Fig 2: Knockout of SEC61A1 suppressed the mycolactone-induced ER stress response and expression of proapoptotic genes.Control EGFP-knockout and SEC61A1-knockout THP-1 cells were treated with 30 ng/mL mycolactone for 6 h (ATF4, DDIT3 and PMAIP1) or 48 h (BBC3 and BCL2L11). (A) The mRNA levels of ER-stress-related genes (ATF4 and DDIT3) and (B) proapoptotic genes (PMAIP1, BBC3 and BCL2L11) were analyzed by real-time RT-PCR. mRNA levels are expressed relative to those of ethanol-treated EGFP-knockout or SEC61A1-knockout THP-1 cells (n = 3). (C) The mRNA levels of unspliced, spliced and total XBP1 using control EGFP-knockout and SEC61A1-knockout THP-1 cells treated with 30 ng/mL mycolactone for 0, 3, 6, 12, and 24 h. mRNA levels are expressed relative to those of mycolactone-treated EGFP-knockout THP-1 cells (n = 3). *: p < 0.05; **: p < 0.01; ***: p < 0.005. (D) Control EGFP-knockout and SEC61A1-knockout THP-1 cells were treated with 30 ng/mL mycolactone for 24 h. ER stress proteins (p-eIF2α, eIF2α, p-PERK, PERK, full length ATF6, cleaved C-ATF6, and IRE1α) and SEC61A1 were evaluated by Western blotting. 1 μM thapsigargin for 24 h was included as a positive control for ER stress.

Fig 3: Extracted rough microsomes (RM) from snap-frozen pancreatic tissue contain ER translocon subunits and associating factors. Western blot analysis of Sec61 translocon subunits and ER translocation-associating factors in the extracted RM samples from 17 different dogs. Selected proteins were identified by western blotting with conditions optimized for each individual primary antibody. Representative images are shown for the following proteins: α subunit of the Sec61 translocon (predicted MW 52 kDa), β subunit of the Sec61 translocon (MW 10 kDa), α subunit of TRAP (MW 32 kDa), BiP (MW 72 kDa), and 60S ribosomal protein L26 (RPL26; MW 17 kDa). The numbers at the top of the figure refer to the ID of each dog sample as in Table 3. The first and last lanes show the MW marker (in kDa).