Cyclin d1

Western blot analysis of the inhibitor-treated cells was performed using a standardized protocol [22 (link)]. The primary human antibodies used in these analyses included NF-κB (p65), Smo, p53, p21, Nestin and β-actin (Santacruz, CA), IκBα, phospho-IκBα, phospho-NF-κB (p65), Bcl-2, S6K, phospho-S6K, 4E-BP1, phospho-4E-BP1, PLK1 and N-Myc (Cell Signaling Technology, MA) and cyclin D1, Bcl-2, and CD133 (BD Biosciences, CA). Immunoreactivity was detected using appropriate peroxidase-conjugated secondary antibodies (Santacruz, CA) and visualized using an enhanced chemiluminescence (ECL) detection system (Pierce, IL).

The cells were washed three times with ice-cold PBS before lysis, and were lysed with a buffer containing 1% Triton X-100, 1% Nonidet P-40 (NP-40), as well as protease and phosphatase inhibitor cocktails (GenDEPOT, Barker, TX). Equal amounts of protein were separated by 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred onto the polyvinylidene fluoride (PVDF) membranes (Millipore, Bedford, MA). Immunostaining was performed by incubating the blots with primary antibodies, followed by horseradish peroxidase (HRP)-conjugated secondary antibody and detected with an enhanced chemiluminescence (ECL) plus system (Amersham Biosciences, Piscataway, NJ). The primary mouse antibodies against the following factors were used: p27 (Cell Signaling Technologies, Danvers, MA), cyclin D1, hypoxia-inducible factor-1α (HIF-1α, BD Biosciences, SanJose, CA), VEGF (Novus Biologicals, Littleton, CO) and β-actin (Abcam, Cambridge, MA). The primary rabbit polyclonal antibodies against the following proteins were also used: cleaved caspase-3, poly (ADP-ribose) polymerase protein (PARP), p-Akt, Akt, p-mTOR, mTOR, p-p70S6K, p70S6K, p-4E-BP1, 4E-BP1 (Cell Signaling Technologies), cleaved caspase-8, procasepase-9 (Santa Cruz Biotechnology, Santa Cruz, CA), and α-tubulin (Abcam). The secondary antibodies were purchased from Amersham Biosciences.

We extracted proteins from Hep3b, SMMC7721 cells, and HCC tissues with mammalian cell lysis buffer M-PER (PIERCE) containing protease and phosphatase inhibitor. Proteins from total cell lysates were resolved with a 4-20% Tris-HCl gradient gel (Bio-Rad), transferred to PVDF membranes, blocked in 5% nonfat milk or BSA in TBS/Tween-20, and blotted with antibodies for PTEN, PDCD4, RECK, c-Jun, JunB, c-Fos, p-AKT, AKT, p-GSK3β, GSK3β, cyclin D1, cyclin E1, CDK2, CDK4, and GAPDH (RECK and c-Jun from BD biosciences, cyclin D1, cyclin E1, CDK2, CDK4, and GAPDH from Abcam, others from CST).

BLM was purchased from Nippon Kayaku Co., Ltd. (Japan), while recombinant TGF-β, Fsp1, cleaved-caspase 3, p-Smad2, p-Smad3, and Smad2/3 antibodies were obtained from Cell Signaling (USA). Collagen I, fibronectin, vimentin, α-SMA, and β-actin antibodies were originated from Santa Cruz Biotechnology (USA). Bax, Bcl-2, p-P85, p-AKT (Ser473 and Thr308), p- mTOR, and cyclin D1 were obtained from BD Bioscience (USA).

The cells were lysed for 30 min and then centrifuged at 13,000× g for 30 min at 4 °C. The quantified protein was mixed in proportion with the protein loading buffer and boiled at 95 °C for 10 min. The sample was electrophoresed and then transferred to a polyvinylidene difluoride (PVDF) membrane. The skim milk powder was diluted with Tween-20 Tris buffered saline (TBST) for 1 h at room temperature, then incubated with the primary antibody overnight, washed with TBST, and then incubated with the corresponding secondary antibody for 1 h at room temperature. The reaction was visualized using electrochemiluminescence (ECL, Bio-Rad, Hercules, CA, USA) and detected by exposure to autoradiographic film. The densitometric reading/intensity ratio for each strip was included in all western blots (Figures S3 and S4).
The primary antibodies used included CyclinD1 from BD Pharmingen(SanDiego, CA, USA); p-IĸB, p-NFĸB1, Bcl-2, Bax, p-TAK1, p-IKK, caspase3, and cleaved caspase3 from Cell Signaling Technology(Danvers, MA, USA); actin from Transgen Biotech(Beijing, China); C20orf27 and p-TGFβR1 from Abcam(Cambridge, MA, USA); Bay11-7082 from TargetMol(Boston, MA, USA); and PP1c, GADD34, and TAK1 from Santa Crus (CA, USA).