Fig. 3

Trehalose induced autophagy-associated gene expression through reducing THBS1 expression and function in hTM cells. To study the effect of trehalose on THBS1 expression, cultured hTM cells were treated with 100 mM trehalose and vehicle control for 48 h. Quantification of relative mRNA levels was performed by RT-qPCR, and western blot was used for quantification of protein levels. (a) Trehalose (100 mM) reduced THBS1 mRNA expression by 57% in hTM cells. (b) Representative of three individual western blots of THSB1 expression in hTM cells after trehalose treatment. The original blot is presented in Supplementary Fig. S5. (c) The normalised THBS1 protein level (n = 3 different cell strains) as shown in (b). Trehalose (100 mM) reduced THBS1 protein level by 58% in hTM cells. To investigate the relevance of THBS1 in trehalose-induced autophagy in hTM cells, cultured hTM cells were transfected with 100 pmol of siCtl or siTHBS1 for 24 h, followed by 48 h of 100 mM trehalose treatment. Gene expression of (d) THBS1, (e) ATG5, (f) ATG7, (g) NFE2L2 and (h) SQSTM1 in siCtl- and siTHBS1-silenced hTM cells, with or without trehalose treatment, were evaluated by RT-qPCR. Data represent mean ± SEM. Statistical significance relative to vehicle control (unpaired Student’s t-test) is indicated by *(P < 0.05), ***(P < 0.01) and ***(P < 0.001). Tre: trehalose; siCtl: negative control siRNA; siTHBS1: THBS1-siRNA.