β actin

To further explore the effects of psoralen on the transcriptional activation of Wnt/β-catenin signaling pathway, western blot analysis was conducted according to the previous protocol^{20 (link)}. Using β-actin as an internal reference, the membrane was subsequently incubated at 4 °C overnight with primary antibodies against β-catenin, phospho-β-catenin and Fra-1 diluted at 1:3000, β-actin diluted at 1:5000 (Proteintech Group, CHI, USA), and enhanced chemiluminescence (ECL) plus kit (Millipore, America) was applied for visualization. The β-catenin expression and intracellular localization were analyzed using immunofluorescence. MDA-MB-231 cells and MCF-7 cells were fixed and stained with β-catenin antibodies (1:200 dilution). Images were captured by fluorescent microscopy.

BCA Protein Colorimetric Assay Kit (Elabscience Biotechnology Co., Ltd., China) was used to quantify the total protein of brain cortex tissues and SY5Y cells. Samples with an equal amount of protein were loaded and separated via sodium dodecyl sulfate‐polyacrylamide gel electrophoresis (SDS‐PAGE). The proteins were transferred to the appropriate polyvinylidene fluoride membrane (Millipore, USA), blocked with 5% skimmed milk for 1 h, and thens incubated with diluted antibodies at 4°C overnight. For the in vivo experiments, Drp1 (1:2000), Fis1 (1:1500), MFF (1:8000), OPA1 (1:2000), Mfn1 (1:1000), Mfn2 (1:2000), and β‐actin (1:5000) antibodies were purchased from Proteintech Group, Inc., China. Meanwhile, for the in vitro experiments, the antibodies included Drp1 (1:800; AiFang Biological, China), Fis1 (1:1500; Bioss, China), OPA1 (1:1000; AiFang Biological, China), Mfn1 (1:1000; Affinity, China), Mfn2 (1:500; AiFang Biological, China), and SENP6 (1:1000; Abcam, USA). Furthermore, MFF (1:2000), SUMO1 (1:2000), SUMO2/3 (1:500), SENP1 (1:2000), SENP2 (1:1000), SENP3 (1:800), SENP5 (1:3000), and β‐actin (1:5000) were obtained from Proteintech Group, Inc., China. Then, the membranes were washed and incubated with secondary antibodies for 2 h at room temperature. After rewashing with TBST buffer, the membranes were added ECL reagent to enable the detection of the protein bands.

U87 or U251 cells 6 h after transfection were lysed by lysis buffer with RIPA on ice for 30 min and centrifuge at 4°C for 20 min at 12000g/min. The total protein concentration of cell lysates was assessed using the BCA Protein Assay kit. Subsequently, 20 μg protein samples were separated on a 10% SDS-PAGE by the use of a Bio-Rad Bis-Tris Gel system (Bio-Rad, CA, USA), transferred onto polyvinylidene difluoride membranes (Millipore, Danvers, MA, USA), blocked with 5% nonfat milk for 60 min at room temperature. Primary antibodies were WEE1 (1 : 1000, Proteintech, USA) and β-actin (1 : 5000, Proteintech, USA); corresponding secondary antibodies were anti-mouse and anti-rabbit (Proteintech, USA). Primary antibodies against WEE1 were purchased from Bioss Co., Ltd. (Beijing, China) and the primary antibodies against N-cadherin, vimentin, and β-actin (Proteintech, USA). Finally, the proteins were detected by enhanced chemiluminescence (ECL) and then exposed about 60 s for scanning and measuring.

Dendrobium officinale was purchased from Zhejiang Shouxiangu Pharmaceutical Co., Ltd. (Jinhua, China), and PMA and LPS were purchased from Sigma (Saint Louis, MI, USA). Recombinant human interleukin-4 (IL-4) was purchased from PeproTech, Inc. (Rocky Hill, NJ, USA). TNF-α and IL-6 ELISA kits were purchased from Lianke Biological Technology (Hangzhou, China). Cell Counting Kit-8 (CCK-8) Assay Kit was received from MCE (Romulus, NJ, USA). Total Protein Extraction kit was purchased from Beyotime (Shanghai, China). The MiNiBEST Universal RNA Extraction Kit, PrimeScript RT Reagent kit, and SYBR Premix Ex Taq II Kit were purchased from TaKaRa (Dalian, China). Transwell polycarbonate membrane had an 8 μm pore size (Corning City, NY, USA). The primary antibodies E-cadherin, N-cadherin, Vimentin, Caspase-3, Bax, Bcl-2, Ki67, and β-actin were purchased from Proteintech (Wuhan, China). The primary antibodies ARG1, TGM2, and Cleaved-NOTCH1 were purchased from Cell Signaling Technology (Danvers, MA, USA). The primary antibodies STAT6, p-STAT6, PPAR-r, JAGGED1, Cleaved-NOTCH1, and NOTCH1 were purchased from Abcam (Cambridge, UK). β-actin was purchased from Proteintech (Wuhan, China). The ECL Plus Western Blotting Detection Kit was purchased from Technology Co., Ltd. (Beijing, China). Anti-CD80-FITC and Anti-CD206-PE were obtained from Thermo Fisher (Waltham, MA, USA).

BDE-47 (≥99.9%) was obtained from CSNpharm (Chicago, IL, USA). ISL (≥98%, HPLC) and LCB (≥98%, HPLC) were obtained from Yuanye (Shanghai, China). DCFH-DA and DMSO were obtained from Solarbio (Beijing, China). An apoptosis kit was obtained from Multi science (Hangzhou, China). A DAPI dye and neutral red staining solution were obtained from Beyotime Biotechnology (Shanghai, China). ELISA kits were obtained from Mlbio (Shanghai, China) (TNF-α Cat. No. ml002095; IL-6 Cat. No. ml063159; IL-1β Cat. No. ml301814). CAT, SOD, and GSH kits were obtained from Solarbio (Beijing, China). β-Actin, Nrf2, Keap1, HO-1, NQO1, IKBKB, IκB-Alpha, Bax, Bcl-2 antibodies, and the ECL luminescent reagent were obtained from Proteintech (Wuhan, China) (β-Actin Cat. No. 66009-1-lg; Nrf2 Cat. No. 16396-1-AP; Keap1 Cat. No. 60027-1-lg; HO-1 Cat. No. 66743-1-lg; NQO1 Cat. No. 67240-1-lg; IKBKB Cat. No. 15649-1-AP; IκB-Alpha Cat. No. 10268-1-AP; Bax Cat. No. 50599-2-lg; Bcl-2 Cat. No. 26593-1-AP). Caspase-3 antibody was obtained from BBI (Shanghai, China) (Cat. No. D16009). NF-κB and p-NF-κB antibodies were obtained from Abcam (Cambridge, UK) (NF-κB Cat. No. ab76302; p-NF-κB Cat. No. ab32536). P-Nrf2 antibody was obtained from Abclonal (Wuhan, China) (Cat. No. AP1133). Real-time fluorescent quantitative reagents were obtained from TransGen Biotech (Beijing, China).