Er tracker blue white dpx

We evaluated the ER and mitochondrial co-localisation using confocal microscopy. For this, SH-SY5Y cells were seeded in 35-mm laser confocal dishes and labelled with 100 nM Mito-Tracker Red CMXRos (Beyotime, Shanghai, China) and 1 μM ER-Tracker Blue-White DPX (Yeason, Shanghai, China) at 37 °C for 30 min. Dual-colour images were captured using a confocal microscope (LSM780, Carl Zeiss Microscopy Cambridge, MA), and mitochondria–ER co-localisation was assessed using Mander’s overlap coefficient, which is commonly used to quantify the degree of co-localisation [25 (link)]. Mitochondrial membrane potential (MMP) was measured using tetramethyl rhodamine methyl ester (TMRM; Beyotime, Shanghai, China) according to the manufacturer’s instructions. TMRM signals were captured using an LSM780 laser scanning confocal microscope (Zeiss), and fluorescence intensities were quantified using Zen Blue software (Zeiss). The images were quantified by two independent investigators blinded to the groups.

Ox-LDL was purchased from Yiyuanbiotech (YB-002, Guangzhou, China). The anti-CDC42 antibody, anti-Rac1/2/3 antibody, anti-PAK antibody, anti-pCDC42 antibody, anti-pPKA antibody, and Phalloidin-iFluor 488 were purchased from Abcam (Oxford, UK). The anti-Unc5b antibody was purchased from CST (Danvers, USA). The PAK inhibitor (FRAX486) and CDC42 inhibitor (ML141) were purchased from MCE (MCE, USA). Biotin-labeled LTL, biotin-labeled LCA, biotin-labeled AAL, biotin-labeled UEA1, biotin-labeled SNA, biotin-labeled MAL1, biotin-labeled VVL, and biotin-labeled PHA-L were purchased from Vector Laboratories (Peterborough, UK). ER-Tracker Blue‒White DPX was purchased from Yeasen Biotech Co., Ltd. (Shanghai, China). Oil Red O solution was purchased from Solarbio Science & Technology Co., Ltd. (Beijing, China). FBS was purchased from BI (Israel).

The cells were seeded into cell culture plates and stained with JC1 staining solution (40705ES03, Yeasen Biotechnology, Shanghai, China) following the instruction manual. The fluorescence was observed with Leica DMi8 fluorescence microscope. Reactive Oxygen Species were detected using the Reactive Oxygen Species Assay Kit (50101ES01, Yeasen Biotechnology, Shanghai, China), and visualized with Leica DMi8 fluorescence microscope. The quantification of fluorescence results was done using the Image J software. Additionally, ER-Tracker Green (BODIPY FL Glibenclamide) (40763ES20, Yeasen Biotechnology, Shanghai, China), ER-Tracker Blue-White DPX (40761ES50, Yeasen Biotechnology, Shanghai, China) and MitoTracker Red CMXRos (40741ES50, Yeasen Biotechnology, Shanghai, China) were used to stain the endoplasmic reticulum or mitochondria of live cells. The staining was visualized using laser scanning confocal microscopy (Leica, TCS SP8 DIVE, Germany), and the co-localization was quantified using the co-localization Finder plugin of ImageJ software. The experimental protocols were conducted as per the standards.