Fig. 3
From: Novel insights into PORCN mutations, associated phenotypes and pathophysiological aspects
Immunological studies of ER-homeostasis and protein clearance on fibroblasts: A Immunoblot studies revealed increase level of GRP170, GRP94, BiP and peIF2a in protein extracts derived from the PORCN-patient compared to controls. Immunoblot studies of PORCN revealed an increase along with the presence of an additional band of lower molecular weight compared to the analysis of the PORCN protein in control fibroblasts. Additionally, immunoblot studies of protein-ubiquitination revealed an increase in patient-derived fibroblasts along with a decrease of LC3-I to LC3-II conversion. Coomassie blue staining was performed to demonstrate equal protein loading. B Immunofluorescence studies of SEC62, SEC63, two ER-membrane resident proteins, revealed a blurred immunoreactivity in fibroblasts derived from the patient compared to the respective protein distributions observed on control cells. Immunofluorescence of GRP170 revealed a cytoplasmic punctuate accumulation in patient-derived fibroblasts (white arrow) compared to the reticular protein distribution detected in control cells. Immunofluorescence studies of PORCN showed focal cytoplasmic accumulations in patient-derived cells (white arrows) compared to the observed immunoreactivity in control cells. Scale bars = 30 µm. C Immunoblot-based investigation of ER-stress response of Tunicamycin treatment (− = without treatment, + = with treatment) focussing on the three major transducers of the UPR revealed increased phosphorylation of PERK and IRE1 in patient-derived fibroblasts whereby on PERK-phosphorylation was enhanced after Tunicamycin-treatment. Along this line, increased proteolytic cleavage of ATF6 toward its activation is visible in patient-derived cells. This effect is not significantly further pronounced in patient-derived cells after Tunicamycin-treatment. Controls were merged in the diagrams. D Immunoblot-based investigation of ER-stress response of Tunicamycin-treatment (− = without treatment, + = with treatment) focussing on down-stream factors revealed a more pronounced increase of BiP and GRP170 in patient-derived cells in treated cells. Whereas GRP94 showed an increased in stressed control fibroblasts, patient-derived cells did not present with elevated level after stressing. Along this line, increase of Calnexin in stressed patient fibroblasts was less pronounced compared to control cells. In stressed patient-derived fibroblasts VAPB-level were decreased whereas in control cells level remained unchanged after Tunicamycin-application. Controls were merged in the diagrams. E Results of MTT-assay based studies focussing on cellular metabolic activity. Left panel visualizes the metabolic activity of control and patient-derived cells under basal conditions (white box plots) and after Tunicamycin-treatment (grey box plots). AU: arbitury unit. Right panel: Tunicamycin-treatment results in a slight decrease of cytotoxicity in control fibroblasts (most likely based on the activation of compensatory mechanisms) whereas a profound increase of cytotoxicity is detectable in PORCN-patient derived cells. F Studies of proteasomal activity in control (blue and orange) and patient-derived (grey and yellow) cells reveal an activity-decrease under basal conditions. In comparison to control cells, patient-derived cells do not show a fundamental decrease of this activity after MG132-treatment. Y-axis: 350/440 nm; X-axis: time points in 5 min intervals between measurements