Cyclin d

Proteins were extracted from mandibles and quantitated using a protein assay kit (Bio‐Rad, Mississauga, Ontario, Canada). Protein samples (20 μg) were fractionated by SDS‐PAGE and transferred to nitrocellulose membranes. Immunoblotting was carried out as described¹⁹ using antibodies against Cyclin D (1:300, sc‐8396, Santa Cruz), CDK4 (1:300, sc‐23896, Santa Cruz), p27 (1:500, sc‐1641, Santa Cruz), p53 (1:500, sc‐126, Santa Cruz) and β‐actin (1:1000, AP0714, Bioworld Technology). Bands were visualized using ECL chemiluminescence (Amersham) and quantitated by Scion Image Beta 4.02 (Scion Corporation, NIH).

The protocol of western blot was followed according to a previous study [10 (link)]. Briefly, adipocytes were split by radioimmunoprecipitation assay (RIPA) buffer (Beyotime, China) by adding protease inhibitor (Pierce, WA, USA). The total protein sample was separated in the SDS-polyacrylamide gel. Then, it was transferred into a PVDF membrane (Millipore, Bedford, MA, USA). Next, the membrane was blocked in 5% defatted milk for 2 h. After that, the membrane was incubated with primary antibodies at 4 °C overnight followed by a secondary antibody at room temperature for 1.5 h. The antibodies Cyclin B, Cyclin D, Cyclin E, PPARγ, and AP2 were purchased from Santa Cruz (CA, USA); C/EBPα and SREBP-1 were purchased from Abcam; p62, LC, ATGL, and HSL were purchased from CST (Boston, MA, USA); and β-tubulin was purchased from Sungene Biotech (Shanghai, China).

Briefly, A375 and SK-MEL-28 cells were treated with 0.625 μM or/and 1.25 μM ABZ for 48 h respectively. Then the total proteins were collected and subjected to 10% SDS–PAGE gel for western blot analysis. For western blot analysis, the following antibody dilutions were used: CDK1 (Santa Cruz, sc-54), Cyclin B1 (Santa Cruz, sc-245), p53 (Santa Cruz, sc-126), p-RB1 (Cell Signaling Technology, 8516), RB1 (Cell Signaling Technology, 9313), Cleaved Caspase-3 (Cell Signaling Technology, 9661), CDK4 (Santa Cruz, sc-238969), Cyclin D (Santa Cruz, sc-8396), phospho Histone H3 (Hunan AiFang biological, Co., Ltd, AF00582) and GAPDH (Santa Cruz, sc-47724).

Quercetin (>98% pure), and Green tea were obtained from Sigma. The CYCLIN D, cyclin E, CYCLIN A, CDK4, CDK2, CDK6, P21, P27, BCL-XL, BAX, BCL-2, cytocrome c, MCL-1, actin, and GAPDH from Santa Cruz Biotechnology. BECLIN-1 and PI3K class III from Cell Signaling Technology. pJNK from Invitrogen. ATG5-ATG12, ATG7 and LC3I/II from Abcam Inc. Anti-rabbit, anti-mouse, and anti-goat peroxidase–conjugated antibodies from KPL, Inc. The HL-60 cell line, derived from a 36-year-old woman with acute promyelocytic leukemia, was obtained from ATCC, Philadelphia, PA. Cytogenetic analyses of the cell line, during the procedures of this study, identified the same alterations as previously described^{27 (link)} in 100% of metaphases analysed.

Hygromycin, puromycin, zeocin, collagen, fibronectin, and laminin substrates were purchased from Life Technologies. Matrigel was obtained from Corning. Transwell inserts (CLS3422) were purchased from Sigma. Antibodies used in our studies were: Vimentin (1:4000 WB; 1:500 IF; Abcam), E-Cadherin (1:250 IF in 2D culture; 1:50 IF in 3D culture; BD Biosciences), Snail (1:500 WB; Cell Signaling), Slug (1:500 WB; Cell Signaling), Calnexin (1:1000 WB; Enzo), Bim (1:250 WB; Enzo), laminin V (1:50 IF in 3D culture; EMD Millipore), Claudin1 (1:500 WB; Invitrogen), Twist1 (1:500 WB; Santa Cruz), PCNA (1:250 WB; Santa Cruz), CyclinD (1:250 WB; Santa Cruz), and VRK1 (1:1000 WB; Sigma).

Cells were washed twice with PBS and harvested in RIPA buffer (with 1 mM phenylmethylsulfonyl fluoride, Biosesang, Seongnam, Gyeonggi, Korea). Total protein amounts were measured using the bicinchoninic acid assay (Pierce, Rockford, IL, USA). Appropriate amounts of protein were subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis (10–12% gels) and transferred to polyvinylidene fluoried membranes. Membranes were blocked with 10% nonfat dry milk in TBST (50 mM Tris/HCl and 150 mM NaCl)/0.1%Tween 20 and incubated with primary antibodies overnight. The primary antibodies used included those against Smad2/3, Zo-1, ZEB1, Cyclin E, Cyclin D, Keratin 14, EEA1, p67phox, p47phox (Santa Cruz Biotechnology, Dallas, TX. USA), slug, snail, vimentin, AKT, pAKT, p38, pp38, ERK, pERK, pSmad2, pSmad3, p21, p27, Src, LC3, pSrc (Y416), p40phox, pp40phox, GAPDH (Cell Signaling Technology, Beverly, MA, USA), ZEB2, YB-1, FAK, pFAK(Y397) (Abcam, Cambridge, UK), NOX4 (Novus Biologicals, Littleton, CO, USA), and NOX2 (BD Biosciences, San Diego, CA, USA).