Fig. 7: Chemical inactivation of PGRMC1 stabilizes WT and mutant proinsulin, enhancing WT proinsulin export from the ER.

a INS-832/13 cells expressing A16P-Myc were treated with either DMSO or AG-205 for 2 h, and subsequently treated with cycloheximide for 0, 1, 2, 3 h. Cells were lysed, and the lysate subjected to SDS-PAGE and immunoblotting as indicated. N = 3 independent experiments. b Quantification of the steady-state A16P-Myc level (at T = 0) from a relative to DMSO (lane 5 relative to lane 1). Data are represented as mean ± SD. N = 3 independent experiments. One-tailed Standard Student’s t-test was used to determine statistical significance. c Quantification of the A16P-Myc level relative to T = 0 for DMSO and AG-205 treatment. Data are represented as mean ± SD. N = 3 independent experiments. d INS-832/13 cells expressing WT proinsulin-sfGFP and co-transfected with either empty vector or A16P-Myc were treated with AG-205 or PGRMC1 siRNA, as indicated. Media and cells (WCL) were collected and subjected to SDS-PAGE and immunoblotted as indicated. N = 3 independent experiments. e Quantification of C-peptide-sfGFP band intensity from the media fraction from d relative to control conditions (lane 1). Data are represented as mean ± SD. N = 3 independent experiments. One-tailed Standard Student’s t-test was used to determine statistical significance. f Quantification of WT proinsulin-sfGFP band intensity from the media fraction from D relative to control conditions (lane 1). Data are represented as mean ± SD. N = 3 independent experiments. One-tailed Standard Student’s t-test was used to determine statistical significance. g Quantification of C-peptide-sfGFP band intensity in the WCL from d relative to control conditions (lane 1). Data are represented as mean ± SD. N = 3 independent experiments. One-tailed Standard Student’s t-test was used to determine statistical significance. h As in d, except cells were transfected with B18A-Myc. N = 3 independent experiments. i–k Quantification of h as in e–g. N = 3 independent experiments. l Extracts derived from INS1E β-cells treated with or without AG-205 (“intracellular”) were subjected to nonreducing SDS-PAGE and immunoblotted as indicated. N = 3 independent experiments. m The secreted materials from L (“extracellular”) were subjected to reducing SDS-PAGE and immunoblotted with a proinsulin antibody. Quantification of the secreted proinsulin level. Data are represented as mean ± SD. N = 3 independent experiments. One-tailed Standard Student’s t-test was used to determine statistical significance. n A model depicting the role of PGRMC1 in RTN3-mediated ER-phagy of low-molecular-weight (LMW) protein aggregates. *P ≤ 0.05; **P ≤ 0.005. See also Fig. S6.