protein (BiP panel in the calnexin lane). Knockdown of BiP had almost no effect on calnexin levels (calnexin panel in the BiP lane). Knockdown of both calnexin and BiP decreased the expression of component proteins in the ER, Golgi apparatus, and cytoplasm. Histone H3 was used as the control. Quantification of band intensities detected with anti-calnexin (H), anti-BiP (I), anti-GOPC (J), and anti-GAPDH (K) antibodies in three categories of GC-2spd(ts) cells: 72-h transfection with control, calnexin or BiP siRNA. The error bars are presented as mean ± SEM (n = 3). ***p < 0.001.

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