Anti cyclind1

Spautin-1(S7888), 3-methyladanine (3-MA, S2767), Z-VAD-FMK (S7023), SP600125 (S1460), SB230580 (S1076), LY3214996 (S8534), Gefitinib (S1025) and Enzalutamide (MDV3100, S1250) were purchased from Sellectchem (Houston, TX, USA). SKP2-C25 (M60136) was obtained from Xcessbio Biosciences, Inc. (San Diego, CA). USP10 (sc-76,811), USP13 (sc-76,815) and Glut1 (sc-35,493) siRNAs were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Antibodies: anti-Glut1 (ab652), anti-USP13 (ab109264) (Abcam, Cambridge, MA); anti-GAPDH (BS60630), anti-Ki67 (BS1454) (Bioworld Technology, Inc., Louis Park, MN, USA); anti-USP10(#8501), anti-SKP2(#2652), anti-p27(#3686), anti-CDK4(#12790), anti-CDK2(#2546), anti-CyclinD1(#2978), anti-p15(#4822), anti-p21(#2947), anti-PARP(#9532), anti-Bim(#2933), anti-Bax(#14796), anti-Bcl-2(#15071), anti-activated Caspase-3(#9664), anti-phospho-JNK (#9255), anti-JNK(#9252), anti-phospho-ERK1/2(#4370), anti-ERK1/2(#4695), anti-phospho-p38(#4511), anti-p38(#8690), anti-phospho-MKK4(#4514), anti-MKK4(#9152), anti-phospho-MEK1/2 (#9154), anti-MEK1/2(#4694), anti-phospho-EGFR (Tyr1173)(#4407), anti-phospho-EGFR (Tyr1068)(#3777), anti-EGFR(#2085) (Cell Signaling Technology, Beverly, MA, USA).

Cells were lysed and homogenized in RIPA buffer supplemented with complete protease inhibitors. Equal amount of proteins (200 µg) was resolved in 12% SDS-PAGE. The proteins were then transferred onto PVDF membranes following electrophoresis. After blocked in 5% (w/v) non-fat milk for 1 h at room temperature, the membranes were then incubated at their respective appropriate dilutions of specific primary antibodies overnight at 4°C. The sources of primary antibodies were: anti-Oct4, anti-Sox2, anti-Vimentin and anti-phospho-STAT3 (Signalway Antibody, USA), anti-GAPDH (Kangcheng, Shanghai, China), anti-E-cadherin, anti-ERK1/2, anti- phospho-ERK1/2 and anti-N-cadherin (Santa Cruz Biotechnology, Santa Cruz, CA, USA), anti-PCNA, anti-STAT3, anti-NF-κB, anti-phospho-NF-κB, anti-CyclinD1, anti-VEGF and anti-C-Jun (Bioworld Technology, Louis Park, MN, USA), anti-C-myc (ProteinTech Group, Chicago, IL, USA).

Immunohistochemistry (IHC) was performed in primary cancer and paired adjacent tissue samples. The procedure was performed as described previously [32 ]. NKD2 antibody (Novus Biology, CO, USA) was used at a 1/500 dilution overnight at 4°C. Anti- phospho-β-catenin (Bioworld Technology, MN, USA) and anti-cyclin D1 (Bioworld Technology, MN, USA) at a 1/150 dilution were incubated overnight at 4°C. The staining intensity and extent of the staining area were scored using the German semi-quantitative scoring system as previously described [32 ].

TCN was provided by Kunming Institute of Botany, the Chinese Academy of Sciences (purity > 99%, HPLC analysis) as previously described [41 (link)] (Fig. 4a). Dimethyl sulphoxide (DMSO, Sigma) was used to dissolve TCN. The final concentration of DMSO in the culture media was kept less than 0.1% v/v which had no significant effect on the cell growth. Elaidic acid and Oleic acid were purchased from MedChemExpress (NJ, USA).
The antibodies against β-actin and CDK4 were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). The antibodies against RB, pRB and Ki67 were obtained from Cell Signaling Technologies (Danvers, MA, USA). The antibody to detect DHRS2 (PA5–25258) was from Thermo Fisher Scientific. Anti-cyclin D1 was from Bioworld Technology.
pEZ-Lv105-DHRS2 was from GeneCopeia. The open reading frame of DHRS2 gene was inserted into the lentivirus vector pEZ-Lv105 using Gateway® recombination technology.

We performed western blot according to the previous study [21 (link)]. Protein lysates were prepared, subjected to SDS-PAGE, transferred onto PVDF membranes and blotted according to standard methods, using anti-GSK3β, anti-phospho-β-catenin (Ser9), (Cell Signaling Technology), anti-CyclinD1, anti-c-Myc (Bioworld Technology, St. Louis Park, MN, USA)), anti-Ki-67 (Abcam, Cambridge, MA, USA), anti-MMP7, anti-SFRP2 (BD Biosciences, San Diego, CA, USA), anti-β-catenin (BD Biosciences, San Diego, CA, USA). Anti-α-Tubulin monoclonal antibody (Sigma, St Louis, MO, USA) served as a loading control.

Proteins were isolated, subjected to SDS-page, transferred onto PVDF membranes and incubated with antibodies anti-SIAH1 (Abcam, Cambridge, MA, USA), anti-SFRP2 (Cell Signaling Technology, Danvers, MA, USA), anti-KRAS (Proteintech, USA), anti-Ki-67 (Abcam, Cambridge, MA, USA), anti-p27, anti-p21, anti-cyclinD1 (Bioworld Technology Inc. St. Louis Park, MN, USA). LamB1 and a-tubulin (Sigma, Saint Louis, MO, USA) were used as loading controls. Then, the membranes were detected by chemiluminescence. All operations above were performed in accordance with standard methods.

The siRNA targeting YAP and the non-targeting siRNA were purchased from Ruibobio (Guangzhou, China). The target sequences for YAP siRNA are 5′-GCGUAGCCAGUUACCAACA dTdT-3′, 5′- CAGUGGCACCUAUCACUCU dTdT-3′ and 5′- GGUGAUACUAUCAACCAAA dTdT-3′. IM was obtained from Novartis (Basel, Switzerland) and VP from Selleckchem (Houston, TX). Following antibodies were used in this study: anti-YAP(S127), anti-YAP(S397), anti-Bax, anti-caspase-3, anti-PARP, anti-p21 (Cell Signaling Technology, USA); anti-YAP and the HRP-conjugated secondary antibodies (Santa Cruz Biotechnology, USA); anti-CyclinD1, anti-survivin and anti-c-Myc (Bioworld Technology Inc, USA); anti-β-Actin (Zhong shan jin qiao, China).