Bapta am

Lipopolysaccharide (LPS), from Escherichia coli strain 0111:B4 (Sigma, L4391) was dissolved in sterile saline and injected subcutaneously (50 μl, 1 mg/kg body weight) close to the midline of the lower lip. At 12 h, 24 h or 1 month following a single LPS injection, a craniotomy was performed in preparation for in vivo imaging. Pharmacological compounds were applied directly to the cortex on the intact dura prior to mounting the cranial window. Imaging was initiated 30 min after application and drugs were maintained in the preparation during the entire session. BAPTA-AM, PPADS (pyridoxal-5-phosphate-6-azophenyl-2′4-disulfonic acid) and bicuculline were purchased from Tocris. Stock solutions were diluted with sterile saline to a final concentration of 5 μg/ml, 5 mM, and 250 μM, respectively.

J774A.1 cells were seeded in 96-well plates at a concentration of 10⁵ cells/well and incubated overnight. Cells were primed for 4 h with 1 μg/ml E. coli O111:B4 LPS and subsequently stimulated for 30 min with 3 mM ATP in 100 μl medium. Where indicated, cells were treated with 100 μM BAPTA-AM (Tocris, Minneapolis, MN, USA) for 15 min before ATP treatment. Supernatants were collected and released IL-1β was evaluated with ELISA using the R&D Systems DuoSet kit according to the manufacturer's protocol. Developed plates were read on a Biotek Synergy H4 mutli-mode plate reader with Gen5 software.

The squaramide compound SQ1, its analogue SQ2, and the control compound SQ3 (with no chloride transport activity) were previously described^{18 (link),19 (link)}. The following chemicals and antibodies were used in this study: Torin 1, bafilomycin A1, BAPTA AM, Cyclosporin A (Tocris Bioscience, Bristol, UK), Cycloheximide, Cyclosporin H, N-Acetyl-L-cysteine, L-Glutathione reduced (Sigma-Aldrich) oleate from Larodan (Solna, Sweden).
Anti-beta actin (Abcam 8226, Cambridge, UK), LC3 (#3868), mTOR (#2983), Phospho-mTOR (Ser2448) (#5536), LAMP1 (#9091), p70 S6 Kinase (#9202), Phospho-p70 S6 Kinase (Thr389) (#9205), SQSTM1/p62 (#7695) antibody were all purchased from Cell Signaling Technology, IF antibody Alexa Fluor® 647 anti-human CD107a (LAMP-1) Antibody from BioLegend.

Auranofin, A23187 and BAPTA-AM were from Tocris (R & D Systems, Abingdon, UK), Q-VD-OPh (non-O-methylated) was from Calbiochem (Merck Millipore, Nottingham, UK). CM-H₂DCFCA and Nu-PAGE sample buffer were from Invitrogen (Life Technologies, Paisley, UK). Anti-caspase-3 and anti-gelsolin antibodies were from Cell Signaling Technology (Beverley, MA, USA). Annexin V-FITC was from Abcam (Cambridge, UK). Fluo-2 LR-AM (acetoxymethylester) was from Teflabs (Cambridge Bioscience, Cambridge, UK). The lactate dehydrogenase (LDH) assay kit was from BioVision inc. (Milpitas, CA, USA). All other reagents, including the TRXR assay kit (CS0170), were from Sigma (Poole, UK), and were of analytical grade.

To test the impact of inhibiting nuclear export on the steady state distribution Chm7-GFP, Chm7-MGM4, chm7_OPEN-GFP, GFP, NES1_CHM7-GFP, NES2_CHM7-GFP, or NES1-2_CHM7-GFP, we used KWY175 (a gift from B. Montpetit and Karsten Weis) in which the genomic deletion of XPO1 is covered with pRS413 expressing the xpo1-T539C allele that confers sensitivity to Leptomycin B (LMB). These strains were grown in YPAR and galactose (final concentration of 1%) was added to the growth medium to induce the expression of the GFP fusion proteins for 2 hr before the addition of 2% D-glucose to repress protein production. Cultures were then treated with 50 ng/mL LMB dissolved in 7:3 MeOH:H₂O solution (Sigma) for 45 min alongside a control of the equivalent volume of MeOH before imaging.
To test if Ca²⁺ plays a role in the physiological recruitment of Chm7 to the nuclear envelope, apq12Δ cells expressing Chm7-GFP (DTCPL567) were cultured overnight at 30°C, diluted to an OD₆₀₀ of 0.2 and grown for an additional 2 hr at RT. Cells were treated with either 25 µM (Li et al., 2011 (link)) of the cell permeable calcium chelater BAPTA-AM (Tocris Bioscience) dissolved in DMSO or DMSO alone for 30 min followed by a 45 min incubation at either RT or 37°C before imaging.

The EGFR kinase domain inhibitors were purchased from Santa Cruz (Gefitinib), Selleckchem (PD153035), Tocris Bioscience (AG1478) and Sigma (GW2974). Marimastat, GI-253023X, glibenclamide and Bapta-AM were purchased from Tocris Bioscience. Inhibitors were reconstituted in DMSO and aliquoted for appropriate freezer temperature storage. TR146 cells were incubated with inhibitors for 1 h prior to C. albicans infection, candidalysin exposure or mock treatment. Phospho-EGFR Tyr1068 (#3777), phospho-EGFR Tyr845 (#6963S), c-Fos (#2250S) and phospho-MKP1 (#2857S) antibodies were purchased from Cell Signalling Technology. Mouse anti-human α-actin antibody was purchased from Millipore (UK) (#MAB1501), goat anti-mouse (#115-035-062) and anti-rabbit (#111-035-144) horseradish peroxidase (HRP)-conjugated antibodies were purchased from Jackson Immunologicals (Stratech Scientific, UK). Fluorescent ErbB receptor affibodies were a kind gift from the Science and Technologies Facilities Council for use with confocal imaging work. APC anti-human EGFR antibody was purchased from BioLegend (# 352905) for use with Imagestream analyses. Biologically active EREG and EPG were purchased from Peprotech and used at 10, 25 and 50 ng/mL either individually or together at the same concentration.

ACET, picrotoxin, TPMPA, strychnine, DNQX, W-7, calmidazolium (CMZ), CALP1, ML-9, KN-62, MMPX, ascomycin, BAPTA-AM, thapsigargin (Tg), YM-58483 were obtained from Tocris. L-AP4 was purchased from Tocris or Cayman. Meclofenamic acid (MFA) and I4AA were obtained from Sigma. TTX was purchased from Alomone Labs. Drugs were dissolved in dimethylsulfoxide (DMSO) where appropriate and then diluted into the bath solution. The final concentrations of DMSO were <0.1% (v/v) in all experiments.