lentivirus of mutant yap 5sa  (Addgene inc)

Journal: Cancers

Article Title: Rac1 GTPase Regulates the βTrCP-Mediated Proteolysis of YAP Independently of the LATS1/2 Kinases

doi: 10.3390/cancers16213605

Figure Lengend Snippet: YAP degradation after Rac1 inhibition requires the SCF βTrCP E3 ubiquitin ligase. ( A ) Time course of YAP decline after Rac1 inhibition. HPAF/CD18 cells were harvested at the indicated time point after the addition of EHT-1864 (50 μM). Levels of YAP and S127-phosphorylated YAP were quantified by immunoblotting. GAPDH was used as an internal standard. ( B ) MG132 blocks the degradation of YAP elicited by the inhibition of Rac1. After 12 h of exposure to EHT-1864 (50 μM; +) or vehicle control (−), HPAF/CD18 cells were exposed or not to MG132 (20 μg/mL) for 4 h prior to harvesting. ( C ) Schematic description of YAP structure showing the position and sequence of critical degrons and phosphorylation sites. Upper drawing shows the structure of YAP, including its heterodimerization domain (TEAD), WD40 domains (WW), transactivation domain (TAD), putative degrons (βTrCP1/2, FBXW7), and LATS1/2 phosphorylation sites (S61, S109, S127, S128, S131, S163, S164, and S381). Lower left panel: Sequence of the putative FBXW7 phosphodegron highlighting its consensus and its potentially required phosphoserine group (S351). Changes introduced by the S351A/P352A mutation are also shown. Lower right panel: Sequence of the βTrCP degron of YAP highlighting its consensus and its required CK1 (S384, S387) and LATS1/2 (S381) phosphorylation sites. Changes introduced by the D383A/S384A mutation are also shown. ( D ) Detection of the Flag-YAP proteins in retrovirally infected Panc1 cells. Panc1 cells infected with pLXSH viruses carrying no insert (Empty), Flag-tagged YAP (WT), or its various mutant versions (5SA, D383A/S384A, S351A/P352A) were analyzed for the presence of Flag-YAP. GAPDH was used as an internal control. ( E ) The βTrCP degron is needed for YAP degradation after Rac1 inhibition, but not the S381 LATS1/2 phosphorylation site. In duplicate, Panc1 cells expressing the different mutants of Flag-YAP were exposed to EHT-1864 (50 μM). Sixteen hours later, Flag-tagged proteins were quantified using western blotting. Again, GAPDH was used as an internal control. ( F ) The siRNA-mediated knockdown of Skp1 blocks YAP degradation after Rac1 inhibition. Panc1 cells were transfected with skp1 siRNA or with a non-targeting siRNA. Forty-eight hours later, transfected cells were treated with EHT-1864 (50 μM; +) or vehicle control (−) for 16 h prior to western blot analysis. ( G ) The siRNA-mediated knockdown of the βTrCP1/2 proteins blocks YAP degradation after Rac1 inhibition. Panc1 cells were transfected with a non-targeting siRNA or with siRNA against βTrCP1, βTrCP2, or both proteins. Forty-eight hours later, transfected cells were treated with EHT-1864 (50 μM; +) or vehicle control (−) for 16 h prior to western blot analysis. GAPDH was again used as an internal standard. For all panels, densitometry readings for the indicated intensity ratios (pYAP/GAPDH, YAP/GAPDH, Skp1/GAPDH, and FLAG/GAPDH) are shown below the lanes of each western blot.

Article Snippet: PCR products encoding Flag-tagged YAP and its 5SA mutant were respectively amplified from vectors p2xFlag CMV2-YAP2 (a gift from Marius Sudol’s lab; purchased from Addgene cat# 19045) and pCMV2-flag YAP2 5SA (a gift from Kunliang Guan’s lab; purchased from Addgene cat# 27371) using the same forward (5′-GTACGC GTCGAC AGTGAACCGTCAGAATTGATCTA-3′; Sal1 site underlined) and reverse (5′-CATGGA AGATCT CTATAACCATGTAAGAAAGCTT-3′; Bgl2 site underlined) primers.

Techniques: Inhibition, Ubiquitin Proteomics, Western Blot, Control, Sequencing, Phospho-proteomics, Mutagenesis, Infection, Expressing, Knockdown, Transfection

Journal: Cancers

Article Title: Rac1 GTPase Regulates the βTrCP-Mediated Proteolysis of YAP Independently of the LATS1/2 Kinases

doi: 10.3390/cancers16213605

Figure Lengend Snippet: YAP degradation after Rac1 inhibition is LATS1/2-independent but requires CK1. ( A ) The silencing of LATS1/2 fails to prevent YAP degradation after Rac1 inhibition. Panc1 cells were transfected with a non-targeting siRNA or with siRNA against LATS1, LATS2, or both proteins. Forty-eight hours later, transfected cells were treated with EHT-1864 (50 μM; +) or the vehicle control (−) for 16 h prior to western blot analysis. GAPDH was again used as an internal standard. ( B ) Detection of LATS1 and LATS2 in the LATS1/2-proficient and -deficient HeLa cells. Cells were probed with the indicated antibodies. ( C , D ) LATS1/2 are not needed for YAP degradation after Rac1 inhibition. LATS1/2-proficient and -deficient cells HeLa cells were exposed to 50 μM EHT-1864 ( C ) or the DMSO vehicle ( D ) for 16 h, after which YAP levels were measured. ( E , F ) Panc1 cells expressing the Flag-YAP protein ( E ) or its 5SA mutant ( F ) were exposed or not to CK1 inhibitor IC-261 (10 μM), after which YAP levels were measured. GAPDH was used as an internal standard. For all panels, densitometry readings for the indicated intensity ratios (pYAP/GAPDH, YAP/GAPDH, LATS1/GAPDH, and FLAG/GAPDH) are shown below the lanes of each western blot.

Article Snippet: PCR products encoding Flag-tagged YAP and its 5SA mutant were respectively amplified from vectors p2xFlag CMV2-YAP2 (a gift from Marius Sudol’s lab; purchased from Addgene cat# 19045) and pCMV2-flag YAP2 5SA (a gift from Kunliang Guan’s lab; purchased from Addgene cat# 27371) using the same forward (5′-GTACGC GTCGAC AGTGAACCGTCAGAATTGATCTA-3′; Sal1 site underlined) and reverse (5′-CATGGA AGATCT CTATAACCATGTAAGAAAGCTT-3′; Bgl2 site underlined) primers.

Techniques: Inhibition, Transfection, Control, Western Blot, Expressing, Mutagenesis

Journal: Nature Communications

Article Title: Metabolic and transcriptomic reprogramming during contact inhibition-induced quiescence is mediated by YAP-dependent and YAP-independent mechanisms

doi: 10.1038/s41467-024-51117-y

Figure Lengend Snippet: a Luciferase assays with the 8×GTIIC-Lux reporter in WT and 5SA cells in the P and Q states. Data are normalized to WT P cells and are presented as the mean ± SEM of 4–5 biologically independent samples. P values by unpaired two-tailed student’s t -test are indicated except for **** P < 0.0001. b , c The extracellular acidification rate (ECAR) and individual glycolysis parameters in WT and 5SA cells in the P and Q states. Data are presented as the mean ± SEM of 3–4 biologically independent samples. P values by unpaired two-tailed student’s t -test are indicated. Glc glucose, Oligo oligomycin, 2DG 2-deoxyglucose. d Immunoblots and protein quantification of glycolytic enzymes and p27 kip1 in WT and 5SA cells in the P and Q states. Vinculin was monitored as a loading control. Representative immunoblots of four independent experiments are shown. Values are the mean ± SEM of 3-4 biologically independent experiments. P values by unpaired two-tailed student’s t -test are indicated except for **** P < 0.0001 and ns (PFK1 P = 0.0667, ALDOA P = 0.1451, P p27 kip1 P = 0.7940, Q p27 kip1 P = 0.9048). HK hexokinase, PFK1 phosphofructokinase 1, PKM pyruvate kinase M, ALDOA aldolase A, LDHA lactate dehydrogenase A. e Comparison of the quantified key extracellular metabolic fluxes (estimated flux ± SD) in WT and 5SA cells in the P and Q states. The flux rates of glucose uptake and lactate excretion are plotted. P values by unpaired two-tailed student’s t-test are indicated in the figure except for **** P < 0.0001 and ns P values (Glucose Cons. Q WT P = 0.3440, Lactate Pro P 5SA P = 0.0557) f Metabolic flux maps of WT and 5SA cells in the P and Q states. The flux maps were determined using 13 C-MFA of multiple datasets at isotopic steady state as described in Methods. g Comparison of key intracellular glycolytic fluxes (estimated flux ± SD) in WT and 5SA cells in the P and Q states. All flux fits passed the chi-square goodness-of-fit test. P values by unpaired two-tailed student’s t -test are indicated except for ns P = 0.5742.

Article Snippet: The cDNA for human YAP WT (pBABEpuro-Flag-YAP2, Addgene plasmid #27472) and the constitutively active mutant YAP 5SA (pCMV-flag YAP2 5SA, Addgene plasmid #27371) were cloned into the pBABE Hygro vector using Gibson Assembly according to the manufacturer’s protocol (NEB).

Techniques: Luciferase, Two Tailed Test, Western Blot, Control, Comparison

Journal: STAR Protocols

Article Title: Lentiviral-mediated ectopic expression of YAP and TAZ in YAP off cancer cell lines

doi: 10.1016/j.xpro.2021.100870

Figure Lengend Snippet: Proposed experimental set-up for flow cytometry analysis

Article Snippet: Plasmids expressing wild type YAP, a TEAD-binding mutant (Serine 94 mutated to Alanine; YAP S94A ) ( ) and an overactive YAP mutant (all five LATS Serine phosphorylation sites mutated to Alanine; YAP 5SA ) ( , ) are described and can be obtained from Addgene.

Techniques: Flow Cytometry

Journal: STAR Protocols

Article Title: Lentiviral-mediated ectopic expression of YAP and TAZ in YAP off cancer cell lines

doi: 10.1016/j.xpro.2021.100870

Figure Lengend Snippet: Example calculations for quantifying YAP expression by western blotting

Article Snippet: Plasmids expressing wild type YAP, a TEAD-binding mutant (Serine 94 mutated to Alanine; YAP S94A ) ( ) and an overactive YAP mutant (all five LATS Serine phosphorylation sites mutated to Alanine; YAP 5SA ) ( , ) are described and can be obtained from Addgene.

Techniques: Expressing, Western Blot

Journal: Scientific Reports

Article Title: Non-cell autonomous promotion of astrogenesis at late embryonic stages by constitutive YAP activation

doi: 10.1038/s41598-020-63890-z

Figure Lengend Snippet: Constitutive YAP activation forms SOX2 − cell clusters in the VZ at E18.5. ( A ) Schematic representation of the retroviral vector MSIG used in this study. Internal ribosome entry site (IRES) allows bicistronic expression of YAP 5SA and GFP, and MSIG expressing only GFP without an insert gene was used as a control. LTR, long terminal repeat; MCS, multicloning site. ( B, D ) Fluorescent microscopy of coronal sections of E16.5 embryonic brains that were intraventricularly injected at E13.5 with retroviral vectors expressing YAP 5SA. Gene-transferred cells were labeled with ( B ) anti-GFP antibody alone, or ( D ) a combination of anti-GFP (green) and anti-SOX2 (red) antibodies. ( F, H ) E18.5 brains injected at E13.5 were labeled using ( F ) only anti-GFP or ( H ) anti-GFP (green) and anti-SOX2 (red) primary antibodies. ( C, E, G ) Quantification of ( B, D, F ). Scale bars, 50 μm for ( B, D, H ) and 100 μm for ( F ). LV, lateral ventricle; VZ, ventricular zone; SVZ, subventricular zone; IZ, intermediate zone; CP, cortical plate; MZ, marginal zone. Error bars represent SD. Student’s t -test was used to determine statistical significance. ** P < 0.01, *** P < 0.001; n ≥ 3.

Article Snippet: The wild-type and constitutively active mutant (YAP 5SA) YAP vectors were purchased from Addgene (plasmids #33091 and #33093, respectively) (Cambridge, MA), and subcloned into the MluI site of the retroviral vector MSIG .

Techniques: Activation Assay, Retroviral, Plasmid Preparation, Expressing, Control, Microscopy, Injection, Labeling

Journal: Scientific Reports

Article Title: Non-cell autonomous promotion of astrogenesis at late embryonic stages by constitutive YAP activation

doi: 10.1038/s41598-020-63890-z

Figure Lengend Snippet: Constitutive YAP activation induces productive GFAP + cell generation at late embryonic periods in a non-cell autonomous fashion. ( A, B, D ) Neocortex or ( C ) ganglionic eminence ( G, E ) regions of E18.5 brains transduced with YAP 5SA retroviruses at E13.5 were double-labeled with the indicated antibodies. ( D ) Single plane confocal image of YAP 5SA-transduced E18.5 neocortex stained with anti-GFP (green) and anti-GFAP (red) antibodies, and the corresponding 14 μm z-stack view (top) at the position of the red line. Scale bars, 25 μm for ( D ), 50 μm for ( A, C ), and 100 μm for ( B ). LV, lateral ventricle.

Article Snippet: The wild-type and constitutively active mutant (YAP 5SA) YAP vectors were purchased from Addgene (plasmids #33091 and #33093, respectively) (Cambridge, MA), and subcloned into the MluI site of the retroviral vector MSIG .

Techniques: Activation Assay, Transduction, Labeling, Staining

Journal: Scientific Reports

Article Title: Non-cell autonomous promotion of astrogenesis at late embryonic stages by constitutive YAP activation

doi: 10.1038/s41598-020-63890-z

Figure Lengend Snippet: Heat-labile soluble factor(s) mediates YAP 5SA-induced astrogenesis in vitro . ( A ) Immunostaining using anti-GFAP after in vitro differentiation of co-cultured cells. E13.5 neural progenitor cells transduced with YAP 5SA retroviruses were mixed with untransduced neural progenitor cells at a ratio of 1:5 (transduced:untransduced) and then cultured in differentiation medium for 3 days. Quantification of ( A ) is shown in ( B ). ( C ) GFP (green) and GFAP (red) double immunostaining of cells differentiated under the same experimental conditions as ( A ). ( D ) Untransduced E13.5 neural progenitor cells were cultured in differentiation medium prepared by mixing conditioned medium (CM) of YAP 5SA-transduced neural progenitor culture and fresh differentiation medium in a 1:1 ratio. CM HI , heat-inactivated (56 °C for 30 min) CM. ( E ) Quantification of ( D ). The DAPI nuclear counterstain is shown in blue in ( A, D ). Scale bars, 100 μm for ( A, D ), and 200 μm for ( C ). Student’s t -test was used to determine statistical significance. ** P < 0.01, *** P < 0.001; n ≥ 3.

Article Snippet: The wild-type and constitutively active mutant (YAP 5SA) YAP vectors were purchased from Addgene (plasmids #33091 and #33093, respectively) (Cambridge, MA), and subcloned into the MluI site of the retroviral vector MSIG .

Techniques: In Vitro, Immunostaining, Cell Culture, Transduction, Double Immunostaining

Journal: Scientific Reports

Article Title: Non-cell autonomous promotion of astrogenesis at late embryonic stages by constitutive YAP activation

doi: 10.1038/s41598-020-63890-z

Figure Lengend Snippet: The ability of YAP 5SA to induce astrogenesis is nuclear localization-dependent. ( A ) Neocortical regions of E18.5 brains injected with YAP retroviruses at E13.5 were double-labeled with anti-GFP (green) and anti-Myc tag (red) antibodies. White arrowheads indicate GFP + /Myc tag − cells ( B ) Expression pattern of endogenous YAP (top) and phosphorylated form of YAP proteins (bottom) in the VZ at E14.5 were analyzed by immunostaining. ( C ) Schematic diagram showing the domain structures of YAP 5SA. ( D ) Expression of Myc-tagged YAP 5SA and PDZ-binding motif-deleted YAP 5SA (YAP 5SAΔPDZ) genes in transduced HEK 293 T cells was confirmed by Western blotting. ( E, F ) E18.5 brains transduced with YAP 5SA retroviruses at E13.5 were harvested and immunostained using ( E ) anti-Myc tag antibody to test nuclear localization, or ( F ) combination of anti-GFP and anti-GFAP antibodies to test astrogenic ability. ( G ) Immunostaining using anti-GFAP antibody after an in vitro differentiation assay. E13.5 neural progenitor cells transduced with YAP retroviruses were mixed with untransduced neural progenitor cells at a ratio of 1:5 (transduced: untransduced) and then cultured in differentiation medium for 3 days. Quantification is shown in ( H ). The DAPI nuclear counterstain is shown in blue in ( B, E, G ). Scale bars, 10 μm for ( E ), 25 μm for ( A, F ), 50 μm for ( B ), and 200 μm for ( G ). Student’s t -test was used to determine statistical significance. ** P < 0.01; n ≥ 3.

Article Snippet: The wild-type and constitutively active mutant (YAP 5SA) YAP vectors were purchased from Addgene (plasmids #33091 and #33093, respectively) (Cambridge, MA), and subcloned into the MluI site of the retroviral vector MSIG .

Techniques: Injection, Labeling, Expressing, Immunostaining, Binding Assay, Western Blot, Transduction, In Vitro, Differentiation Assay, Cell Culture

Journal: Scientific Reports

Article Title: Non-cell autonomous promotion of astrogenesis at late embryonic stages by constitutive YAP activation

doi: 10.1038/s41598-020-63890-z

Figure Lengend Snippet: TEAD-dependent transcriptional activation is associated with astrogenic activity of YAP 5SA. ( A ) Schematic diagram showing the location of mutations (red arrows) disrupting interaction with TEAD family transcription factors and WW domain functions in the YAP gene. ( B ) Expression of Myc-tagged YAP 5SA derivative mutants in transduced HEK 293 T cells was confirmed by Western blotting using an anti-Myc tag antibody. ( C ) Confocal microscopy images of wild-type and mutant YAP 5SA-transduced E18.5 neocortices stained with anti-GFP (green) and anti-GFAP (red) antibodies, and the corresponding Z-stack view (bottom) at the position of the red line. ( D ) An in vitro differentiation assay was performed after mixing retrovirally transduced E13.5 primary neural progenitor cells with untransduced cells at a ratio of 1:5, and GFAP immunostaining was carried out at 3 days postdifferentiation. ( E ) Quantification of ( D ). Scale bars, 25 μm for ( C ) and 100 μm for ( D ). Student’s t -test was used to determine statistical significance. *** P < 0.001; n ≥ 3.

Article Snippet: The wild-type and constitutively active mutant (YAP 5SA) YAP vectors were purchased from Addgene (plasmids #33091 and #33093, respectively) (Cambridge, MA), and subcloned into the MluI site of the retroviral vector MSIG .

Techniques: Activation Assay, Activity Assay, Expressing, Western Blot, Confocal Microscopy, Mutagenesis, Staining, In Vitro, Differentiation Assay, Immunostaining

Journal: Scientific Reports

Article Title: Non-cell autonomous promotion of astrogenesis at late embryonic stages by constitutive YAP activation

doi: 10.1038/s41598-020-63890-z

Figure Lengend Snippet: YAP 5SA-expressing cells produce CNTF. ( A ) E13.5 primary neural progenitor cells were transduced with retroviral vectors expressing YAP 5SA and incubated in differentiation medium. After 2 days, mRNA expression levels of indicated astrogenesis-related ligand genes were measured by qPCR. ( B ) Western blot analysis of YAP 5SA-transduced neural progenitor cell lysates using an anti-CNTF antibody. ( C ) E18.5 brains transduced with YAP 5SA retroviruses at E13.5 were harvested and double-immunostained using anti-GFP (green) and anti-CNTF (red) antibodies. ( D ) 14 μm z-stack view (right) at the position of the red line. ( E, F ) The ability of indicated YAP 5SA mutants to induce CNTF expression was tested by qPCR under the same experimental conditions as in ( A ). ( G ) Untransduced E13.5 neural progenitor cells were cultured in the differentiation medium containing conditioned medium of YAP 5SA-transduced neural progenitor culture with or without neutralizing antibodies against CNTF. ( H ) Quantification of ( F ). Scale bars, 25 μm for ( D ), 50 μm for ( C ), and 100 μm for ( G ). Student’s t -test was used to determine statistical significance. * P < 0.05, ** P < 0.01, *** P < 0.001; n ≥ 3.

Article Snippet: The wild-type and constitutively active mutant (YAP 5SA) YAP vectors were purchased from Addgene (plasmids #33091 and #33093, respectively) (Cambridge, MA), and subcloned into the MluI site of the retroviral vector MSIG .

Techniques: Expressing, Transduction, Retroviral, Incubation, Western Blot, Cell Culture

Journal: Scientific Reports

Article Title: Non-cell autonomous promotion of astrogenesis at late embryonic stages by constitutive YAP activation

doi: 10.1038/s41598-020-63890-z

Figure Lengend Snippet: YAP 5SA expression ectopically produces SMA + cells. ( A, B ) E13.5 neural progenitor cells transduced with YAP 5SA retroviruses were cultured in differentiation medium for 2 days, and ( A ) qPCR analysis of SMA and FN1 genes and ( B ) coimmunostaining using anti-GFP (green) and anti-SMA (red) antibodies were carried out. Cortical regions of E18.5 brains transduced with YAP 5SA-expressing retroviruses at E13.5 were labeled with ( C ) anti-GFAP (red) and anti-SMA (blue) antibodies, or ( D ) anti-GFP (green) antibody together with anti-SMA (red) or anti-FN1 (red) antibodies. Z-stack views at the position of the red line are shown on the right. LV, lateral ventricle. Scale bars, 20 μm for ( C ), 50 μm for ( D ), and 100 μm for ( B ). Student’s t -test was used to determine statistical significance. ** P < 0.01, *** P < 0.001; n ≥ 3.

Article Snippet: The wild-type and constitutively active mutant (YAP 5SA) YAP vectors were purchased from Addgene (plasmids #33091 and #33093, respectively) (Cambridge, MA), and subcloned into the MluI site of the retroviral vector MSIG .

Techniques: Expressing, Transduction, Cell Culture, Labeling

Journal: Hepatology (Baltimore, Md.)

Article Title: HMGB1 Controls Liver Cancer Initiation through YAP-dependent Aerobic Glycolysis

doi: 10.1002/hep.29663

Figure Lengend Snippet: (A) Indicated mouse and human HCC cells were treated with glycyrrhizin (0.625, 1.25, 2.5, 5, 10 mM) or verteporfin (2.5, 5, 10, 20, and 40 µM) for 24 hours. Cell viability was assayed (n=3, *p<0.05 versus control untreated group). (B) Clonogenic cell survival assay determined the reproductive ability of Hepa1-6 and HuH7 cells following treatment with glycyrrhizin (2.5 mM) or verteporfin (10 µM). A representative image is shown in the left panel. Relative reproductive ability is semi-quantified in the right panel (n=3, *p < 0.05 versus control untreated group). (C) Q-PCR analysis of mRNA expressions of indicated genes in HCC cells after glycyrrhizin (2.5 mM) or verteporfin (10 µM) treatment for 24 hours (n=3, *p<0.05). (D–F) ECAR levels in indicated HCC cells (n=3, *p < 0.05). (G) HIF1α DNA binding activity in indicated HCC cells with or without glycyrrhizin (2.5 mM) or verteporfin (10 µM) treatment for 24 hours (n=3, *p<0.05 versus control in the treated group). (H) Immunoprecipitation analysis of HIF1α-YAP complex in hepatocytes from DEN-induced Hmgb1f/f mice or primary human HCC (pHCC) cells or HuH7 cells. (I–K) YAP-knockdown HuH7 cells were transfected with YAP-cDNA or YAP-5SA-S94A mutant for 48 hours, and then treated with hypoxia (1% O2) for 24 hours. HIF1α DNA binding activity (I), gene expression (J), and YAP-HIF1α complex (K) were assayed.

Article Snippet: Expression plasmids for mouse HMGB1-cDNA and YAP-cDNA (pCMV6) were purchased from OriGene Technologies, Inc. YAP-5SA-S94A mutant (pCMV) and PGC1α-cDNA (pcDNA3.1) were obtained from Addgene.

Techniques: Clonogenic Cell Survival Assay, Binding Assay, Activity Assay, Immunoprecipitation, Transfection, Mutagenesis, Expressing