Anti yap1

Molecular subtypes of medulloblastomas were detected, as recommended by the 2016 WHO classification:

WNT-activated tumors were identified by the presence of at least two features: CTNNB1 mutation, immunohistochemical positive nuclear reaction against β-catenin (#760-4242, clone 14, Cell Margue, dilution 1.68 µg/ml) and the presence of chromosome 6 monosomy detected by multiplex ligation-dependent probe amplification (MLPA).

SHH-activated tumors were determined by the presence of immunohistochemical positive reaction with anti-GAB1 (Abcam, Cambridge, USA, #ab27439 and/or ab #59362, dilution 1:100) and anti-YAP1 (Santa Cruz Biotechnology, Dallas, USA, #sc-101199, dilution 1:50) antibodies, as described by Ellison et al. [12 (link)].

The remaining tumors that were negative for the above features were assigned as non-WNT, non-SHH tumors.

Procedures for the detection of mutations in exon 3 of CTNNB1 and monosomy of chromosome 6 by MLPA are described elsewhere [13 (link)].

Cells were grown in six‐well plates to 80% confluency and harvested. Cell pellet was snap frozen and lysed in 100 μl lysis buffer (0.5% NP40, 50 mM HEPES (pH 7.5), 150 mM NaCl, 50 mM NaF, 400 nM Na₃VO₄, 1 mM PMSF and protease inhibitor cocktail). The cell lysate was cleared by centrifugation (15,000 g for 20 min), boiled for 5 min after the addition of 3× Laemmli sample buffer, loaded on NuPAGE 4–12% Bis‐Tris SDS–PAGE gels (Invitrogen) for gel electrophoresis and then transferred onto nitrocellulose membranes (Trans‐Blot Turbo, BioRad). The following primary antibodies were used: anti‐PTPN14 (#13808, Cell Signaling), anti‐actin (#179467, Abcam), anti‐YAP1 (#15407, Santa Cruz), anti‐YAP1phosphoS127 (#4911, Cell Signaling), and anti‐HA (HA.11,901513, BioLegend). Proteins were detected by enhanced chemiluminescence (ECL, Amersham) using horseradish‐peroxidase‐coupled secondary antibodies (Rabbit #7074, Cell Signaling and Mouse #115035003, Jackson ImmunoResearch).

After cells were seeded overnight, TPM1, TPM2, TPM3, YAP1, and TP53 siRNA (Tsingke) and Lipofectamine RNAiMAX (Invitrogen) were mixed for transfection on the following day. Control cells were transfected with scrambled siRNA (Tsingke). The knockdown efficiency was verified on the 3rd day, and the cells were then used for further experiments. For immunoblotting, cells were lysed in radioimmunoprecipitation assay buffer supplemented with 1% phenylmethanesulfonyl fluoride, and protein quantification was performed using the bicinchoninic acid method. The extracted proteins were separated by 10% gel electrophoresis and transferred to polyvinylidene fluoride membranes. The membranes were incubated with the following primary antibodies at 4 °C overnight: anti-GAPDH (Proteintech, dilution 1:50 000), anti-BAX (Proteintech, dilution 1:2 000), anti-Caspase-3 (Proteintech, dilution 1:1 000), anti-Caspase-9 (CST, dilution 1:1 000), anti-TPM1 (Proteintech, dilution 1:1 000), anti-TPM2 (Proteintech, dilution 1:1 000), anti-TPM3 (Proteintech, dilution 1:500), anti-YAP1 (Santa Cruz, dilution 1:500), anti-p-YAP1 (CST, dilution 1:1 000), and anti-TP53 (Proteintech, dilution 1:1 000). Primary antibody binding was detected using enhanced chemiluminescence with appropriate horseradish peroxidase-conjugated secondary antibodies.

Cells were fixed with 4% paraformaldehyde (Affymetrix Inc.) for 10 min, followed by permeabilization with 0.1% Triton X‐100 (MilliporeSigma) for 15 min and blocking for 1 h with a solution that contained 1% BSA (MilliporeSigma), 2% goat serum (Vector Laboratories), and 0.1% Triton X‐100. Next, anti‐Yap1 (1:50, Santa Cruz Biotechnology) and anti‐Histone H3 (1:400, Cell Signaling) antibodies were applied to cells overnight at 4 °C. Cells were then incubated with Alexa Flour 488 goat anti‐rabbit (1:100, Thermo Fisher Scientific), Alexa Flour 568 goat‐anti mouse (1:200, Thermo Fisher Scientific), and Hoechst 33 342 (1:2500, Biotium) for 2 h. Primary and secondary antibodies were dissolved in 1% BSA (MilliporeSigma)/0.3% Triton X‐100 (MilliporeSigma). Samples incubated only with secondary antibodies served as negative controls. For Phalloidin staining, cells were blocked with a solution containing 5% goat serum and 0.1% Triton X‐100, and stained with Phalloidin (1:100, Thermo Fisher Scientific) and Hoechst 33 342 (1:2500, Biotium) for 1 h.