Anti sp1 antibody

Sp1 binding activity to the CPT1A promoter region in intact cells was confirmed using a ChIP assay kit (Beyotime). Briefly, 2 × 10⁷ HepG2 cells were treated with D, G200, Q20, K20 or I8 for 6 hours and then cross-linked in 1% formaldehyde solution for 10 minutes at 37°C. Cross-linking was stopped by the addition of glycine to a final concentration of 125 mM. After 5 minutes, the cells were washed twice with 1 × PBS and harvested with PBS containing 100 mM PMSF. The cells were lysed and sonicated on ice with a Sonifier (Measuring and Scientific Equipment, UK) at 7 W for nine 10-second pulses and then centrifuged. After centrifugation, 20 μl of supernatants were used to measure total input chromatin (input control) and the rest were incubated in a rotor with 4 μg of anti-Sp1 antibody (Abcam) or normal-rabbit IgG (Santa Cruz Biotechnology) at 4°C overnight. Fifty μl of Dynabeads® Protein A beads (Invitrogen) were added for further 1.5 hours incubation the next day. Then the immunoprecipitated DNA-protein complexes were washed, eluted, and purified to conduct PCR for forty cycles. The PCR primers for ChIP assay (forward, 5′-CTCGGCGTCCCCACAG-3′; reverse, 5′-TTCCCAGGGCTCTTCG-3′) were designed to flank the Sp1 binding sites of the −50 to −5 of CPT1A promoter region, and the PCR products were analyzed on 2% agarose gel.

Total proteins from hMSCs cells, treated for 10 days with MM-EVs, were isolated and analyzed by SDS-PAGE followed by western blotting. Antibodies used in the experiments were as follows: anti-SP1 antibody (Abcam) and anti-GAPDH (Santa Cruz Biotechnology, CA, USA).

Nuclear extract was prepared from 143B cells and incubated with ³²P dATP‐labeled double‐stranded oligonucleotide containing either native or mutated BS1 binding sequence of FOXC1 to Sp1, in the absence or presence of recombinant human Sp1 protein (Abcam) or anti‐Sp1 antibody (Abcam). Band shifts were detected on 4% polyacrylamide gel.

Nuclear extracts of Hep2 cells were prepared using a nuclear extract kit (Pierce, USA) following the manufacturer's instructions. Oligonucleotides used in EMSA were synthesized by Sangene (Beijing, China), and their sequences were as follows: SP1 wild type: 5′-CTCTGGGGGCGGGGGGGTCGG-3′ and mutant: 5′-CTCTGGAGAATAAGAGGTCGG-3′. The oligonucleotides were labeled using the biotin 3′ end DNA Labeling Kit (Pierce, USA). EMSA was performed by LightShift Chemiluminescent EMSA kit (Pierce, USA) according to the protocol provided. In brief, nuclear protein extracts were incubated with 3′-end-biotin-labeled probes in binding buffer for 20 min on ice, separated on a 6% nondenaturing polyacrylamide gel, and then transferred onto a nylon membrane and fixed by ultraviolet cross-linking. Protein-DNA complexes were visualized by streptavidin-horseradish peroxidase followed by chemiluminescent detection (Pierce, USA). For competition assays, nuclear protein extracts were incubated with a 100-fold excess of the unlabeled wild type and mutated oligonucleotide duplex competitors, respectively. For supershift reaction, anti-SP1 antibody (Abcam, USA) was incubated with nuclear extracts for 1 h at 4°C prior to the addition of the biotin-labeled DNA probes.

ChIP assay was performed by using the ChIP Assay Kit (17–295, Millipore) according to the instructions. Briefly, 5 × 10⁶ pBMSCs were crosslinked with 1% formaldehyde for 10 min and quenched by 200 mM glycine, crosslinked cells were re‐suspended with lysis buffer containing proteinase inhibitors (Sigma) and subjected to sonicate chromatin into 200–1000 bp fragments by a Sonicator (SONICS, VCX 130). Then samples were immunoprecipitation (IP) with 5 μg anti‐SP1 antibody (Abcam, ab13370) at 4°C overnight. The antibody‐bind chromatin was then reversed by incubating with 0.1% SDS and 1 mg/mL Proteinase K (Sigma) at 65°C for 4 h. The ChIP DNA was extracted by using ChIP‐DNA clean and a concentrator kit (Zymo research) and then using for semi‐PCR and qPCR analysis. The primer sequence for ChIP‐PCR is listed in Tables S1.

We used the BCA Protein Detection Kit (Pierce Biotechnology, USA) to determine the concentration of each protein sample. Western blotting was then carried out in accordance with standard procedures. Membranes were incubated overnight withspecific primary antibodies, as follows: anti-Fn antibody (Abcam, 1:1500), anti-CoL-1 antibody (Affinity, 1:1000), anti-Sp1 antibody (Abcam, 1:5000), and anti-β-actin antibody (Proteintech, 1:3000). The next morning, the membranes were washed and incubated for 1 h with peroxidase-conjugated goat anti-rabbit immunoglobulin G (secondary antibody). Membrane binding to the antibodies was detected with the enhanced chemiluminescence system. Finally, band intensity was evaluated by Image J software (National Institutes of Health, Bethesda, MD).