Fig 2: Expression of caspase-3 and Bcl-2 in Jurkat cells lacking vimentin or overexpressing vimentin. Jurkat cells were transfected with control siRNA, vimentin-specific siRNA or a plasmid expressing vimentin as described in the Materials and Methods. Cells were then cultured in serum-free DMEM in the presence or absence of LPS (10 µg/mL) for 24 h. Total cell lysates were analyzed via immunoblotting for caspase-3, Bcl-2 and ß-actin (as a loading control) as described in the Materials and Methods. Panel A: Representative immunoblots. Panel B: Semiquantitative comparison of target proteins with normalization. Vertical axes: protein level expressed as “Ratio versus control”, which was obtained as follows: (1). The densities of target protein versus internal control ß-actin were obtained. (2). The group of cells transfected with Con-siRNA and cultured in SF-DMEM was set as a reference (“Control = 1”), and the ratio of other groups versus this control group were obtained. Horizontal axes: cells transfected with control siRNA (Con-siRNA), vimentin-specific siRNA (Vim-siRNA) or vimentin expressing plasmid (Vim-vector). Open bar: cells were cultured in serum-free DMEM (SF-DMEM); closed bar: cells treated with 10 µg/mL LPS. #p < 0.05; *p < 0.05 compared to cells transfected with Con-siRNA and cultured in SF-DMEM. Data represent an average of 3 separate experiments.

Fig 3: Expression of mitochondrial membrane proteins and apoptosis- or autophagy-associated proteins in the HFL-1 cells lacking vimentin or overexpressing vimentin. The HFL-1 cells were transfected with vimentin-specific siRNA or a plasmid expressing vimentin as described in the methods section. The cells were then cultured in serum-free DMEM in the presence or absence of LPS (1 µg/mL) for 24 h. Total cell lysates were subjected to immunoblotting for COX II, TOM20, TIM 23, caspase-3, Bcl-2, and P62, with GAPDH as the loading control, as described in the methods section. (A) Representative immunoblotting. (B) Semiquantitative comparison of COX II. (C) Semiquantification of TOM20 expression. (D) Semiquantification of TIM23 expression. (E) Semiquantification of Bcl-2 expression. (F) Semiquantification of caspase-3 expression. (G) Semiquantification of P62 expression. (H) Semiquantification of LC3II expression. Vertical axes: grayscale of targeted proteins vs. GAPDH. Horizontal axes: HFL-1 control cells, cells treated with LPS, cells transfected with vimentin-specific siRNA (VIM-siRNA) or vimentin-expressing plasmid (VIM-pCMV3) plus LPS. Data are presented with significance denoted by *P<0.05.