Diamide

To study band 3 spatial distribution during diamide exposure, we utilized a technique that labels band 3 via the lysine-430 of their cytoplasmic domain [53 (link)]. Mouse RBCs were washed in 5% PBS glucose solution with 1 mM EDTA solution for preparation. For the diamide samples, RBCs were incubated in 0.125 mM diamide (from Sigma, St. Lewis, Missouri, USA) for 30 minutes at 37°C. Control and diamide-treated RBCs were then fixed using 4% paraformaldehyde, labeled with 0.5 mM eosin-5-maleimide (EMA; from Molecular Probes, Eugene, Oregon USA), and washed three times with PBS. Images were collected using an inverted microscope (Ti-U, Nikon) with a 100x 1.49 NA oil-immersion objective lens, an EMCCD camera with 512x512-pixel chip (iXon3, Andor Technology, Belfast, UK) and a fiber-coupled 488 nm laser. Eosin was excited by a 488 nm laser and fluorescence was collected through a 525/45 nm bandpass filter. NIS elements software (Nikon, Tokyo, Japan) was used for image acquisition and ImageJ software was used for image processing and final figure preparation.

At 48 h after transfection with Flag-tagged PGK1, cells were treated with 0.5 mM H₂O₂, 1 mM H₂O₂, 0.5 mM diamide (a thiol-oxidizing compound, Sigma-Aldrich) or 1 mM diamide for 30 min, respectively.
Hypoxic treatment was performed in a sealed hypoxia chamber (Proox Model 110, BioSpherix) filled with 1% O₂, 5% CO₂, and 94% N₂ at 37 °C and 70% cell confluency for 12 h. For glucose treatment assays, HEK293T cells were cultured in low glucose DMEM medium (5 mM, Hyclone) or high glucose DMEM medium (25 mM) for 24 h after transfected with the Flag-PGK1 construct.

Gel retardation assays were done with in vitro generated proteins using the TnT^™ SP6-Coupled Reticulocyte Lysate system (Promega) according to the manufactures instructions and ³⁵S-labeled methionine was added to enable protein synthesis and detection (S1 Fig). For DNA probe preparation, purchased oligonucleotides (S1A Table) were labeled with γ-P³² at the 5’ ends using a T4 polynucleotide kinase (Thermo Scientific) according to the manufactures instructions. EMSAs were performed as described in [20 (link)] with a reaction mix with or without DTT (redox EMSA). For redox EMSAs, deviating from [20 (link)], either DTT (0.9 mM, 20 mM) (Roth) or 2 mM diamide (Sigma-Aldrich) was added. Probes were incubated for 30 min in the dark prior to DNA application. The potential re-reduction of previously diamide-treated reaction mixtures was achieved by addition of 20 mM DTT after the first incubation step followed by a second 30 min incubation step in the dark prior to DNA probe addition. Complex formation was detected by autoradiography after overnight exposure using a Storage Phosphor Screen (GE Healthcare) and visualized with a Storm^™820^® PhosphorImager (Molecular Dynamics, Amersham). All EMSA experiments were repeated at least three times with different in vitro translation products.

To assess the susceptibility to H₂O₂ and other oxidants, we used disk diffusion assay as previously described [10 (link)]. Briefly, an overnight culture of bacteria was diluted in LB to McFarland 0.5 using a Densimat (bioMérieux, Marcy-l’Étoile, France) and LB agar plates were inoculated using a sterile cotton swab. Sterile cellulose disks (5 mm diameter) were placed on the plate and 10 μL of 1 M H₂O₂ diluted in sterile water was added to the center of the disk. Other oxidants were used at the following concentrations: methylhydroquinone (Sigma-Aldrich) MHQ 0.5 M in water; methyl viologen dichloride hydrate, also called paraquat, (Sigma-Aldrich) PQ 1 M in water; diamide (Sigma-Aldrich) DI 0.2 M in water; menadione (Sigma-Aldrich) K3 360 mM in DMSO; cumene hydroperoxide (Sigma-Aldrich) CHP 0.25 M in DMSO; sodium hypochlorite (Sigma-Aldrich) NaOCl 5%; ciprofloxacin (Sigma-Aldrich) CIP 0.5 µg/µL in water; ampicillin AMP 1µg/µL in water.
Plates were incubated at 37 °C for 18 h and the diameter of inhibition was measured in mm. The area of inhibition was calculated as: [diameter of inhibition/2]² × 3.14. To compare the effect of different oxidants, data were normalized as following: [area of inhibition of the interested mutant] × 100/[area of inhibition of the WT].

To better distinguish between the oxidized and the reduced form of PDI, a modified N-ethylmaleimide (NEM) alkylation assay^{31 (link)} using methoxy polyethylene glycol 5000 maleimide (mPEG-mal₅₀₀₀, Sigma) was performed as follows. After seeding HCT116-shERp57 cells in 6-well plates, ERp57 KD was induced and the cells were grown for 96 h. In control dishes, cells were incubated for 15 min with either 10 mM DTT (AppliChem, Darmstadt, Germany) to fully reduce or 5 mM diamide (Sigma) to fully oxidize PDI. The growth medium was removed and the cells were incubated for 20 min on ice in PBS containing 20 mM NEM (Sigma) that binds to SH-groups and makes them inaccessible for further modification. After washing with PBS, the cells were lysed in RIPA buffer (50 mM Tris pH 7.5, 2 mM EDTA, 150 mM NaCl, 1% Nonidet P40, 0.1% SDS, 0.5% sodium desoxycholate and protease/phosphatase inhibitor cocktail (#5872, Cell Signaling, ZA Leiden, The Netherlands)). Next, 12 mM TCEP solution (Thermo Scientific, Darmstadt, Germany) was added to completely reduce the remaining disulfide bonds. After 20 min at RT, the reduced thiols were alkylated during incubation with 15 mM mPEG-mal₅₀₀₀ for 60 min at RT. After adding SDS sample buffer, lysates were boiled for 5 min and used for SDS-PAGE and further western blot.