Cct020312

The PTP1B inhibitor, sc-222227, was purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA, USA). The autophagy inhibitor, N(3)-methyladenine (3-MA) and the ER stress inhibitor, 4-phenylbutylamine (4-PBA), were purchased from Sigma (Sigma, St. Louis, MO, USA). The protein kinase R-like endoplasmic reticulum kinase (PERK) activator, CCT020312, was purchased from Merck Millipore (Billerica, MA, USA). The PERK inhibitor, GSK2606414, was purchased from Sigma. Antibodies were obtained from the following sources: CD11b/c, PTP1B, inositol requiring enzyme-1 (IRE1), p-IRE1, activating transcription factor 6 (ATF6), neuronal nuclear antigen (NeuN), ionized calcium binding adapter molecule 1 (Iba-1), heavy chain binding protein (Bip), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), and β-actin were purchased from Abcam (Cambridge, MA, USA); LC3-I/II and beclin-1 were purchased from Cell Signaling (Danvers, MA, USA); glial fibrillary acidic protein (GFAP) was purchased from Santa Cruz Biotechnology, Inc.; primary antibody for p-PERK (Thr982), PERK, p-eIF2α (Ser51), eIF2α, CD16/32, CD206, and secondary antibodies were purchased from Invitrogen (Carlsbad, CA, USA); and donkey anti-rabbit IgG, rabbit anti-goat IgG, and goat anti-mouse IgG were purchased from Invitrogen.

HeLa cells, NIH3T3 and both wild-type and eIF2αS51A MEF cells (kindly provided by P. Fafournoux from INRA, Unité de nutrition Humaine, France) were cultivated in DMEM media (Sigma Aldrich, Saint-Quentin Fallavier France) supplemented with 10% FCS, 1% glutamine and antibiotics. Cells were transfected using the JetPEI transfection reagent (Polyplus Transfection), according to the manufacturer’s instructions. 24 hours after transfection with the bicistronic constructs, cells were treated with DTT, the PERK activator CCT020312 (Merck Millipore, Fontenay sous Bois, France) or the PERK inhibitor GSK2606414 (Merck Millipore) for various concentrations, and were then harvested. For hypoxia, cells were incubated at 37 °C at 1% O₂.
Human Umbilical Vein Endothelial Cells (HUVEC) pooled from 6 donors were prepared by digestion of umbilical veins with 0.1 g/L collagenase A (Roche, Meylan, France) and cultivated in the specific Endothelial Cell Growth Medium 2 (PromoCell, Heidelberg Germany). Cells were transfected using the JetPEI-HUVEC transfection reagent (Polyplus Transfection), according to the manufacturer’s instructions.

Cells were stressed with various compounds for indicated times. At the start of treatment, we replaced the culture medium with DMEM/F12 medium supplemented with UPR activators tunicamycin (2.5 µg/ml; Sigma) and thapsigargin (0.33 and 1 µM; Sigma) or GCN2 activator halofuginone (10 nM; Cayman Chemical Company)^{39 (link)}, HRI activator BTdCPU (KM09748SC) (6 µM; Thermofisher)^{38 (link)}, PKR activator BEPP (10 µM; Sigma)^{37 (link)} or PERK activator CCT020312 (10 µM; Millipore) which specifically activate the ISR^{40 (link)}. All compounds were dissolved in DMSO. Control cultures were simultaneously treated with equal amounts of DMSO (vehicle control).

Neuro2a and mouse embryonic fibroblast (MEF) cells were maintained in DMEM supplemented with 10% foetal- calf serum at 37 °C and 5% CO₂. Japanese encephalitis virus (Vellore strain) and West Nile virus were grown in Porcine kidney cells (PS) or Vero cells as described earlier23 (link). For the purpose of infection, virus stocks were diluted in DMEM-2% FCS and incubated with cells for 1 hour. At the end of infection, the inocula were discarded and complete growth medium overlaid on infected cells.
For treatment with different drugs complete media supplemented with 1μM of Thapsigargin (Sigma) either alone or supplemented with DMSO (appropriate concentration) or 150 nM of PERKi (Millipore) or 50μM of STF083010 (Sigma) or 1μg/ml of Actinomycin-D (Sigma), was overlaid on cells and incubated for the time indicated. For PERK activation cells were treated with complete media supplemented with 10μM CCT020312 (Millipore) for the indicated time.

Primary microglial cells and stable GFP-LC3-expressing BV-2 cells were maintained in glucose-free medium in an anaerobic chamber (Thermo Fisher Scientific, Waltham, MA, USA) filled with 94% N₂, 1% O₂, and 5% CO₂ at 37° C for 3 h. Then, cells were returned to normal cell culture incubators (95% air and 5% CO₂) with normal medium. After 24 h of re-oxygenation, the OGD/R-treated conditioned medium was collected, and the supernatant without OGD/R treatment served as the sham control. The PTP1B inhibitor, sc-22227 (1 and 2 μM; Santa Cruz Biotechnology, Inc.), the PERK activator, CCT020312 (200 nM; Millipore), the PERK inhibitor, GSK2606414 (100 nM; Sigma), 4-PBA (4 mM; Sigma), and 3-MA (1 mM; Sigma) were added to microglial cultures 2 h before OGD/R treatment.