Ab76252

The paraffin-embedded sections were dewaxed, dehydrated, and washed under tap water for 2 min. After reaction with 3% methanol-H₂O₂ for 20 min, the sections were rinsed with distilled water for 2 min and treated with 0.1 M PBS for 3 min, followed by antigen retrieval. Thereafter, the sections were blocked utilizing normal goat serum (C-0005, Shanghai Haoran Biotechnology Co., Ltd., Shanghai, China) and allowed to maintain at ambient temperature for 20 min. After that, the sections were probed with primary antibodies against STK4 (1 : 150, ab51134, Abcam) and p-YAP1 (ab76252, 1 : 200, Abcam) overnight at 4°C, and then re-probed with biotin-labeled goat anti-rabbit IgG (ab97051, dilution ratio of 1 : 2000, Abcam) at room temperature for 40 min. Subsequently, the samples were stained with 3,3′-diaminobenzidine (DA1010, Solarbio, Beijing, China) for 10 min, followed by 1 min counter-staining by hematoxylin (H8070, Solarbio), dehydration by gradient alcohol, clearing with xylene and mounting with neutral gum. The primary antibody was substituted with PBS as NC. Finally, the sections were observed under an optical microscope (CX41-12C02, Olympus) in 5 randomly selected high-power visual fields. The positive cells were those presenting with brown yellow granules, and the percentage of positive cell was calculated by 2 people blinded to the study.

Total cell lysates were prepared from HUVECs exposed to various experimental conditions. Equal amount of total proteins (8 μg) were loaded and separated on 4–12% NuPAGE gradient gel (Life Technologies), transferred to PVDF membrane and detect with Enhanced Chemiluminescence substrate (Pierce) as per the manufacturer’s instructions. Primary antibodies for YAP (14,074, Cell Signaling), YAP-PhosphoSer127 (ab76252, Abcam), VCAM1 (ab134047, Abcam), eNOS (610,297, BD Biosciences), Rabbit anti-CYR61 (144,795, Cell Signaling) and ARHGAP18 clone 2A3-F3 (in-house), were used at 1:500–1000 and Actin-HRP (Abcam) was used at 1:10,000 dilution in PBS/Tween20 containing 1% BSA.

HGC-27 and MKN-28 cells were harvested and extracted using lysis buffer (100 mM Tris-HCl, 2% SDS, 1 mM Mercaptoethanol, 25% Glycerol). Cell extracts were boiled in loading buffer and equal amount of cell extracts were separated on 15% SDS-PAGE gels. Separated protein bands were transferred into polyvinylidene fluoride (PVDF) membranes. The primary antibodies-anti-LATS1 (ab70561, Rabbit polyclonal antibody, Abcam, Cambridge, MA, USA), anti-YAP (ab52771, Rabbit monoclonal antibody, Abcam, Cambridge, MA, USA), anti-p-YAP (S127) (ab76252, Rabbit monoclonal antibody, Abcam, Cambridge, MA, USA) and anti-GAPDH (ab153802, Rabbit polyclonal antibody, Abcam, Cambridge, MA, USA) were diluted at a ratio of 1:1000 according to the instructions and incubated overnight at 4 °C. Horseradish peroxidase-linked secondary antibodies were added at a dilution ratio of 1:10,000, and incubated at room temperature for 1 h. The membranes were washed with PBS for three times and the immunoreactive bands were visualized using ECL-PLUS/Kit (GE Healthcare, Piscataway, NJ, USA) according to the kit’s instruction.

A total of 37 formalin-fixed, paraffin-embedded tissues were obtained from patients diagnosed with SCLC by bronchofiberscopy or biopsy between January 2012 and March 2015. All patients received care and follow-up in our hospital. Informed consent was obtained under the protocol approved by our hospital's ethics committee. The clinical characteristics are summarized in Table 1.
The endogenous peroxidase activity was blocked by soaking the deparaffinized specimens in 3.3% H₂O₂. The specimens were then incubated with primary antibodies (MST2, 1 : 200, ab87322, abcam; pMST2 (phosphor Thr180), 1 : 200, PA5-104616, Invitrogen; YAP1, 1 : 100, ab205270, abcam; pYAP1 (phosphor S127), 1 : 100,ab76252, abcam; CD133, 1 : 100, ab216323, Abcam) and the corresponding secondary antibodies [20 (link)]. Semiquantitative results were obtained by using the German semiquantitative scoring method, as previously described [21 (link)].

Antibodies plasmids and chemicals used in the study were listed: GAPDH (sc-47724, Santa cruz biotechnology, for western blotting), YAP1 (ab205270, abcam, for western blotting; sc-376830, Santa cruz biotechnology, for Immunofluorescence (IF) and Immunohistochemistry), p-YAP(Ser127) (ab76252, abcam, for western blotting), CYR61 (26689-1-AP, proteintch, for western blotting), c-Myc (ab32072, abcam, for western blotting), CyclinD1 (ab16663, abcam, for western blotting), LaminB1 (PB9611, Boster, for western blotting), RhoA (#2117, CST, for western blotting), ROCK1 (ab134181, abcam, for western blotting), PKA C-ɑ(#4782, CST, for western blotting), TGR5 (ab72608, abcam, for western blotting, for IF and Immunohistochemistry), and Ki67 (#9449, CST, for Immunohistochemistry). Ursodeoxycholic acid was obtained from Target Mol (Shanghai, China), while INT-777 was obtained from Medchemexpress. TGR5 receptor antogonist SBI-115, H89, and SQ22536 were purchased from Selleck, while KT5720 was purchased from Sigma.

Metafectene Pro transfection reagent was obtained from Biontex. siRNAs targeting 14-3-3σ (sc-29590), YAP1 (sc-38637) were purchased from Santa Cruz Biotechnology. Antibodies against GFP (ab290), YAP1 (ab52771), pYAP1 (ab76252) were from Abcam. Antibodies against 14-3-3σ (05-632), RRM1 (MABE567) and ChIP Assay kit (17-295) were purchased from EMD Millipore. Antibody against RRM2 was generated in-house [31 (link)]. Lipofectamine, pcDNA3.1(+) plasmid, and G418 were from Invitrogen. RNeasy Mini kit and Qiagen Blood and Cell Culture DNA Kit were from Qiagen. The iScript™ cDNA synthesis kit and the SYBR Green PCR master mix were from Bio-Rad and Applied Biosystems, respectively. Gemcitabine were purchased from Besse Medical