4 pba

For intra-ACC drug administration, ACC catheterization was performed under anesthesia with pentobarbital sodium (60 mg/kg) according to our previous report [27 (link)]. Briefly, the head was fixed on a stereotactic apparatus in the prone position, and a 1-cm longitudinal incision was made in the middle of the head to expose the skull. Two holes were drilled on each side (Bregma forward, 1.7 mm; lateral, 0.6 mm) and a trocar (Shenzhen Ruiwode Life Science and Technology Co., Ltd, Guangdong, China) was inserted. Two small screws were installed superficially in the occipital bone, dental methyl methacrylate was used to fix the trocar with screws, and the rats were administered a subcutaneous injection of 80,000 U penicillin to prevent infection. CCI was performed 7 days after catheterization. The BIP and IRE-1 inhibitors 4-PBA and Kira-6 were purchased from Selleck (Houston, TX, USA) and dissolved in 10% dimethyl sulfoxide (DMSO). The rats were randomly assigned to the following four groups: CCI, CCI + DMSO, CCI + 4-PBA, and CCI + Kira-6. Both 4-PBA and Kira-6 were dissolved in 10% DMSO at a concentration of 20 µg/µL. Drugs or vehicle were bilaterally injected into the ACC (1 µL per side) from days 0 to 6 after CCI surgery.

The PROTEOSTAT Aggresome Detection kit (Enzo Life Sciences, Farmingdale, NY, USA) was used to detect protein aggregates in NPCs or NGN2-neurons according to the manufacturer’s instructions. Briefly, protein aggregates in NPCs or neurons were evaluated using red fluorescent molecular rotor dye to specifically detect denatured protein cargo within aggresome and aggresome-like inclusion bodies in fixed and permeabilized cells. Nuclei were counterstained with Hoechst 33342. The cytoplasm was stained with HCS CellMask Deep Red Stain. For drug treatment experiments, cells were treated with vehicle, 4-PBA (100 µM), TMP-269 (0.1 µM; Selleck, Houston, TX, USA), tasquinimod (0.1 µM, Selleck), or valproic acid (10 µM; Wako) from days 0 to 14, or with 3-methyladenine (3-MA, 10 µM; Sigma) for 24 h prior to aggregation analysis. All images were taken by the IN Cell Analyzer 6000 and were analysed using the IN Cell Developer Toolbox 1.9. Nuclei and cells were defined by Hoechst 33342 and CellMask, and the relative intensities of aggregates per cell were evaluated.

Irisin was purchased from Cayman Chemical (Ann Arbor, MI, USA). Angiotensin II (Ang II), BAPTA-AM and 4-PBA were purchased from Selleck Chemicals (Houston, TX, USA). Alezet Osmotic pump (2004) was purchased from Alzet (Cupertino, CA, USA). Transwell for migration was purchased from Corning (NY, USA). CCK8 was purchased from Biomake (Houston, TX, USA). The Irisin ELISA kits were purchased from Phoenix Pharmaceuticals Inc. (Burlingame, CA, USA). Compound C and HY-W007355 were purchased from MedChemExpress (NJ, USA). Edu cell proliferation kit, Fluo-3 AM Calcium probe and Crystal violet dye were obtained from Beyotime Biotechnology (Shanghai, China). anti-RYR2 was purchased from Abcam (Waltham, MA, USA); anti-IP3R, anti-SERCA, anti-p-SERCA, anti-PLN, anti-p-PLN were obtained from Abclonal (Wuhan, China); anti-p-CaMK II, anti-CaMKII were purchased from Santa Cruz (Dallas, TX, USA); anti-Ki67, anti-Bip, anti-ATF6, anti-IRE1α, anti-p-eif2α, anti-eif2α, anti-p-AMPK, anti-AMPK, anti-p-ERK, anti-ERK, anti-p-p38, anti-p38, anti-p-JNK, anti-JNK and anti-β-tubulin were obtained from Cell Signaling Technology (Danvers, MA, USA); anti-αV/β5 antibody was obtained from Millipore (Boston, MA, USA). All the secondary antibodies used in this study were provided by Zhongshan Golden Bridge Biotechnology (Beijing, China). All chemicals and reagents used were of analytically pure.

Mouse macrophage cell line, RAW264.7 cells were cultured in DMEM (ThermoFisher, USA) with 10% fetal bovine serum (FBS; ThermoFisher, USA) at 37 °C in a 5% CO₂ incubator. SiNPs were diluted by DMEM to appropriate concentrations, and an ER stress inhibitor, 4-phenylbutyric acid (4-PBA; Selleck, USA) was applied (3 mM, 6 h). The dosage of SiNPs was set according to the cell viability analysis by using MTT assay. Since a significant acute toxicity (24 h) was seen in SiNPs-treated group at a concentration of 50 μg/ml, while simultaneously with cell viability > 70%, the exposure mode of SiNPs (50 μg/ml, 24 h) was used in the subsequent in vitro experiments. Similarly, the application of 4-PBA was set up through MTT assay and also verified as evidenced by an efficient inhibition on the up-regulated expressions of Bip and CHOP induced by SiNPs. See details in the supplementary Fig. S4. After SiNPs with or without 4-PBA treatment, cells were harvested for the following measurement.