Anti yap

For immunofluorescence assay, A549 cells were collected 48 h posttransfection and then seeded as 2 × 10³ cells/well in eight-well chamber slide (Millipore, Darmstadt, Germany). After incubation for 24 h, cells were washed with PBS and fixed with -20°C precold methanol for 10 min at -20°C. Then, cells were washed with PBS and incubated in 0.2% Triton for 10 min at room temperature. Cells were blocked in 5% fetal bovine serum for 1 h at room temperature and incubated with rabbit polyclonal anti-YAP (1 : 50, Proteintech) over night at 4°C and shaking. Next, cells were washed with PBS and incubated in a secondary antibody for 45 min. At last, cells were washed and stained with DAPI for 10 min. The images were taken by using a fluorescence microscope (Olympus BX51, Tokyo, Japan). ImageJ software was used to calculate the colocalization rate.

The whole proteins were extracted using cell lysis buffer. Then, SDS-PAGE was used to separate proteins with different sizes. After that, protein was transferred to a PVDF membrane (Millipore) and target protein was immunoblotted with specific primary antibody. Primary antibodies were listed as follows: anti-E-cadherin (1:5000, mouse anti-human, Cell Signaling Technology Inc.), anti-Vimentin (1:2000, mouse anti-human, Abcam), anti-HPV16 E6 + HPV18 E6 (1:1000, mouse anti-human papillomavirus, Abcam), anti-HPV16 E7 (1:1000, mouse anti-human papillomavirus, Abcam), anti-YAP (1:2000, mouse anti-human, Proteintech), anti-TAZ (1:2000, mouse anti-human, Proteintech), anti-CCL2 (1:2000, mouse anti-human, Proteintech), anti-flag-YAP (1:1000, mouse, Abbkine), anti-GAPDH (1:2000, rabbit anti-human, Sigma-Aldrich). After incubating goat anti-rabbit or goat anti-mouse secondary antibody (MultiSciences), immunoblots were visualized using the chemiluminescence (ECL) reagent (Beyotime Biotechnology) and ChemiDoc XRS+ System (Bio-Rad).

RIP was performed using Magna RIP RNA Binding Protein Immunoprecipitation Kit (Millipore, MA, USA) according to the manufacturer’s protocols. Antibodies including anti-IgG (Millipore, MA, USA), anti-YAP (Proteintech, Wuhan, China) and anti-14-3-3ζ(Proteintech, Wuhan, China) were listed in Table S4. The relative interaction between circRNA and proteins were assessed by qPCR, and normalized to input.

Total protein of cells was extracted by RIPA buffer (Thermo Fisher Scientific) with protease and phosphatase inhibitors (Thermo Fisher Scientific). We measured total protein concentration using a BCA protein assay kit. In total, 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis was used to separate the proteins. Then the electrophoresed proteins were transferred to the polyvinylidene fluoride membrane. Primary antibodies were incubated with the membrane at 4 °C overnight after blocking the non-specific sites with 5% skim milk for 2 h at room temperature. Primary antibodies were included anti-YAP (cat no. 13584-1-AP; 1:2000; Proteintech), anti-E-cadherin (cat No. 20874-1-AP; 1:5000; Proteintech), anti-Desmin (cat no. ab32362; 1:2000; abcam), anti-Vimentin (cat no. ab92547; 1:1000; abcam), anti-Snail (cat no. 13099-1-AP; 1:1000; Proteintech), anti-Type I collagen (cat no. ab260043; 1:1000; abcam), anti-α-SMA (cat no. ab32575; 1:1000; abcam) and anti-GAPDH (cat no. ab8245; 1:1000; abcam). On the next day, the membrane was incubated with secondary antibody for 1 h at room temperature. GAPDH acted as an internal reference.

Protein from cell or tissue lysates was separated by SDS-polyacrylamide gel electrophoresis (PAGE) and electro-transferred onto a polyvinylidene difluoride (PVDF) membrane (Pall Corp, Port Washington, NY). Then the membranes were blocked with 5% skimmed milk and incubated using primary antibodies anti-CSRP2 (1:800 dilution, Sigma), anti-GAPDH, anti-p130Cas, anti-p-p130Cas (Tyr410), anti-PAK, anti-p-PAK, anti-LIMK, anti-p-LIMK, anti-cortactin, anti-p-cortactin, anti-E-cadherin, anti-N-cadherin, anti-vimentin, anti-β-catenin (Cell Signaling Technology, Beverly, MA), anti-LATS1, anti-Yap (Proteintech), anti-ERK1/2, anti-p-ERK1/2 (Thr202/Tyr204), anti-p-Yap (Ser127) (Absci), anti-p-LATS1 (Thr1079/1041) (Bioss) at 4 °C. Then the membranes were incubated with appropriate secondary antibodies (anti-rabbit IgG, 1:3000 dilution, #7074; cell signaling). Enhanced chemiluminescence (Pierce, Rockford, 1L, USA) was used to detect the signal.

We used a high-resolution Micro-CT scanner (skyscan 1072; Skyscan, Aartselaar, Belgium) to scan the amount of new bone around the implant. 0.5 μm thickness of bone around the implant was defined as the new bone and analyzed. We measured the microstructure index of trabecular bone mineral density (BV/TV), trabecular thickness (TB. Th), trabecular number (TB. N), and trabecular spacing (TB. SP). For histological evaluation, pre-fixed femoral specimens were decalcified in 10% ethylenediaminetetraacetic acid (EDTA). After decalcification, the implant was gently removed and embedded into paraffin to perform tissue section (5 μm). For histological evaluation, tissue sections were stained with hematoxylin and eosin (H&E). To evaluate immunohistochemistry, tissue sections were dewaxed in xylene and standard alcohol gradients and then washed with PBS. Then, nonspecific binding sites were blocked with 10% goat serum. The sections were incubated with primary antibodies (anti-OCN, anti-Piezo1, and anti-Yap purchased from Proteintech) at 4°C overnight. The next day, sections were washed with PBS and incubated with the appropriate HRP-labeled secondary antibody (Abcam) at RT for 1 h and then further developed with diaminobenzidine solution.