Fig 2: Cleavage of raptor at the C-term DDADD (amino acids 939–943) residue and at the N-term DEADLTD (amino acids 17–23) residue. (a) Cartoon of raptor and its putative C-term caspase-mediated cleavage residues (UniProtKB: Q8N122). The recombinant N-term raptor protein (amino acids 1–379) is represented by the arrow. Different N-term tags (HA or GST) were used according to the raptor constructs. (b) Jurkat T cells were transfected with GFP, WT, DDAAA or AVAAKA raptor and treated with STS for 2 h. (c) Jurkat T cells were transfected with GFP, WT or DDAAA raptor and treated with FasL for 2 h. (d) Recombinant N-term raptor was incubated with recombinant caspase-6 for 2 h in the presence (or in the absence) of z-VAD-fmk. (e) Recombinant raptor was incubated with recombinant caspase-6 and directly inactivated at 95 °C for 10 min or incubated for 3 h at 37 °C. The samples were analyzed by tandem MS.

Fig 3: Dose-dependent effect of stearic acid (SA) on cell growth and viability in 1.1B4 cells (A). Effect of 1 mM SA on the level of caspase-2 (C2), cleaved caspase-9 (C9), cleaved caspase-8 (C8), cleaved caspase-7 (C7), cleaved caspase-6 (C6), cleaved caspase-3 (C3), and cleaved PARP in 1.1B4 cells, (B). Effect of 1 mM SA on the level of BiP and CHOP in 1.1B4 cells (C). Cells incubated without SA represented control cells. When assessing cell growth and viability (A), the number of living cells was determined after 48 h of incubation. Each column represents the mean of four separate cultures ± SEM. * p < 0.05 when comparing the number of control cells and cells treated with SA. The dotted line represents the number of cells of the inoculum. After 24 h of the incubation (B, C), the levels of individual proteins were determined using western blot analysis and the relevant antibodies (see “Materials and Methods”). Monoclonal antibody against human actin was used to confirm equal protein loading. The data shown were obtained in one representative experiment from at least three independent experiments

Fig 4: Representative western blots of selected markers of ER stress (CHOP, GRP78) and apoptosis (cleaved caspase-6, -7, -8, -9, and PARP).The picture shows the expression of chosen proteins in NES2Y cells exposed to DMSO as a solvent control (con) and 150 μM p,p‘-DDT for 24 and 30 hours. We used actin as a loading control. CHOP means C/EBP homologous protein, GRP78 means 78kDa glucose-regulated protein, and PARP means poly (ADP-ribose) polymerase.

Fig 5: Gene expression of microglial phenotype markers in WT primary microglia following LPS (10 ng/ml), Q-VD-OPh (20 µm), Q-VD-OPh + LPS, and caspase-6 KO control and LPS. Expression of genes indicative of M1: Cox-2 (A) and iNOS (B); M2a: CD206 (C) and IGF-1 (D), and M2b: IL-1ra (E) and SOCS3 (F), was normalised to WT control (black checked bars) for WT LPS (black bars), WT Q-VD-OPh (dark grey checked bars), WT Q-VD-OPh + LPS (dark grey bars) and caspase-6 KO LPS (white bars) was normalised to caspase-6 KO control (checked bars). For iNOS (B) gene expression, only LPS-treated cells were detected, so data were normalised to WT LPS. Data were analysed with a two-way ANOVA (for time and treatment) and, when significant, a Dunnett's post hoc analysis was conducted comparing with WT LPS treatment. ND = Not detected. Data are mean ± SEM (n = 4-5 independent experiments; iNOS: n = 3-4 independent experiments). Dotted line indicates 1. * p < 0.05.