Journal: Clinical cancer research : an official journal of the American Association for Cancer Research

Article Title: Diverse resistance mechanisms to the third-generation ALK inhibitor lorlatinib in ALK-rearranged lung cancer

doi: 10.1158/1078-0432.CCR-19-1104

Figure Lengend Snippet: Clinical and molecular features of patients with tumor molecular profiling on biopsies obtained upon resistance to ALK inhibitors in the MATCH-R study.

Article Snippet: NF2 gene knock-out was performed with the CRISPR/Cas9 KO Plasmid (h) from Santa Cruz Biotechnology (sc-400504).

Techniques: Biomarker Discovery, Sequencing, RNA Sequencing

Journal: Clinical cancer research : an official journal of the American Association for Cancer Research

Article Title: Diverse resistance mechanisms to the third-generation ALK inhibitor lorlatinib in ALK-rearranged lung cancer

doi: 10.1158/1078-0432.CCR-19-1104

Figure Lengend Snippet: NF2 loss of function mediates resistance to lorlatinib. A, Clinical course of patient MR135 and mutational profile of samples obtained on lorlatinib progression (PD, progressive Disease). B, Cell survival assay assessed with Cell Titer Glo of MR135 lorlatinib resistant cells from biopsy 1 (MR135-R1) treated for 7 days with the indicated concentrations of lorlatinib and vistusertib (AZD2014) alone or in combination. C, Immunoblot analysis from cell lysates of MR135-R1 treated for 24hs with the specified doses of lorlatinib, vistusertib (AZD2014) and ponatinib alone or in combination using indicated antibodies. D, Athymic nude mice bearing MR135-R2 PDX were administered lorlatinib or vistusertib 20 mg/kg orally. Tumor volumes, mean ±SD (n =8); (*** p < 0.001). E, Cell lysates from H3122 parental and H3122 cells with NF2 heterozygous deletions or homozygous deletions, generated by CRISPR-CAS9 gene editing, were immunoblotted to detect merlin expression. H3122 cells with bi-allelic NF2 knock-out lacked merlin expression. F, Cell survival assay of H3122 parental and H3122 NF2 knock-out (NF2 KO) cells treated with lorlatinib for 7 days. Cell survival was assessed by Cell Titer Glo. G, Cell proliferation assay of H3122 parental and H3122 NF2 KO cells untreated and treated with lorlatinib measured at baseline, day 2, day 5 and day 7. Cell viability was assessed with Cell Titer Glo. H, Caspase 3/7 activation (Caspase 3/7-Glo assay) relative to the number of live cells simultaneously assessed in the cell proliferation assay previously described. I, H3122 parental and NF2 KO cells were treated with the indicated doses of lorlatinib for 24hs. Cell lysates were immunoblotted to detect the selected proteins.

Article Snippet: NF2 gene knock-out was performed with the CRISPR/Cas9 KO Plasmid (h) from Santa Cruz Biotechnology (sc-400504).

Techniques: Clonogenic Cell Survival Assay, Western Blot, Generated, CRISPR, Expressing, Knock-Out, Proliferation Assay, Activation Assay, Glo Assay

Journal: The Journal of Biological Chemistry

Article Title: The tumor suppressor NF2 modulates TEAD4 stability and activity in Hippo signaling via direct interaction

doi: 10.1016/j.jbc.2024.107212

Figure Lengend Snippet: NF2 decr eases the protein levels of TEADs independently of LATS1/2 and YAP. A , the protein levels of HA-NF2 (IB: HA), YAP, TAZ, p-YAP, TEAD4, and GAPDH were determined via western blotting in HEK293T cells overexpressing HA-NF2. B , RT‒PCR analysis of TEAD1/2/3/4, NF2, and YAP mRNA levels was performed in HeLa cells overexpressing HA-NF2. ns: not significant (one-way ANOVA). C , MCF-10A cells were transfected with control siRNA or NF2 siRNA-1. Then, 100 μg/ml cycloheximide (CHX) was added, and the cells were harvested at the indicated time points. The protein levels of endogenous TEAD4, YAP, p-YAP, LATS-1/2 and tubulin were determined via western blotting. D , Wild-type (WT) and YAP knockout (KO) HeLa cells were transfected with or without NF2 siRNA-1. The protein levels of NF2, TEAD4, YAP, Cyr61, and GAPDH were determined via western blotting. E , WT and LATS1/2 KO HeLa cells were transfected with the control siRNA or NF2 siRNA-1/2. The protein levels of NF2, TEAD4, LATS1, YAP, p-YAP, Cyr61 and GAPDH were determined via western blotting.

Article Snippet: Antibodies used: TEAD4 (abcam, ab58310, 1:1000), TEAD2 (abcam, ab273017, 1:1000), LATS1 (abcam, ab70561, 1:1000), LATS2 (abcam, ab243657, 1:1000), YAP (abcam, ab52771, 1:1000), TAZ (abcam, ab110239, 1:1000), p-YAP (abcam, ab76252, 1:500), Cyr61 (abcam, ab228592, 1:1000), GAPDH (Proteintech, 60004-1-Ig, 1:2000), beta-Actin (abcam, ab8227, 1:3000), NF2 (ABclonal, A2456, 1:1000), ATP2 (ABclonal, A15792, 1:000), ABHD17A (Proteintech, 15854-1-AP, 1:500), His×6 (Proteintech, 66005-I-Ig, 1:1000), GST (ABclonal, AE001, 1:1000), Flag (Proteintech, 20543-I-AP, 1:1000), Myc (Cell Signaling Technology, 71D10, 1:1000), HRP-Goat Anti-Mouse Recombinant Secondary Antibody (Proteintech, RGAM001, 1:3000), HRP-Goat Anti-Rabbit Recombinant Secondary Antibody (RGAR001, 1:3000), IRDye 800CW, Steptavidin (LI-COR, D10114–10, 1:2000), IRDye 800CW Goat anti-Mouse IgG Secondary Antibody (LI-COR, 926–32210, 1:5000), IRDye 680RD Goat anti-Rabbit IgG Secondary Antibody (LI-COR, 926–68071, 1:3000).

Techniques: Western Blot, Transfection, Control, Knock-Out

Journal: The Journal of Biological Chemistry

Article Title: The tumor suppressor NF2 modulates TEAD4 stability and activity in Hippo signaling via direct interaction

doi: 10.1016/j.jbc.2024.107212

Figure Lengend Snippet: NF2 interacts with TEAD4 through the FERM domain and C-terminal tail. A , the GST pull-down assay was performed to assess the interaction between SUMO-tagged TEAD4-YBD and GST-tagged NF2 truncations. TEAD4 was indicated by western blotting, and schematic views of NF2 organization are shown in the right panel . B , coimmunoprecipitation of endogenous NF2 with TEAD4 was performed in HEK293T cells. The cell lysates were incubated with Protein-A Magnetic beads and TEAD4 antibodies or IgG. Immunoprecipitated samples were determined by western blotting. C , in vitro binding assay of GST-tagged NF2 C-terminal fragments with His-TEAD4-YBD. The GST-NF2 C-terminal fragments were pre-bound to GST beads, and beads were further incubated with TEAD4-YBD alone or the mixture of TEAD-YBD (His-tag) and FERM domain (His-tag). The FERM and TEAD4 on the beads were analyzed by western blotting. Quantitative analysis of the relative protein binding activity of NF2 to TEAD4 is shown in the right panel . Mean ± sd. N = 3, ∗∗ p < 0.01 (unpaired two-tailed t test with Welch’s correction). D , in vitro pull-down assay of MBP-NF2 WT and the A585W mutant with His-TEAD4-YBD to assess the interaction between TEAD4 and NF2. The level of TEAD4 was determined by western blotting. Quantitative analysis of the relative protein binding activity of NF2 to TEAD4 is shown in the right panel . Mean ± sd. N = 3, ∗∗ p < 0.01 (unpaired two-tailed t test with Welch’s correction). E , a cartoon model of NF2 binding to TEAD4 through the FERM domain and C-terminal tail.

Article Snippet: Antibodies used: TEAD4 (abcam, ab58310, 1:1000), TEAD2 (abcam, ab273017, 1:1000), LATS1 (abcam, ab70561, 1:1000), LATS2 (abcam, ab243657, 1:1000), YAP (abcam, ab52771, 1:1000), TAZ (abcam, ab110239, 1:1000), p-YAP (abcam, ab76252, 1:500), Cyr61 (abcam, ab228592, 1:1000), GAPDH (Proteintech, 60004-1-Ig, 1:2000), beta-Actin (abcam, ab8227, 1:3000), NF2 (ABclonal, A2456, 1:1000), ATP2 (ABclonal, A15792, 1:000), ABHD17A (Proteintech, 15854-1-AP, 1:500), His×6 (Proteintech, 66005-I-Ig, 1:1000), GST (ABclonal, AE001, 1:1000), Flag (Proteintech, 20543-I-AP, 1:1000), Myc (Cell Signaling Technology, 71D10, 1:1000), HRP-Goat Anti-Mouse Recombinant Secondary Antibody (Proteintech, RGAM001, 1:3000), HRP-Goat Anti-Rabbit Recombinant Secondary Antibody (RGAR001, 1:3000), IRDye 800CW, Steptavidin (LI-COR, D10114–10, 1:2000), IRDye 800CW Goat anti-Mouse IgG Secondary Antibody (LI-COR, 926–32210, 1:5000), IRDye 680RD Goat anti-Rabbit IgG Secondary Antibody (LI-COR, 926–68071, 1:3000).

Techniques: Pull Down Assay, Western Blot, Incubation, Magnetic Beads, Immunoprecipitation, In Vitro, Binding Assay, Protein Binding, Activity Assay, Two Tailed Test, Mutagenesis

Journal: The Journal of Biological Chemistry

Article Title: The tumor suppressor NF2 modulates TEAD4 stability and activity in Hippo signaling via direct interaction

doi: 10.1016/j.jbc.2024.107212

Figure Lengend Snippet: NF2 decreases TEAD4 protein levels via direct interaction. A , GST pull-down assay of GST-NF2-FERM WT and mutants with His-TEAD4-YBD was performed to assess the interaction between TEAD4 and NF2-FERM. The level of TEAD4 was determined by western blotting. B , GST pull-down assay of GST-NF2-507-595 WT and mutants with His-TEAD4-YBD was performed to assess the interaction between TEAD4 and NF2 C-terminal tail. The level of TEAD4 was determined by western blotting. C , HEK293T cells were transfected with Myc-tagged NF2 WT/mutants and Flag-tagged TEAD4. Cell lysates were incubated with anti-Flag affinity beads, Protein-A magnetic beads and Myc antibodies. Myc-NF2 (IB: Myc) and Flag-TEAD4 (IB: Flag) on the beads was determined by western blotting. D , TEAD4 protein levels in NF2 KO HEK293A cells stably expressing GFP-NF2 WT or mutants. Protein expressions were determined via western blotting. Quantitative analysis of the relative protein levels of TEAD4 are shown in the right panel . Mean ± sd. N = 3, ∗∗∗∗ p < 0.0001 (one-way ANOVA). E , competitive binding assay was performed to evaluate the binding effect of the YAP-TEAD4 complex with increasing doses of NF2. A schematic diagram of the competition assay is shown in the left panel . TEAD4 were pre-incubated with GST-YAP, after which NF2 was added as the competitor. The protein concentration on the beads was determined via SDS‒PAGE. ∗ Degradation.

Article Snippet: Antibodies used: TEAD4 (abcam, ab58310, 1:1000), TEAD2 (abcam, ab273017, 1:1000), LATS1 (abcam, ab70561, 1:1000), LATS2 (abcam, ab243657, 1:1000), YAP (abcam, ab52771, 1:1000), TAZ (abcam, ab110239, 1:1000), p-YAP (abcam, ab76252, 1:500), Cyr61 (abcam, ab228592, 1:1000), GAPDH (Proteintech, 60004-1-Ig, 1:2000), beta-Actin (abcam, ab8227, 1:3000), NF2 (ABclonal, A2456, 1:1000), ATP2 (ABclonal, A15792, 1:000), ABHD17A (Proteintech, 15854-1-AP, 1:500), His×6 (Proteintech, 66005-I-Ig, 1:1000), GST (ABclonal, AE001, 1:1000), Flag (Proteintech, 20543-I-AP, 1:1000), Myc (Cell Signaling Technology, 71D10, 1:1000), HRP-Goat Anti-Mouse Recombinant Secondary Antibody (Proteintech, RGAM001, 1:3000), HRP-Goat Anti-Rabbit Recombinant Secondary Antibody (RGAR001, 1:3000), IRDye 800CW, Steptavidin (LI-COR, D10114–10, 1:2000), IRDye 800CW Goat anti-Mouse IgG Secondary Antibody (LI-COR, 926–32210, 1:5000), IRDye 680RD Goat anti-Rabbit IgG Secondary Antibody (LI-COR, 926–68071, 1:3000).

Techniques: Pull Down Assay, Western Blot, Transfection, Incubation, Magnetic Beads, Stable Transfection, Expressing, Competitive Binding Assay, Binding Assay, Protein Concentration

Journal: The Journal of Biological Chemistry

Article Title: The tumor suppressor NF2 modulates TEAD4 stability and activity in Hippo signaling via direct interaction

doi: 10.1016/j.jbc.2024.107212

Figure Lengend Snippet: NF2 induces the cytoplasmic retention of TEAD4 via interaction. A , BiFC assays were performed to determine the location of the NF2-TEAD4 complex. HEK293T cells were transfected with TEAD4-nYFP, NF2-cYFP, NF2-cYFP and TEAD4-nYFP, NF2-cYFP and LATS1 -nYFP, and NF2-cYFP and NF2-nYFP. The fluorescence signals of intact YFP were captured. Representative images are shown here. Scale bar = 10 μm. B , quantification of flow cytometry data from the BiFC assay ( A ). Mean ± sd. ∗∗∗∗ p < 0.0001 (one-way ANOVA). C , immunofluorescence staining for endogenous TEAD4 was performed in NF2 KO HEK293A cells stably expressing WT or mutants GFP-NF2 WT and mutants at a high cell density. Scale bar = 10 μm. The co-localization analysis of NF2 and TEAD4 were shown in the right panel , and the fluorescence intensity and distance were measured using ImageJ.

Article Snippet: Antibodies used: TEAD4 (abcam, ab58310, 1:1000), TEAD2 (abcam, ab273017, 1:1000), LATS1 (abcam, ab70561, 1:1000), LATS2 (abcam, ab243657, 1:1000), YAP (abcam, ab52771, 1:1000), TAZ (abcam, ab110239, 1:1000), p-YAP (abcam, ab76252, 1:500), Cyr61 (abcam, ab228592, 1:1000), GAPDH (Proteintech, 60004-1-Ig, 1:2000), beta-Actin (abcam, ab8227, 1:3000), NF2 (ABclonal, A2456, 1:1000), ATP2 (ABclonal, A15792, 1:000), ABHD17A (Proteintech, 15854-1-AP, 1:500), His×6 (Proteintech, 66005-I-Ig, 1:1000), GST (ABclonal, AE001, 1:1000), Flag (Proteintech, 20543-I-AP, 1:1000), Myc (Cell Signaling Technology, 71D10, 1:1000), HRP-Goat Anti-Mouse Recombinant Secondary Antibody (Proteintech, RGAM001, 1:3000), HRP-Goat Anti-Rabbit Recombinant Secondary Antibody (RGAR001, 1:3000), IRDye 800CW, Steptavidin (LI-COR, D10114–10, 1:2000), IRDye 800CW Goat anti-Mouse IgG Secondary Antibody (LI-COR, 926–32210, 1:5000), IRDye 680RD Goat anti-Rabbit IgG Secondary Antibody (LI-COR, 926–68071, 1:3000).

Techniques: Transfection, Fluorescence, Flow Cytometry, Bimolecular Fluorescence Complementation Assay, Immunofluorescence, Staining, Stable Transfection, Expressing

Journal: The Journal of Biological Chemistry

Article Title: The tumor suppressor NF2 modulates TEAD4 stability and activity in Hippo signaling via direct interaction

doi: 10.1016/j.jbc.2024.107212

Figure Lengend Snippet: NF2 inhibits TEAD4 palmitoylation via direct interaction. A , schematic model of the in vitro auto-palmitoylation assay of recombinant TEAD4. B , TEAD4 was pre-incubated with NF2 or YAP. Then, in vitro auto-palmitoylation assay was performed to evaluate the palmitoylation of TEAD4 via streptavidin-mediated immunoblotting. C , quantitative analysis of the palmitoylated TEAD4 (Pal-TEAD4) panel in ( B ). D , schematic model of the in vivo palmitoylation assay with acyl resin-assisted capture methods in cells. E , in vivo palmitoylation assay to evaluate the palmitoylation levels of endogenous TEAD4 in NF2 KO HEK293A cells stably expressing the NF2 WT and mutants. The concentration of input TEAD4 was adjusted to a constant level for the palmitoylation capture assay, and the level of TEAD4 was determined via western blotting. The quantitative analysis of palmitoylated TEAD4 (Pal-TEAD4) is shown in the lower panel . Mean ± sd. N = 3, ∗∗ p < 0.01, ∗∗∗∗ p < 0.0001 (one-way ANOVA). F , in vitro ubiquitination assay was performed with purified recombinant E1, UbcH5b (the E2 ubiquitin-conjugating enzyme), and the E3 ligase CHIP to detect the ubiquitination levels of TEAD4 WT and 2CS. G , in vitro pull-down assay of MBP-NF2 with TEAD4 WT and 2CS. The two lanes were spliced from the same blot/gel, and the border was labeled with black dashed line .

Article Snippet: Antibodies used: TEAD4 (abcam, ab58310, 1:1000), TEAD2 (abcam, ab273017, 1:1000), LATS1 (abcam, ab70561, 1:1000), LATS2 (abcam, ab243657, 1:1000), YAP (abcam, ab52771, 1:1000), TAZ (abcam, ab110239, 1:1000), p-YAP (abcam, ab76252, 1:500), Cyr61 (abcam, ab228592, 1:1000), GAPDH (Proteintech, 60004-1-Ig, 1:2000), beta-Actin (abcam, ab8227, 1:3000), NF2 (ABclonal, A2456, 1:1000), ATP2 (ABclonal, A15792, 1:000), ABHD17A (Proteintech, 15854-1-AP, 1:500), His×6 (Proteintech, 66005-I-Ig, 1:1000), GST (ABclonal, AE001, 1:1000), Flag (Proteintech, 20543-I-AP, 1:1000), Myc (Cell Signaling Technology, 71D10, 1:1000), HRP-Goat Anti-Mouse Recombinant Secondary Antibody (Proteintech, RGAM001, 1:3000), HRP-Goat Anti-Rabbit Recombinant Secondary Antibody (RGAR001, 1:3000), IRDye 800CW, Steptavidin (LI-COR, D10114–10, 1:2000), IRDye 800CW Goat anti-Mouse IgG Secondary Antibody (LI-COR, 926–32210, 1:5000), IRDye 680RD Goat anti-Rabbit IgG Secondary Antibody (LI-COR, 926–68071, 1:3000).

Techniques: In Vitro, Recombinant, Incubation, Western Blot, In Vivo, Stable Transfection, Expressing, Concentration Assay, Ubiquitin Proteomics, Purification, Pull Down Assay, Labeling

Journal: The Journal of Biological Chemistry

Article Title: The tumor suppressor NF2 modulates TEAD4 stability and activity in Hippo signaling via direct interaction

doi: 10.1016/j.jbc.2024.107212

Figure Lengend Snippet: TEAD4 interaction is required for NF2 function to suppress tumor cell proliferation. A , NF2 KO HeLa or stably expressing NF2-WT/5A/A585W cells were subjected to BrdU incorporation assay. The fluorescence signals of BrdU and DAPI were detected. Representative images are shown here. Scale bar = 20 μm. B , the percentage of BrdU-positive cells in ( A ) was quantified. Mean ± sd. N = 3, ∗∗∗∗ p < 0.0001 (one-way ANOVA). C , NF2 KO HeLa or stably expressing NF2-WT/5A/A585W cells were subjected to cell counting kit-8 (CCK-8), and the optical density at 450 nm was quantified. Mean ± sd. N = 3, ∗∗∗ p < 0.001, ∗∗∗∗ p < 0.0001 (one-way ANOVA). D , coimmunoprecipitation of Myc-tagged NF2 WT and mutants with HA-tagged LATS2 was performed in HEK293T cells. Cell lysates were incubated with Protein-A Magnetic beads and HA antibodies. Myc-NF2 (IB: Myc) and HA-LATS2 (IB: HA) expression on the beads was determined via western blotting. The quantitative analysis of the binding of NF2 to LATS2 is shown. Mean ± sd. N = 3, ∗∗∗∗ p < 0.0001 (one-way ANOVA). E , working model for the molecular mechanism of NF2 function to suppress tumor cell proliferation through LATS1/2 ( right ) and directly through TEAD4 ( left ). NF2 directly binds to and downregulates TEAD4 activity could complement classical regulation through the Hippo pathway.

Article Snippet: Antibodies used: TEAD4 (abcam, ab58310, 1:1000), TEAD2 (abcam, ab273017, 1:1000), LATS1 (abcam, ab70561, 1:1000), LATS2 (abcam, ab243657, 1:1000), YAP (abcam, ab52771, 1:1000), TAZ (abcam, ab110239, 1:1000), p-YAP (abcam, ab76252, 1:500), Cyr61 (abcam, ab228592, 1:1000), GAPDH (Proteintech, 60004-1-Ig, 1:2000), beta-Actin (abcam, ab8227, 1:3000), NF2 (ABclonal, A2456, 1:1000), ATP2 (ABclonal, A15792, 1:000), ABHD17A (Proteintech, 15854-1-AP, 1:500), His×6 (Proteintech, 66005-I-Ig, 1:1000), GST (ABclonal, AE001, 1:1000), Flag (Proteintech, 20543-I-AP, 1:1000), Myc (Cell Signaling Technology, 71D10, 1:1000), HRP-Goat Anti-Mouse Recombinant Secondary Antibody (Proteintech, RGAM001, 1:3000), HRP-Goat Anti-Rabbit Recombinant Secondary Antibody (RGAR001, 1:3000), IRDye 800CW, Steptavidin (LI-COR, D10114–10, 1:2000), IRDye 800CW Goat anti-Mouse IgG Secondary Antibody (LI-COR, 926–32210, 1:5000), IRDye 680RD Goat anti-Rabbit IgG Secondary Antibody (LI-COR, 926–68071, 1:3000).

Techniques: Stable Transfection, Expressing, BrdU Incorporation Assay, Fluorescence, Cell Counting, CCK-8 Assay, Incubation, Magnetic Beads, Western Blot, Binding Assay, Activity Assay

Journal: The Journal of Biological Chemistry

Article Title: The tumor suppressor NF2 modulates TEAD4 stability and activity in Hippo signaling via direct interaction

doi: 10.1016/j.jbc.2024.107212

Figure Lengend Snippet:

Article Snippet: Antibodies used: TEAD4 (abcam, ab58310, 1:1000), TEAD2 (abcam, ab273017, 1:1000), LATS1 (abcam, ab70561, 1:1000), LATS2 (abcam, ab243657, 1:1000), YAP (abcam, ab52771, 1:1000), TAZ (abcam, ab110239, 1:1000), p-YAP (abcam, ab76252, 1:500), Cyr61 (abcam, ab228592, 1:1000), GAPDH (Proteintech, 60004-1-Ig, 1:2000), beta-Actin (abcam, ab8227, 1:3000), NF2 (ABclonal, A2456, 1:1000), ATP2 (ABclonal, A15792, 1:000), ABHD17A (Proteintech, 15854-1-AP, 1:500), His×6 (Proteintech, 66005-I-Ig, 1:1000), GST (ABclonal, AE001, 1:1000), Flag (Proteintech, 20543-I-AP, 1:1000), Myc (Cell Signaling Technology, 71D10, 1:1000), HRP-Goat Anti-Mouse Recombinant Secondary Antibody (Proteintech, RGAM001, 1:3000), HRP-Goat Anti-Rabbit Recombinant Secondary Antibody (RGAR001, 1:3000), IRDye 800CW, Steptavidin (LI-COR, D10114–10, 1:2000), IRDye 800CW Goat anti-Mouse IgG Secondary Antibody (LI-COR, 926–32210, 1:5000), IRDye 680RD Goat anti-Rabbit IgG Secondary Antibody (LI-COR, 926–68071, 1:3000).

Techniques:

Journal: Oncogene

Article Title: KIBRA exhibits MST-independent functional regulation of the Hippo signaling pathway in mammals.

doi: 10.1038/onc.2012.196

Figure Lengend Snippet: Figure 4. KIBRA expression antagonises YAP-induced transcriptional regulation. (a) mRNA expression of the YAP target genes BMP2, FGF1, RASSF4 and PDGFb was assessed by quantitative real-time PCR in MCF10A-YAP cells overexpressing KIBRA, Willin or Merlin. mRNA levels were compared with the empty vector control MCF10A-YAP cells. Willin and KIBRA overexpression increased BMP2 and RASSF4 mRNA levels and decreased FGF1 and PDGFb mRNA levels. Means were calculated from Ct values from two analyses conducted in triplicate. Error bars represent ±s.e. (n ¼ 6). MCF10A-YAP-vector vs MCF10A-YAP-KIBRA or MCF10A-YAP-Willin for all the analysed genes: *Po0.05, **Po0.01, ***Po0.001 Student’s t-test. MCF10A-YAP-vector vs MCF10A-YAP-Merlin: CTGF (P ¼ 0.129), FGF1 (P ¼ 0.005), BMP2 (P ¼ 0.585), RASSF4 (P ¼ 0.307), PDGFb (P ¼ 0.06), Student’s t-test. (b) Immunoblot analysis of KIBRA, Willin or Merlin overexpression in MCF10A-YAP cells, showing 2.7, 1.8 and 8.6-fold increases compared to empty vector control. b-actin was used as a loading control.

Article Snippet: All experiments were carried out at 80–90% confluency. pBABE Nf2 (Johnson et al.45) was obtained from Addgene (plasmid 14116).

Techniques: Expressing, Real-time Polymerase Chain Reaction, Plasmid Preparation, Control, Over Expression, Western Blot