Ab252930

MDA-MB-231 cells were treated with vehicle or 1 μM CB for 48 h and then harvested for ChIP assays using a Magna ChIP™ A/G Chromatin Immunoprecipitation Kit (Millipore, Cat #.17-10085). ChIP assays were performed using antibodies against HMGA1 (ab252930, Abcam) and normal IgG antibodies, according to the manufacturer’s protocol. qPCR was performed with primers flanking the cyclin E promoter. Relative fold change of HMGA1 recruitment on cyclin E promoter was determined by calculating the fold enrichment of target antibodies over IgG, followed by normalization of CB-treated samples over the vehicle.

Antibodies against HMGA1 (ab129153), HMGA1-ChIP Grade (ab252930), and FKBP12 (ab2918) were purchased from Abcam. Antibodies against HMGA1 (sc-393213) and FKBP12 (sc-133067) and mouse IgG (sc-2025) were purchased from Santa Cruz Biotechnology. Antibodies against S6 ribosomal protein (#64108) and phospho-S6 ribosomal protein (#81736) and rabbit IgG (#2729), mTOR (#2983), phospho-mTOR (#5536), eIF4EBP1(#9452), and phospho-eIF4EBP1(#9456) were purchased from Cell Signaling Technology (Danvers, MA). Antibody against beta actin (81115-1-RR) was purchased from Protein Tech (Wuhan, China). Rapamycin was obtained from MedChemExpress (HY-10219).

Primary tumors generated from transplanted MDA-MB-231 cells were fixed in 4% paraformaldehyde overnight and embedded in paraffin. Primary tumor sections were dewaxed with xylene and rehydrated with ethanol (100% to 70%). Antigen retrieval was performed by boiling the specimens in Immunosaver (Nissin EM, Tokyo, Japan) diluted 1:200 for 45 min at 98°C. The sections were permeabilized with 0.1% Triton X-100 (Sigma-Aldrich, St Louis, MO, USA) for 15 min. After blocking with Dako blocking reagent (Vector Laboratories, Newark, CA, USA) for 30 min, sections were incubated with primary antibodies for 1 h at room temperature. Sections were then incubated with the primary antibodies, anti-CD44 (60224-1-IG, Proteintech Group, Rosemont, IL, USA; diluted 1:100) and anti-HMGA1 (ab252930, Abcam, Cambridge, UK; diluted 1:200), and secondary anti-mouse Alexa Fluor 488-labeled antibody (A-11001, Thermo Fisher Scientific, Waltham, MA, USA; diluted 1:1000) and anti-rabbit Alexa Fluor 594-labeled antibody (A-212-7, Thermo Fisher Scientific; diluted 1:1000). Slides were mounted with VECTASHIELD mounting medium with Hoechst 33342 (Thermo Fisher Scientific). Stained sections were imaged using an FV10i Laser Scanning Microscope (OLYMPUS, Tokyo, Japan).

Total protein was extracted from EPCs using RIPA lysis buffer (#P0013B, Beyotime Biotechnology) according to the instructions. Protein was quantified in each group according to BCA protein assay kit. SDS-PAGE loading buffer (#MB2479, Meilunbio) was mixed. The mixture was heated in a boiling water bath at 100°C for 5 minutes. The protein was adsorbed onto PVDF membrane by gel electrophoresis. Primary antibodies against TSG101 (14497-1-AP, 1 : 1000, Proteintech), CD81 (66866-1-Ig, 1 : 1000, Proteintech), CD63 (25682-1-AP, 1 : 1000, Proteintech), ASC (10500-1-AP, 1 : 2000, Proteintech), Caspase-1 (#4199, 1 : 1000, CST), NLRP3 (19771-1-AP, 1 : 1000, Proteintech), IL-18 (10663-1-AP, 1 : 2000, Proteintech), HMGA1 (ab252930, 1 : 1000, Abcam), RRM2 (11661-1-AP, 1 : 2000, Proteintech), TK1 (15691-1-AP, 1 : 1000, Proteintech), TYMS (15047-1-AP, 1 : 1000, Proteintech), and β-actin (66009-1-IG, 1: 1000, Proteintech) were incubated at 4°C overnight. TBST was washed three times at room temperature and incubated with secondary HRP goat anti-mouse IgG (SA00001-1, 1 : 5000, Proteintech) and HRP goat anti-Rabbit IgG (SA00001-2, 1: 6000, Proteintech). ECL chromogenic exposure was performed. Odyssey Infrared Imaging System (Li-COR Biosciences, Lincoln, NE, USA) was applied to detect protein bands, and β-actin was used as an internal reference to detect expression levels.