Ab180768

Human BMSCs were transfected with miRNA-455 mimics, inhibitors, or negative control (customized and purchased from RIBOBIO, Guangzhou, China) using Lipofectamine RNAiMAX transfection reagent (Invitrogen). Cells were transfected with SOX11 siRNA, FOXO1 siRNA, or control siRNA (Thermo Scientific) using Lipofectamine 3000 (Invitrogen) according to the manufacturer’s instructions. The SOX11 expression plasmid was obtained using the pcDNA™ 3.1/V5-His TOPO™ TA Expression Kit (Invitrogen™). After 48 h of transfection, the expression levels of the target genes were evaluated from the collected cellular lysates by qRT-PCR and western blotting of specific genes in the miR-455/SOX11/FOXO signaling axis (ab185966, ab232628, ab34712, ab39670, ab17091, ab39012, ab32034, ab200738, and ab180768, Abcam, dilution at 1:1000–2000).

Cell lysates were prepared by rinsing cells with 0.5 mL of lysis buffer (125 mM Tris-HCl, 750 mM NaCl, 1% v/v NP40, 10% v/v glycerol, 50 mM MgCl₂, 5 mM EDTA, 25 mM NaF, 1 mM Na₃VO₄, 10 mg/mL leupeptin, 10 mg/mL pepstatin, 10 mg/mL aprotinin, 1 mM phenylmethylsulphonyl fluoride, pH 7.5). Samples were sonicated and centrifuged at 13,000× g at 4 °C for 10 min. The immunoblot analyses were performed on 50 μg of proteins, using the following antibodies: anti-OSCP1 (ab244416, Abcam, Cambridge, UK; dilution 1/2000), anti-EPB41L4A (ab67551, Abcam; dilution 1/500), anti-EIF5 (ab228874, Abcam; dilution 1/1000), anti-ABCB1 (C219, Novus Biologicals, Littleton, CO; dilution 1/250), anti-GADD45A (ab180768, Abcam; dilution 1/500), anti-MYC (10828-1-AP, Proteintech Group Inc., Rosemont, IL, USA; dilution 1/1000), anti-TOP2A (20233-1-AP, Proteintech Group Inc.; dilution 1/1000), followed by a peroxidase-conjugated secondary antibody. Anti-β-tubulin antibody (sc-5274, Santa Cruz Biotechnology Inc.; dilution 1/1000) was used as control of equal protein loading. The proteins were detected by enhanced chemiluminescence (Bio-Rad Laboratories). The relative quantitation of immunoblot was performed with the ImageJ software (https://imagej.nih.gov/ij/; v1.52t). The band density of untreated cells was considered as 1 arbitrary unit.

Cell lysates were prepared with RIPA lysis buffer (P0013B, Beyotime) at 4°C. Protein concentrations were measured by BCA Protein Assay Kit (P0010S, Beyotime). Proteins were fractionated using SDS/PAGE and then electroblotted on to 0.45-μm PVDF membranes and then blocked in 5% nonfat milk dissolved in TBST for 1 h at room temperature. The membranes were incubated with primary antibodies overnight, washed in TBST for 10 min three times, and then incubated with the HRP-conjugated secondary antibody. Primary antibodies against GAPDH (sc-32233) were purchased from Santa Cruz Biotechnology. The antibodies against GOLIM4 was purchased from Abnova (PAB28477). The antibodies against MDM2 (ab38618), CDK6 (ab151247), and GADD45 (ab180768) were purchased from Abcam. Secondary antibody goat anti-rabbit IgG-HRP (sc-2005) were obtained from Santa Cruz Biotechnology. The blots were detected using Pierce™ ECL Western Blotting Substrate kit (M3121, Thermo Scientific).

Whole cell lysates were used for western blotting analysis as previously described [38 (link), 39 (link)], with glyceraldehyde 3-phosphate dehydrogenase (GAPDH) used as a loading control. The anti-CTCF antibody (07–729) was obtained from Millipore, while antibodies against EGR1 (ab54966), TNFAIP3 (ab74037), GADD45A (ab180768), and p65 (ab16502) were obtained from Abcam; a GAPDH monoclonal antibody (SC-32233) and rabbit or mouse secondary antibodies were purchased from Santa Cruz Biotechnology. The anti-phospho-NF-κB p65 (Ser536) (93H1) antibody was obtained from Cell Signaling.

Protein extracts were resolved on 8–15% SDS-PAGE gels and transferred onto PVDF membranes (Millipore, Billerica, MA, USA). The membranes were blocked with 5% nonfat dry milk in TBS containing 0.1% Tween 20 at room temperature for 1 h and incubated with antibodies against human ATF4 (1/1000, ab184909, Abcam, Cambridge, MA, USA), SNAIL2 (1/1000, #9585, Cell Signaling Technology, Danvers, MA, USA), GADD45A (1/1000, ab180768, Abcam, Cambridge, MA, USA), PSAT1 (1/1000, ab96136, Abcam, Cambridge, MA, USA), or GAPDH (1/2000, sc-32233, Santa Cruz Biotechnology, Dallas, TN, USA) overnight at 4°C, followed by incubation with the secondary peroxidase-conjugated anti-mouse (1/5000, #7076, Santa Cruz Biotechnology, Dallas, TN, USA) or rabbit IgG antibodies (1/5000, #7074, Santa Cruz Biotechnology, Dallas, TN, USA). The antigen–antibody reaction was visualized using the Pierce™ ECL Western Blotting Substrate (Thermo Fisher Scientific, Waltham, MA, USA).