Fig. 3.

Piperine reduced ER stress caused by mutant TBP. a, b Stable transfected PC12 cells expressing TBP-13Q (a) or TBP-105Q (b) were treated with different concentrations (0, 2 or 10 μM) of piperine for 48 h. Different concentrations of tunicamycin (0, 2.5 or 10 μM) were then added for 4 h. Western blotting was performed to examine the levels of XBP1s and CHOP. Untreated cells (ctl) were used as controls. GAPDH was used as a loading control. The ratios of XBP1s or CHOP to GAPDH are presented beneath the blots (n = 3, * P < 0.05). c, d PC12 cells expressing TBP-13Q (c) or TBP-105Q (d) were transfected with either MANF (M) or empty vector (C), followed by treatment with 2.5 μM of tunicamycin for 4 h. Western blotting was performed to check the levels of XBP1s and CHOP. Actin was used as a loading control. Quantification of the ratios of XBP1s or CHOP to actin is presented beneath the blots (n = 3, * P < 0.05, ** P < 0.01). e N2a cells were transfected with either MANF-sgRNA and Cas9 (MANF-sgRNA +), or control-sgRNA and Cas9 (MANF-sgRNA -), treated with 2 μM of piperine for 48 h. Different concentrations of tunicamycin (2.5 or 10 μM) were then added for 4 h. Western blotting was performed to examine the expression of MANF and CHOP. Actin was used as a loading control. f Quantification of western blotting result in Fig. 3e. MANF KD (knockdown) was used to indicate cells transfected with MANF-sgRNA and Cas9 (n = 3, * P < 0.05, ** P < 0.01). Results are presented as mean ± SEM