Fig 1: Study of cellular SIL1 immunoreactivity and presence of ER-stress in Hek293 cells overexpressing SIL1 or its variant forms. (A) Immunofluorescence-based investigation of the SIL1-immunoreactivity pattern in Hek293 cell revealed a reticular distribution for WT-SIL1, p.R92W and p.K132Q variant forms of SIL1 but a non-reticular rather “clumpy” immunoreactivity for p.G312R and p.L457P mutant forms of SIL1. For p.V231_I232del mutant SIL1, a reticular distribution with focal accumulations was observed. (B) EM revealed widened and proliferated ER in cells overexpressing the mutant forms p.G312R and p.L457P, but not in cells overexpressing WT-SIL1, p.R92W, p.K132Q, and p.V231_I232del (6 h of induced overexpression) forms of the proteins. In addition, accumulation of electron dense material could occasionally be seen in cells overexpressing p.G312R and p.L457P muatnt SIL1 (black arrows). Along this line, overexpression of these two known missense mutants of SIL1 resulted in the build-up of electron dense material (most likely corresponding to aggregated protein) in the cytoplasm as well as in (autophagic) vacuoles. The white arrow highlights an abnormal centrosome in a Hek293 cell overexpressing p.G312R mutant SIL1. (C) Immunoblot-based studies aimed to investigate the presence of ER-Stress by focussing on marker proteins for activation of the unfolded protein response (GRP170, IRE1, PERK, GRP94, ERj3 & pEif2α), the ER-associated degradation pathway (VCP, DNAJB6) and proteolysis (Beclin). Results revealed a considerable presence of ER-stress in Hek293 cells overexpressing p.L457P mutant SIL1 than in cells overexpressing p.G312R mutant SIL1 where solely pEif2α and Beclin are increased compared to cells overexpressing WT-SIL1. p.R92W, p.K132Q variant SIL1, and p.V231_I232del mutant SIL1 (6 h of induced overexpression) do not show remarkable changes in abundances of the stress markers compared to cells overexpressing WT-SIL1. GAPDH was investigated to demonstrate equal protein loading. The differences in expression of these markers between C-TAP and WT-SIL1 overexpressing Hek293 cells have been described previously (12).