Journal: Cancers

Article Title: FAK Inhibition Induces Glioblastoma Cell Senescence-Like State through p62 and p27

doi: 10.3390/cancers12051086

Figure Lengend Snippet: Inhibition of focal adhesion kinase (FAK) reshapes glioblastoma (GBM) cell morphology and increases cell size. ( A ) Cell lysates from different GBM cell lines (A172, U251-MG, U87-MG, and T98G) were analyzed for PY397 FAK and total FAK. β-actin was used as a loading control. GBM cell lines display active PY397 FAK, with U251-MG and U87-MG showing the highest levels. ( B ) U251-MG and U87-MG cell lysates (control or treated with PF-573228 10 µM) were analyzed for active and total FAK. β-actin was used as a loading control. FAK inhibitor effectively reduced PY397 FAK levels. ( C ) Glial Fibrillary Acidic Protein (GFAP), βIII-tubulin, and Lamin B1 immunostainings performed in U251-MG cells (after 4–5 days of treatment with PF-573228 10 µM). Cytoskeleton remodeling accompanied by cell body enlargement and lobulated/enlarged nuclei is revealed by Lamin B1 immunostaining. Bars = 28 µm.

Article Snippet: Antibodies against the following proteins were used: PY397 FAK/PTK2 (Cell Signaling Danvers, MA, USA; 8556P), FAK (Cell Signaling 13009P), β-actin/ACTB (Merck Sigma-Aldrich A5441), βIII-tubulin/TUBB3 (Covance, Princetown, New Jersey, USA; PRB-435P), GFAP (Millipore, Burlington, MA, USA; AB5804), Ki67/MKI67 (Santa Cruz Biotechnology sc-15402), Lamin B1/LMNB1 (Calbiochem, San Diego, CA, USA; NA12), p27 (Genetex, Alton Pkwy Irvine, CA, USA; GTX100446-25), p62 (Novus Biologicals, Centennial, CO, USA; NBP1-48320), and SKP-2 (Abcam, Cambridge, UK; ab19877).

Techniques: Inhibition, Control, Immunostaining

Journal: Cancers

Article Title: FAK Inhibition Induces Glioblastoma Cell Senescence-Like State through p62 and p27

doi: 10.3390/cancers12051086

Figure Lengend Snippet: Inhibition of focal adhesion kinase (FAK) reshapes glioblastoma (GBM) cell morphology and increases cell size. ( A ) Cell lysates from different GBM cell lines (A172, U251-MG, U87-MG, and T98G) were analyzed for PY397 FAK and total FAK. β-actin was used as a loading control. GBM cell lines display active PY397 FAK, with U251-MG and U87-MG showing the highest levels. ( B ) U251-MG and U87-MG cell lysates (control or treated with PF-573228 10 µM) were analyzed for active and total FAK. β-actin was used as a loading control. FAK inhibitor effectively reduced PY397 FAK levels. ( C ) Glial Fibrillary Acidic Protein (GFAP), βIII-tubulin, and Lamin B1 immunostainings performed in U251-MG cells (after 4–5 days of treatment with PF-573228 10 µM). Cytoskeleton remodeling accompanied by cell body enlargement and lobulated/enlarged nuclei is revealed by Lamin B1 immunostaining. Bars = 28 µm.

Article Snippet: Antibodies against the following proteins were used: PY397 FAK/PTK2 (Cell Signaling Danvers, MA, USA; 8556P), FAK (Cell Signaling 13009P), β-actin/ACTB (Merck Sigma-Aldrich A5441), βIII-tubulin/TUBB3 (Covance, Princetown, New Jersey, USA; PRB-435P), GFAP (Millipore, Burlington, MA, USA; AB5804), Ki67/MKI67 (Santa Cruz Biotechnology sc-15402), Lamin B1/LMNB1 (Calbiochem, San Diego, CA, USA; NA12), p27 (Genetex, Alton Pkwy Irvine, CA, USA; GTX100446-25), p62 (Novus Biologicals, Centennial, CO, USA; NBP1-48320), and SKP-2 (Abcam, Cambridge, UK; ab19877).

Techniques: Inhibition, Control, Immunostaining

Journal: Cancers

Article Title: FAK Inhibition Induces Glioblastoma Cell Senescence-Like State through p62 and p27

doi: 10.3390/cancers12051086

Figure Lengend Snippet: PF-573228 promotes GBM cell senescence. ( A ) Representative SA-β-gal staining from U87-MG and U251-MG cells, control or treated with PF-573228 10 µM for four days. The % of SA-β-gal positive cells significantly increases upon FAK inhibition (*** p < 0.001; n ≥ 3). Bar = 251 µm. ( B ) p27/CDKN1B and p21/CDKN1A mRNA levels did not change significantly between control cells or cells treated with PF-573228 (10 µM, two or four days; n = 3). ( C ) p27 protein levels were analyzed in control cells and cells treated PF-573228 for two or four days. β-actin was used as loading control. Quantification of p27 normalized to β-actin indicates that p27 significantly increases after two days of treatment with PF-573228, and after two and four days in U87-MG and U251-MG cells (* p < 0.05; n ≥ 4). ( D ) SKP2 protein levels were analyzed in control cells or cells treated PF-573228 for two or four days. β-actin was used as loading control. Plot represents SKP2 levels normalized vs. control, which decrease in PF-573228-treated cells (*** p < 0.001; n = 4).

Article Snippet: Antibodies against the following proteins were used: PY397 FAK/PTK2 (Cell Signaling Danvers, MA, USA; 8556P), FAK (Cell Signaling 13009P), β-actin/ACTB (Merck Sigma-Aldrich A5441), βIII-tubulin/TUBB3 (Covance, Princetown, New Jersey, USA; PRB-435P), GFAP (Millipore, Burlington, MA, USA; AB5804), Ki67/MKI67 (Santa Cruz Biotechnology sc-15402), Lamin B1/LMNB1 (Calbiochem, San Diego, CA, USA; NA12), p27 (Genetex, Alton Pkwy Irvine, CA, USA; GTX100446-25), p62 (Novus Biologicals, Centennial, CO, USA; NBP1-48320), and SKP-2 (Abcam, Cambridge, UK; ab19877).

Techniques: Staining, Control, Inhibition

Journal: Cancers

Article Title: FAK Inhibition Induces Glioblastoma Cell Senescence-Like State through p62 and p27

doi: 10.3390/cancers12051086

Figure Lengend Snippet: p62 is repressed upon FAK inhibition. ( A ) p62 was analyzed by Western blot in control cells or cells treated with FAK inhibitors and β-actin was used as a loading control. Plot shows p62 protein levels normalized to β-actin and indicates that p62 significantly decreases after treatment with PF-573228 (10 µM, two and four days; n = 10) compared with untreated cells. ( B ) p62 mRNA relative expression in control U87-MG cells and cells treated with PF-573228 (10 µM, two or four days). p62 mRNA levels decrease after FAK inhibition (** p < 0.01; *** p < 0.001; n = 4).

Article Snippet: Antibodies against the following proteins were used: PY397 FAK/PTK2 (Cell Signaling Danvers, MA, USA; 8556P), FAK (Cell Signaling 13009P), β-actin/ACTB (Merck Sigma-Aldrich A5441), βIII-tubulin/TUBB3 (Covance, Princetown, New Jersey, USA; PRB-435P), GFAP (Millipore, Burlington, MA, USA; AB5804), Ki67/MKI67 (Santa Cruz Biotechnology sc-15402), Lamin B1/LMNB1 (Calbiochem, San Diego, CA, USA; NA12), p27 (Genetex, Alton Pkwy Irvine, CA, USA; GTX100446-25), p62 (Novus Biologicals, Centennial, CO, USA; NBP1-48320), and SKP-2 (Abcam, Cambridge, UK; ab19877).

Techniques: Inhibition, Western Blot, Control, Expressing

Journal: Cancers

Article Title: FAK Inhibition Induces Glioblastoma Cell Senescence-Like State through p62 and p27

doi: 10.3390/cancers12051086

Figure Lengend Snippet: Silencing p62 increases p27 expression. ( A ) Cell lysates of U87-MG cells expressing scrambled (Scr) or p62 shRNAs (shp62) were immunoblotted for p62 and compared with β-actin, used as loading control. Plot represents the p62 levels in cells expressing shRNAs of p62 or Scr (*** p < 0.001; n = 4). ( B ) Representative SA-β-gal staining performed in cells expressing Scr or p62 shRNAs. Plot represents the % of SA-β-gal compared with the total number of cells stained by Hoechst (** p < 0.01; n = 3). Bars = 251 µm. ( C ) Cell lysates of cells expressing Scr or p62 shRNAs were immunoblotted for p27 and β-actin. p27 levels (normalized to β-actin) increase in p62-depleted cells (* p < 0.05; n = 4). ( D ) p27/CDKN1B , p21/CDKN1A , and p16/CDKN2A mRNA levels were measured from cells expressing Scr or p62 shRNAs. p27 expression significantly increases, whereas p21 decreases, in p62-depleted cells ( p16 expression was upregulated in two of three experiments). *** p < 0.001 and ** p < 0.05 (n ≥ 3). ( E ) Proposed model: PF-573228 transcriptionally downregulates p62, decreases SKP2, and post-transcriptionally elevates p27. Both FAK inhibition and p62 silencing (blue lines) increase SA-β-gal activity (represented with an enlarged cell, arrested cell cycle, and β-gal positivity), in the latter case through transcriptional upregulation of p27.

Article Snippet: Antibodies against the following proteins were used: PY397 FAK/PTK2 (Cell Signaling Danvers, MA, USA; 8556P), FAK (Cell Signaling 13009P), β-actin/ACTB (Merck Sigma-Aldrich A5441), βIII-tubulin/TUBB3 (Covance, Princetown, New Jersey, USA; PRB-435P), GFAP (Millipore, Burlington, MA, USA; AB5804), Ki67/MKI67 (Santa Cruz Biotechnology sc-15402), Lamin B1/LMNB1 (Calbiochem, San Diego, CA, USA; NA12), p27 (Genetex, Alton Pkwy Irvine, CA, USA; GTX100446-25), p62 (Novus Biologicals, Centennial, CO, USA; NBP1-48320), and SKP-2 (Abcam, Cambridge, UK; ab19877).

Techniques: Expressing, Control, Staining, Inhibition, Activity Assay

Journal: Cancers

Article Title: FAK Inhibition Induces Glioblastoma Cell Senescence-Like State through p62 and p27

doi: 10.3390/cancers12051086

Figure Lengend Snippet: Inhibition of focal adhesion kinase (FAK) reshapes glioblastoma (GBM) cell morphology and increases cell size. ( A ) Cell lysates from different GBM cell lines (A172, U251-MG, U87-MG, and T98G) were analyzed for PY397 FAK and total FAK. β-actin was used as a loading control. GBM cell lines display active PY397 FAK, with U251-MG and U87-MG showing the highest levels. ( B ) U251-MG and U87-MG cell lysates (control or treated with PF-573228 10 µM) were analyzed for active and total FAK. β-actin was used as a loading control. FAK inhibitor effectively reduced PY397 FAK levels. ( C ) Glial Fibrillary Acidic Protein (GFAP), βIII-tubulin, and Lamin B1 immunostainings performed in U251-MG cells (after 4–5 days of treatment with PF-573228 10 µM). Cytoskeleton remodeling accompanied by cell body enlargement and lobulated/enlarged nuclei is revealed by Lamin B1 immunostaining. Bars = 28 µm.

Article Snippet: Antibodies against the following proteins were used: PY397 FAK/PTK2 (Cell Signaling Danvers, MA, USA; 8556P), FAK (Cell Signaling 13009P), β-actin/ACTB (Merck Sigma-Aldrich A5441), βIII-tubulin/TUBB3 (Covance, Princetown, New Jersey, USA; PRB-435P), GFAP (Millipore, Burlington, MA, USA; AB5804), Ki67/MKI67 (Santa Cruz Biotechnology sc-15402), Lamin B1/LMNB1 (Calbiochem, San Diego, CA, USA; NA12), p27 (Genetex, Alton Pkwy Irvine, CA, USA; GTX100446-25), p62 (Novus Biologicals, Centennial, CO, USA; NBP1-48320), and SKP-2 (Abcam, Cambridge, UK; ab19877).

Techniques: Inhibition, Immunostaining

Journal: Cancer cell

Article Title: A Platform of Synthetic Lethal Gene Interaction Networks Reveals that the GNAQ Uveal Melanoma Oncogene Controls the Hippo Pathway through FAK

doi: 10.1016/j.ccell.2019.01.009

Figure Lengend Snippet: A. Pipeline to identify and discover druggable therapeutic target-genes in UM: molecular screen, survival screen, in vitro screen and druggable screen. B. Table depicting the final 7 gene hits of our analysis. Molecular, clinical, phenotypic and synthetic lethal (SL) scores were calculated via our bioinformatic pipeline (Lee et al., 2018). Cell viability was assessed in vitro in UM cells (OMM1.3) following siRNA-mediated inhibition of each gene (cell viability normalized to OMM1.3 treated with non-targeting siRNA, siRNA-GNAQ used as positive control, mean, n=3). C. Oncoprint depicting the genomic landscape of TCGA uveal melanoma patient cohort (Robertson et al., 2017). Each bar represents one patient and their respective mutation or gene expression status (data were downloaded from TCGA UM cohort from cBioPortal (Gao et al., 2013). MutSig Q value is listed on the right. D. Kaplan-Meier plot depicting overall survival for UM patients stratified against FAK expression. FAK-High and FAK-Low groups are defined as top and bottom 50% of FAK gene expression. p value=0.002. E. UM cell lines (MEL270, 92.1, OMM1.3, OMM1.5 and MEL202 with GNAQ active mutation) are sensitive to FAK inhibition in a dose-dependent manner after treatment with VS-4718, SKCM cells (SK-MEL-28 with BRAF mutation) served as control. Driver oncogenes are indicated. Data are the percent viability normalized to vehicle treatment (mean ± SEM, n=3). F. VS-4718 inhibits FAK activation in OMM1.3 (decrease in pY397- FAK, left panel) and induces apoptosis (increased cleaved PARP, right panel). G. VS-4718 inhibits OMM1.3 colony formation in semisolid media (mean ± SEM, n=3; ***, p<0.001).

Article Snippet: ​ REAGENT or RESOURCE SOURCE IDENTIFIER Antibodies YAP Cell Signaling Technology, MA 14074 pS127-YAP Cell Signaling Technology, MA 4911 pS909-LATS1 Cell Signaling Technology, MA 9157 pT1079-LATS1 Cell Signaling Technology, MA 8654 LATS1 Cell Signaling Technology, MA 3477 P-MST1/MST2 Cell Signaling Technology, MA 3681 MST1 Cell Signaling Technology, MA 3682 GAPDH(14C10) Cell Signaling Technology, MA 2118 α-Tubulin Cell Signaling Technology, MA 3873 pY Cell Signaling Technology, MA 9411 HA-tag-HRP Cell Signaling Technology, MA 2999 HA-tag Cell Signaling Technology, MA 3724 myc-tag Cell Signaling Technology, MA 2278 pY397-FAK Cell Signaling Technology, MA 8556 FAK Cell Signaling Technology, MA 3285 cleaved PARP Cell Signaling Technology, MA 9541 p-ERK1/2 Cell Signaling Technology, MA 4370 ERK1/2 Cell Signaling Technology, MA 4696 MOB1 Cell Signaling Technology, MA 13730 pT35-MOB1 Cell Signaling Technology, MA 8699 Gαq(E-17) Santa Cruz Biotech., CA sc-393 FAK(C-20) Santa Cruz Biotech., CA sc-558 RhoA Cell Signaling Technology, MA 2117 TRIO(H120) Santa Cruz Biotech., CA sc-28564 Rac1 BD Biosciences, CA 610651 pY357-YAP Abcam, MA ab62751 LATS2 Bethyl Laboratories, TX A300–479A pY26-MOB1A Signalway Antibody, MA 12878 flag-tag-HRP Sigma-Aldrich, MO A8592 Ki67 DAKO, CA M724029–2 Bacterial strains DH5alpha Competent E. coli BioPioneer, CA GACC-96 Stbl3 Competent E. coli Thermo Fisher C737303 siRNAs Non-targeting Dharmacon, CO D-001810–0X GNAQ Sigma-Aldrich, MO SASI Hs01 00231793 PTK2 Thermo Fisher, MA s11485 AKT1 Thermo Fisher, MA s659 MGLL Thermo Fisher, MA s22380 MTHFD1 Thermo Fisher, MA s9032 CDK1 Thermo Fisher, MA s464 SIRT1 Thermo Fisher, MA s223591 PSMB5 Thermo Fisher, MA s11354 TRIO Dharmacon, CO L-005047–00-0005 RHOA Dharmacon, CO L-003860–00-0005 RAC1 Dharmacon, CO L-003560–00-0005 LATS1 Sigma-Aldrich, MO Hs01 00046128 LATS2 Sigma-Aldrich, MO Hs01 00158803 DNAs pCMV-myc-MST1 Addgene 8847 pCMV2-FLAG-SAV1 Addgene 18970 pcDNA3-HA-MOB1 Addgene 32835 pcDNA3-HA-Y26F-MOB1 Generated in-lab NA pLENTi-HA-MOB1 Generated in-lab NA pLENTi-HA-Y26F-MOB1 Generated in-lab NA pGEX-HA-MOB1 Generated in-lab NA pGEX-HA-Y26F-MOB1 Generated in-lab NA pLVX-T etOne-FLAG-FAK Generated in-lab NA p2xFLAG-CMV2-LATS1 Addgene 18971 8xGTIIC-luciferase Addgene 34615 REAGENT alamarBlue™ Reagent Grand Island, NY DAL1100 FAK Kinase Enzyme System Promega V1971 YAP1 (Human) Recombinant Abnova H00010413-P01 Protein Glutathione Sepharose 4B GE Healthcare 17–0756-01 N/C Extraction Reagents ThermoFisher 78833 Software and Algorithms ISLE Lee et al 2018 https://www.github.com/jooslee/ISLE Open in a separate window KEY RESOURCES TABLE Despite the central role of Hippo/YAP-regulating mechanisms in uveal melanoma (UM), there are no clinically effective therapeutic targets.

Techniques: In Vitro, Inhibition, Positive Control, Mutagenesis, Expressing, Activation Assay

Journal: Cancer cell

Article Title: A Platform of Synthetic Lethal Gene Interaction Networks Reveals that the GNAQ Uveal Melanoma Oncogene Controls the Hippo Pathway through FAK

doi: 10.1016/j.ccell.2019.01.009

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: ​ REAGENT or RESOURCE SOURCE IDENTIFIER Antibodies YAP Cell Signaling Technology, MA 14074 pS127-YAP Cell Signaling Technology, MA 4911 pS909-LATS1 Cell Signaling Technology, MA 9157 pT1079-LATS1 Cell Signaling Technology, MA 8654 LATS1 Cell Signaling Technology, MA 3477 P-MST1/MST2 Cell Signaling Technology, MA 3681 MST1 Cell Signaling Technology, MA 3682 GAPDH(14C10) Cell Signaling Technology, MA 2118 α-Tubulin Cell Signaling Technology, MA 3873 pY Cell Signaling Technology, MA 9411 HA-tag-HRP Cell Signaling Technology, MA 2999 HA-tag Cell Signaling Technology, MA 3724 myc-tag Cell Signaling Technology, MA 2278 pY397-FAK Cell Signaling Technology, MA 8556 FAK Cell Signaling Technology, MA 3285 cleaved PARP Cell Signaling Technology, MA 9541 p-ERK1/2 Cell Signaling Technology, MA 4370 ERK1/2 Cell Signaling Technology, MA 4696 MOB1 Cell Signaling Technology, MA 13730 pT35-MOB1 Cell Signaling Technology, MA 8699 Gαq(E-17) Santa Cruz Biotech., CA sc-393 FAK(C-20) Santa Cruz Biotech., CA sc-558 RhoA Cell Signaling Technology, MA 2117 TRIO(H120) Santa Cruz Biotech., CA sc-28564 Rac1 BD Biosciences, CA 610651 pY357-YAP Abcam, MA ab62751 LATS2 Bethyl Laboratories, TX A300–479A pY26-MOB1A Signalway Antibody, MA 12878 flag-tag-HRP Sigma-Aldrich, MO A8592 Ki67 DAKO, CA M724029–2 Bacterial strains DH5alpha Competent E. coli BioPioneer, CA GACC-96 Stbl3 Competent E. coli Thermo Fisher C737303 siRNAs Non-targeting Dharmacon, CO D-001810–0X GNAQ Sigma-Aldrich, MO SASI Hs01 00231793 PTK2 Thermo Fisher, MA s11485 AKT1 Thermo Fisher, MA s659 MGLL Thermo Fisher, MA s22380 MTHFD1 Thermo Fisher, MA s9032 CDK1 Thermo Fisher, MA s464 SIRT1 Thermo Fisher, MA s223591 PSMB5 Thermo Fisher, MA s11354 TRIO Dharmacon, CO L-005047–00-0005 RHOA Dharmacon, CO L-003860–00-0005 RAC1 Dharmacon, CO L-003560–00-0005 LATS1 Sigma-Aldrich, MO Hs01 00046128 LATS2 Sigma-Aldrich, MO Hs01 00158803 DNAs pCMV-myc-MST1 Addgene 8847 pCMV2-FLAG-SAV1 Addgene 18970 pcDNA3-HA-MOB1 Addgene 32835 pcDNA3-HA-Y26F-MOB1 Generated in-lab NA pLENTi-HA-MOB1 Generated in-lab NA pLENTi-HA-Y26F-MOB1 Generated in-lab NA pGEX-HA-MOB1 Generated in-lab NA pGEX-HA-Y26F-MOB1 Generated in-lab NA pLVX-T etOne-FLAG-FAK Generated in-lab NA p2xFLAG-CMV2-LATS1 Addgene 18971 8xGTIIC-luciferase Addgene 34615 REAGENT alamarBlue™ Reagent Grand Island, NY DAL1100 FAK Kinase Enzyme System Promega V1971 YAP1 (Human) Recombinant Abnova H00010413-P01 Protein Glutathione Sepharose 4B GE Healthcare 17–0756-01 N/C Extraction Reagents ThermoFisher 78833 Software and Algorithms ISLE Lee et al 2018 https://www.github.com/jooslee/ISLE Open in a separate window KEY RESOURCES TABLE Despite the central role of Hippo/YAP-regulating mechanisms in uveal melanoma (UM), there are no clinically effective therapeutic targets.

Techniques: Generated, Recombinant, Software

Journal: Cancer cell

Article Title: A Platform of Synthetic Lethal Gene Interaction Networks Reveals that the GNAQ Uveal Melanoma Oncogene Controls the Hippo Pathway through FAK

doi: 10.1016/j.ccell.2019.01.009

Figure Lengend Snippet: A. Pipeline to identify and discover druggable therapeutic target-genes in UM: molecular screen, survival screen, in vitro screen and druggable screen. B. Table depicting the final 7 gene hits of our analysis. Molecular, clinical, phenotypic and synthetic lethal (SL) scores were calculated via our bioinformatic pipeline (Lee et al., 2018). Cell viability was assessed in vitro in UM cells (OMM1.3) following siRNA-mediated inhibition of each gene (cell viability normalized to OMM1.3 treated with non-targeting siRNA, siRNA-GNAQ used as positive control, mean, n=3). C. Oncoprint depicting the genomic landscape of TCGA uveal melanoma patient cohort (Robertson et al., 2017). Each bar represents one patient and their respective mutation or gene expression status (data were downloaded from TCGA UM cohort from cBioPortal (Gao et al., 2013). MutSig Q value is listed on the right. D. Kaplan-Meier plot depicting overall survival for UM patients stratified against FAK expression. FAK-High and FAK-Low groups are defined as top and bottom 50% of FAK gene expression. p value=0.002. E. UM cell lines (MEL270, 92.1, OMM1.3, OMM1.5 and MEL202 with GNAQ active mutation) are sensitive to FAK inhibition in a dose-dependent manner after treatment with VS-4718, SKCM cells (SK-MEL-28 with BRAF mutation) served as control. Driver oncogenes are indicated. Data are the percent viability normalized to vehicle treatment (mean ± SEM, n=3). F. VS-4718 inhibits FAK activation in OMM1.3 (decrease in pY397- FAK, left panel) and induces apoptosis (increased cleaved PARP, right panel). G. VS-4718 inhibits OMM1.3 colony formation in semisolid media (mean ± SEM, n=3; ***, p<0.001).

Article Snippet: ​ REAGENT or RESOURCE SOURCE IDENTIFIER Antibodies YAP Cell Signaling Technology, MA 14074 pS127-YAP Cell Signaling Technology, MA 4911 pS909-LATS1 Cell Signaling Technology, MA 9157 pT1079-LATS1 Cell Signaling Technology, MA 8654 LATS1 Cell Signaling Technology, MA 3477 P-MST1/MST2 Cell Signaling Technology, MA 3681 MST1 Cell Signaling Technology, MA 3682 GAPDH(14C10) Cell Signaling Technology, MA 2118 α-Tubulin Cell Signaling Technology, MA 3873 pY Cell Signaling Technology, MA 9411 HA-tag-HRP Cell Signaling Technology, MA 2999 HA-tag Cell Signaling Technology, MA 3724 myc-tag Cell Signaling Technology, MA 2278 pY397-FAK Cell Signaling Technology, MA 8556 FAK Cell Signaling Technology, MA 3285 cleaved PARP Cell Signaling Technology, MA 9541 p-ERK1/2 Cell Signaling Technology, MA 4370 ERK1/2 Cell Signaling Technology, MA 4696 MOB1 Cell Signaling Technology, MA 13730 pT35-MOB1 Cell Signaling Technology, MA 8699 Gαq(E-17) Santa Cruz Biotech., CA sc-393 FAK(C-20) Santa Cruz Biotech., CA sc-558 RhoA Cell Signaling Technology, MA 2117 TRIO(H120) Santa Cruz Biotech., CA sc-28564 Rac1 BD Biosciences, CA 610651 pY357-YAP Abcam, MA ab62751 LATS2 Bethyl Laboratories, TX A300–479A pY26-MOB1A Signalway Antibody, MA 12878 flag-tag-HRP Sigma-Aldrich, MO A8592 Ki67 DAKO, CA M724029–2 Bacterial strains DH5alpha Competent E. coli BioPioneer, CA GACC-96 Stbl3 Competent E. coli Thermo Fisher C737303 siRNAs Non-targeting Dharmacon, CO D-001810–0X GNAQ Sigma-Aldrich, MO SASI Hs01 00231793 PTK2 Thermo Fisher, MA s11485 AKT1 Thermo Fisher, MA s659 MGLL Thermo Fisher, MA s22380 MTHFD1 Thermo Fisher, MA s9032 CDK1 Thermo Fisher, MA s464 SIRT1 Thermo Fisher, MA s223591 PSMB5 Thermo Fisher, MA s11354 TRIO Dharmacon, CO L-005047–00-0005 RHOA Dharmacon, CO L-003860–00-0005 RAC1 Dharmacon, CO L-003560–00-0005 LATS1 Sigma-Aldrich, MO Hs01 00046128 LATS2 Sigma-Aldrich, MO Hs01 00158803 DNAs pCMV-myc-MST1 Addgene 8847 pCMV2-FLAG-SAV1 Addgene 18970 pcDNA3-HA-MOB1 Addgene 32835 pcDNA3-HA-Y26F-MOB1 Generated in-lab NA pLENTi-HA-MOB1 Generated in-lab NA pLENTi-HA-Y26F-MOB1 Generated in-lab NA pGEX-HA-MOB1 Generated in-lab NA pGEX-HA-Y26F-MOB1 Generated in-lab NA pLVX-T etOne-FLAG-FAK Generated in-lab NA p2xFLAG-CMV2-LATS1 Addgene 18971 8xGTIIC-luciferase Addgene 34615 REAGENT alamarBlue™ Reagent Grand Island, NY DAL1100 FAK Kinase Enzyme System Promega V1971 YAP1 (Human) Recombinant Abnova H00010413-P01 Protein Glutathione Sepharose 4B GE Healthcare 17–0756-01 N/C Extraction Reagents ThermoFisher 78833 Software and Algorithms ISLE Lee et al 2018 https://www.github.com/jooslee/ISLE Open in a separate window KEY RESOURCES TABLE Despite the central role of Hippo/YAP-regulating mechanisms in uveal melanoma (UM), there are no clinically effective therapeutic targets.

Techniques: In Vitro, Inhibition, Positive Control, Mutagenesis, Expressing, Activation Assay

Journal: Cancer cell

Article Title: A Platform of Synthetic Lethal Gene Interaction Networks Reveals that the GNAQ Uveal Melanoma Oncogene Controls the Hippo Pathway through FAK

doi: 10.1016/j.ccell.2019.01.009

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: ​ REAGENT or RESOURCE SOURCE IDENTIFIER Antibodies YAP Cell Signaling Technology, MA 14074 pS127-YAP Cell Signaling Technology, MA 4911 pS909-LATS1 Cell Signaling Technology, MA 9157 pT1079-LATS1 Cell Signaling Technology, MA 8654 LATS1 Cell Signaling Technology, MA 3477 P-MST1/MST2 Cell Signaling Technology, MA 3681 MST1 Cell Signaling Technology, MA 3682 GAPDH(14C10) Cell Signaling Technology, MA 2118 α-Tubulin Cell Signaling Technology, MA 3873 pY Cell Signaling Technology, MA 9411 HA-tag-HRP Cell Signaling Technology, MA 2999 HA-tag Cell Signaling Technology, MA 3724 myc-tag Cell Signaling Technology, MA 2278 pY397-FAK Cell Signaling Technology, MA 8556 FAK Cell Signaling Technology, MA 3285 cleaved PARP Cell Signaling Technology, MA 9541 p-ERK1/2 Cell Signaling Technology, MA 4370 ERK1/2 Cell Signaling Technology, MA 4696 MOB1 Cell Signaling Technology, MA 13730 pT35-MOB1 Cell Signaling Technology, MA 8699 Gαq(E-17) Santa Cruz Biotech., CA sc-393 FAK(C-20) Santa Cruz Biotech., CA sc-558 RhoA Cell Signaling Technology, MA 2117 TRIO(H120) Santa Cruz Biotech., CA sc-28564 Rac1 BD Biosciences, CA 610651 pY357-YAP Abcam, MA ab62751 LATS2 Bethyl Laboratories, TX A300–479A pY26-MOB1A Signalway Antibody, MA 12878 flag-tag-HRP Sigma-Aldrich, MO A8592 Ki67 DAKO, CA M724029–2 Bacterial strains DH5alpha Competent E. coli BioPioneer, CA GACC-96 Stbl3 Competent E. coli Thermo Fisher C737303 siRNAs Non-targeting Dharmacon, CO D-001810–0X GNAQ Sigma-Aldrich, MO SASI Hs01 00231793 PTK2 Thermo Fisher, MA s11485 AKT1 Thermo Fisher, MA s659 MGLL Thermo Fisher, MA s22380 MTHFD1 Thermo Fisher, MA s9032 CDK1 Thermo Fisher, MA s464 SIRT1 Thermo Fisher, MA s223591 PSMB5 Thermo Fisher, MA s11354 TRIO Dharmacon, CO L-005047–00-0005 RHOA Dharmacon, CO L-003860–00-0005 RAC1 Dharmacon, CO L-003560–00-0005 LATS1 Sigma-Aldrich, MO Hs01 00046128 LATS2 Sigma-Aldrich, MO Hs01 00158803 DNAs pCMV-myc-MST1 Addgene 8847 pCMV2-FLAG-SAV1 Addgene 18970 pcDNA3-HA-MOB1 Addgene 32835 pcDNA3-HA-Y26F-MOB1 Generated in-lab NA pLENTi-HA-MOB1 Generated in-lab NA pLENTi-HA-Y26F-MOB1 Generated in-lab NA pGEX-HA-MOB1 Generated in-lab NA pGEX-HA-Y26F-MOB1 Generated in-lab NA pLVX-T etOne-FLAG-FAK Generated in-lab NA p2xFLAG-CMV2-LATS1 Addgene 18971 8xGTIIC-luciferase Addgene 34615 REAGENT alamarBlue™ Reagent Grand Island, NY DAL1100 FAK Kinase Enzyme System Promega V1971 YAP1 (Human) Recombinant Abnova H00010413-P01 Protein Glutathione Sepharose 4B GE Healthcare 17–0756-01 N/C Extraction Reagents ThermoFisher 78833 Software and Algorithms ISLE Lee et al 2018 https://www.github.com/jooslee/ISLE Open in a separate window KEY RESOURCES TABLE Despite the central role of Hippo/YAP-regulating mechanisms in uveal melanoma (UM), there are no clinically effective therapeutic targets.

Techniques: Generated, Recombinant, Software

Journal: Cancer cell

Article Title: A Platform of Synthetic Lethal Gene Interaction Networks Reveals that the GNAQ Uveal Melanoma Oncogene Controls the Hippo Pathway through FAK

doi: 10.1016/j.ccell.2019.01.009

Figure Lengend Snippet: A. Pipeline to identify and discover druggable therapeutic target-genes in UM: molecular screen, survival screen, in vitro screen and druggable screen. B. Table depicting the final 7 gene hits of our analysis. Molecular, clinical, phenotypic and synthetic lethal (SL) scores were calculated via our bioinformatic pipeline (Lee et al., 2018). Cell viability was assessed in vitro in UM cells (OMM1.3) following siRNA-mediated inhibition of each gene (cell viability normalized to OMM1.3 treated with non-targeting siRNA, siRNA-GNAQ used as positive control, mean, n=3). C. Oncoprint depicting the genomic landscape of TCGA uveal melanoma patient cohort (Robertson et al., 2017). Each bar represents one patient and their respective mutation or gene expression status (data were downloaded from TCGA UM cohort from cBioPortal (Gao et al., 2013). MutSig Q value is listed on the right. D. Kaplan-Meier plot depicting overall survival for UM patients stratified against FAK expression. FAK-High and FAK-Low groups are defined as top and bottom 50% of FAK gene expression. p value=0.002. E. UM cell lines (MEL270, 92.1, OMM1.3, OMM1.5 and MEL202 with GNAQ active mutation) are sensitive to FAK inhibition in a dose-dependent manner after treatment with VS-4718, SKCM cells (SK-MEL-28 with BRAF mutation) served as control. Driver oncogenes are indicated. Data are the percent viability normalized to vehicle treatment (mean ± SEM, n=3). F. VS-4718 inhibits FAK activation in OMM1.3 (decrease in pY397- FAK, left panel) and induces apoptosis (increased cleaved PARP, right panel). G. VS-4718 inhibits OMM1.3 colony formation in semisolid media (mean ± SEM, n=3; ***, p<0.001).

Article Snippet: REAGENT or RESOURCE SOURCE IDENTIFIER Antibodies YAP Cell Signaling Technology, MA 14074 pS127-YAP Cell Signaling Technology, MA 4911 pS909-LATS1 Cell Signaling Technology, MA 9157 pT1079-LATS1 Cell Signaling Technology, MA 8654 LATS1 Cell Signaling Technology, MA 3477 P-MST1/MST2 Cell Signaling Technology, MA 3681 MST1 Cell Signaling Technology, MA 3682 GAPDH(14C10) Cell Signaling Technology, MA 2118 α-Tubulin Cell Signaling Technology, MA 3873 pY Cell Signaling Technology, MA 9411 HA-tag-HRP Cell Signaling Technology, MA 2999 HA-tag Cell Signaling Technology, MA 3724 myc-tag Cell Signaling Technology, MA 2278 pY397-FAK Cell Signaling Technology, MA 8556 FAK Cell Signaling Technology, MA 3285 cleaved PARP Cell Signaling Technology, MA 9541 p-ERK1/2 Cell Signaling Technology, MA 4370 ERK1/2 Cell Signaling Technology, MA 4696 MOB1 Cell Signaling Technology, MA 13730 pT35-MOB1 Cell Signaling Technology, MA 8699 Gαq(E-17) Santa Cruz Biotech., CA sc-393 FAK(C-20) Santa Cruz Biotech., CA sc-558 RhoA Cell Signaling Technology, MA 2117 TRIO(H120) Santa Cruz Biotech., CA sc-28564 Rac1 BD Biosciences, CA 610651 pY357-YAP Abcam, MA ab62751 LATS2 Bethyl Laboratories, TX A300–479A pY26-MOB1A Signalway Antibody, MA 12878 flag-tag-HRP Sigma-Aldrich, MO A8592 Ki67 DAKO, CA M724029–2 Bacterial strains DH5alpha Competent E. coli BioPioneer, CA GACC-96 Stbl3 Competent E. coli Thermo Fisher C737303 siRNAs Non-targeting Dharmacon, CO D-001810–0X GNAQ Sigma-Aldrich, MO SASI Hs01 00231793 PTK2 Thermo Fisher, MA s11485 AKT1 Thermo Fisher, MA s659 MGLL Thermo Fisher, MA s22380 MTHFD1 Thermo Fisher, MA s9032 CDK1 Thermo Fisher, MA s464 SIRT1 Thermo Fisher, MA s223591 PSMB5 Thermo Fisher, MA s11354 TRIO Dharmacon, CO L-005047–00-0005 RHOA Dharmacon, CO L-003860–00-0005 RAC1 Dharmacon, CO L-003560–00-0005 LATS1 Sigma-Aldrich, MO Hs01 00046128 LATS2 Sigma-Aldrich, MO Hs01 00158803 DNAs pCMV-myc-MST1 Addgene 8847 pCMV2-FLAG-SAV1 Addgene 18970 pcDNA3-HA-MOB1 Addgene 32835 pcDNA3-HA-Y26F-MOB1 Generated in-lab NA pLENTi-HA-MOB1 Generated in-lab NA pLENTi-HA-Y26F-MOB1 Generated in-lab NA pGEX-HA-MOB1 Generated in-lab NA pGEX-HA-Y26F-MOB1 Generated in-lab NA pLVX-T etOne-FLAG-FAK Generated in-lab NA p2xFLAG-CMV2-LATS1 Addgene 18971 8xGTIIC-luciferase Addgene 34615 REAGENT alamarBlueTM Reagent Grand Island, NY DAL1100 FAK Kinase Enzyme System Promega V1971 YAP1 (Human) Recombinant Abnova H00010413-P01 Protein Glutathione Sepharose 4B GE Healthcare 17–0756-01 N/C Extraction Reagents ThermoFisher 78833 Software and Algorithms ISLE Lee et al 2018 https://www.github.com/jooslee/ISLE Open in a separate window KEY RESOURCES TABLE Despite the central role of Hippo/YAP-regulating mechanisms in uveal melanoma (UM), there are no clinically effective therapeutic targets.

Techniques: In Vitro, Inhibition, Positive Control, Mutagenesis, Gene Expression, Expressing, Control, Activation Assay

Journal: Cancer cell

Article Title: A Platform of Synthetic Lethal Gene Interaction Networks Reveals that the GNAQ Uveal Melanoma Oncogene Controls the Hippo Pathway through FAK

doi: 10.1016/j.ccell.2019.01.009

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: REAGENT or RESOURCE SOURCE IDENTIFIER Antibodies YAP Cell Signaling Technology, MA 14074 pS127-YAP Cell Signaling Technology, MA 4911 pS909-LATS1 Cell Signaling Technology, MA 9157 pT1079-LATS1 Cell Signaling Technology, MA 8654 LATS1 Cell Signaling Technology, MA 3477 P-MST1/MST2 Cell Signaling Technology, MA 3681 MST1 Cell Signaling Technology, MA 3682 GAPDH(14C10) Cell Signaling Technology, MA 2118 α-Tubulin Cell Signaling Technology, MA 3873 pY Cell Signaling Technology, MA 9411 HA-tag-HRP Cell Signaling Technology, MA 2999 HA-tag Cell Signaling Technology, MA 3724 myc-tag Cell Signaling Technology, MA 2278 pY397-FAK Cell Signaling Technology, MA 8556 FAK Cell Signaling Technology, MA 3285 cleaved PARP Cell Signaling Technology, MA 9541 p-ERK1/2 Cell Signaling Technology, MA 4370 ERK1/2 Cell Signaling Technology, MA 4696 MOB1 Cell Signaling Technology, MA 13730 pT35-MOB1 Cell Signaling Technology, MA 8699 Gαq(E-17) Santa Cruz Biotech., CA sc-393 FAK(C-20) Santa Cruz Biotech., CA sc-558 RhoA Cell Signaling Technology, MA 2117 TRIO(H120) Santa Cruz Biotech., CA sc-28564 Rac1 BD Biosciences, CA 610651 pY357-YAP Abcam, MA ab62751 LATS2 Bethyl Laboratories, TX A300–479A pY26-MOB1A Signalway Antibody, MA 12878 flag-tag-HRP Sigma-Aldrich, MO A8592 Ki67 DAKO, CA M724029–2 Bacterial strains DH5alpha Competent E. coli BioPioneer, CA GACC-96 Stbl3 Competent E. coli Thermo Fisher C737303 siRNAs Non-targeting Dharmacon, CO D-001810–0X GNAQ Sigma-Aldrich, MO SASI Hs01 00231793 PTK2 Thermo Fisher, MA s11485 AKT1 Thermo Fisher, MA s659 MGLL Thermo Fisher, MA s22380 MTHFD1 Thermo Fisher, MA s9032 CDK1 Thermo Fisher, MA s464 SIRT1 Thermo Fisher, MA s223591 PSMB5 Thermo Fisher, MA s11354 TRIO Dharmacon, CO L-005047–00-0005 RHOA Dharmacon, CO L-003860–00-0005 RAC1 Dharmacon, CO L-003560–00-0005 LATS1 Sigma-Aldrich, MO Hs01 00046128 LATS2 Sigma-Aldrich, MO Hs01 00158803 DNAs pCMV-myc-MST1 Addgene 8847 pCMV2-FLAG-SAV1 Addgene 18970 pcDNA3-HA-MOB1 Addgene 32835 pcDNA3-HA-Y26F-MOB1 Generated in-lab NA pLENTi-HA-MOB1 Generated in-lab NA pLENTi-HA-Y26F-MOB1 Generated in-lab NA pGEX-HA-MOB1 Generated in-lab NA pGEX-HA-Y26F-MOB1 Generated in-lab NA pLVX-T etOne-FLAG-FAK Generated in-lab NA p2xFLAG-CMV2-LATS1 Addgene 18971 8xGTIIC-luciferase Addgene 34615 REAGENT alamarBlueTM Reagent Grand Island, NY DAL1100 FAK Kinase Enzyme System Promega V1971 YAP1 (Human) Recombinant Abnova H00010413-P01 Protein Glutathione Sepharose 4B GE Healthcare 17–0756-01 N/C Extraction Reagents ThermoFisher 78833 Software and Algorithms ISLE Lee et al 2018 https://www.github.com/jooslee/ISLE Open in a separate window KEY RESOURCES TABLE Despite the central role of Hippo/YAP-regulating mechanisms in uveal melanoma (UM), there are no clinically effective therapeutic targets.

Techniques: Generated, Recombinant, Extraction, Software