Journal: OMICS : a Journal of Integrative Biology

Article Title: Legal Agreements and the Governance of Research Commons: Lessons from Materials Sharing in Mouse Genomics

doi: 10.1089/omi.2013.0158

Figure Lengend Snippet: Global Partners in the IKMC and IMPC Networks

Article Snippet: , ○ New phenotyping partners include Model Animal Research Center (MARC), Nanjing University (China), RIKEN BioResource Center (BRC) and Japan Mouse Clinic (Japan), Korea Mouse Phenotype Consortium, and the Australian Phenomics Network (APN, including the APF and APB). In addition, the consortium is seeking industrial partners. Taconic Inc, a commercial mouse model provider, is a corporate sponsor..

Techniques: Knock-Out, Mutagenesis, Functional Assay, Produced, Zinc-Fingers, Homologous Recombination, Modification

Journal: Oncogene

Article Title: Sirtuin 3 inhibits hepatocellular carcinoma growth through the glycogen synthase kinase-3β/BCL2-associated X protein-dependent apoptotic pathway.

doi: 10.1038/onc.2015.121

Figure Lengend Snippet: Figure 3. Functional effect of SIRT3 on cell apoptosis. (a, b) SIRT3 overexpression induced HCC cell apoptosis. SK-Hep1, SMMC-7721 and PLC5 cells were transduced with lentiviruses containing plenti6-SIRT3 or empty vector for 4 days. Cell apoptosis was analyzed by flow cytometry with annexin V/PI (a) and by PARP cleavage analysis (b). *Po0.01. (c) SIRT3 knockdown sensitized L02 cells to epirubicin treatment. L02 cells transfected with small interfering RNA targeting SIRT3 or siCont were treated with 0.2 μM epirubicin for 72 h. Cells were harvested for analysis by flow cytometry with annexin V/PI.

Article Snippet: SIRT3 (#3627), PARP (#9542), Bax (#5023), BCL-2 (#2870), GSK-3β (#9315), p-GSK-3β (#9323), AKT (#2938), and p-AKT (#9271) antibodies were obtained from Cell Signaling Technology (Danvers, MA, USA), whereas the β-actin antibody (SC-1616) was obtained from Santa Cruz Biotechnology (Santa Cruz, CA, USA).

Techniques: Functional Assay, Over Expression, Transduction, Plasmid Preparation, Cytometry, Knockdown, Transfection, Small Interfering RNA

Journal: Oncotarget

Article Title: Multiplicity of acquired cross-resistance in paclitaxel-resistant cancer cells is associated with feedback control of TUBB3 via FOXO3a-mediated ABCB1 regulation.

doi: 10.18632/oncotarget.9118

Figure Lengend Snippet: Figure 1: Overexpression profiles of TUBB3 and FOXO3a in a panel of cancer cells with ABCB1-associated acquired drug resistance. (A) Characterization of indicated parental or drug-resistant phenotype cell lines for TUBB3 expression at both mRNA (upper panel) and protein (lower panel) levels. (B) Characterization of indicated parental or drug-resistant phenotype cell lines for FOXO3a expression at both mRNA (upper panel) and protein (lower panel) levels. (C) Identification of ABCB1-association expression in a panel of indicated parental or drug resistant cancer cell lines at the mRNA level. (D) Confirmation of P-gp protein overexpression in drug resistant cancer cell lines. (E) Intracellular distribution and localization of P-gp expression in both wild-type and drug-resistant phenotype cell lines. Cells were stained with human P-gp antibody and DAPI and analyzed through confocal microscopy. Images shown were magnified at ×200.

Article Snippet: For ABCB1 silencing, cells were transfected with scramble siRNA (FITC-conjugate) or human-specific ABCB1 (MDR1) siRNA duplexes (Santa Cruz Biotechnology) using Lipofectamine 3000 [80].

Techniques: Over Expression, Expressing, Staining, Confocal Microscopy

Journal: Oncotarget

Article Title: Multiplicity of acquired cross-resistance in paclitaxel-resistant cancer cells is associated with feedback control of TUBB3 via FOXO3a-mediated ABCB1 regulation.

doi: 10.18632/oncotarget.9118

Figure Lengend Snippet: Figure 2: Occurrence of cross-resistance in PTX-resistant cancer cells is highly associated with ATP-dependent P-gp/ ABCB1 efflux activity. (A and B) Fraction of drug-intolerant A549, A549-PacR (A) and HEK293, HEK293/ABCB1 (B) cells. Cells were treated with PTX, 5-FU, DCT, or CIS for 24 hr, and the cell viability was determined by MTT assay. Data are represented as means ± SEM. (C) Representative drug intolerant cell colonies (right) and quantified colony numbers of A549-PacR cells (left). Cells were exposed to 5-FU-conditioned media (CM) and continuously grown for > 7 days and formed colonies of established PTX-resistant cells were stained with sapphire 700. Data are represented as means ± SEM. (D and E) ATP-dependent P-gp efflux activity. Cells were treated with 60 µM 5-FU for 24 hr and assayed for Rho-123 incorporation. Flow cytometry was used to quantify Rho-123 fluorescence (D). Drug-resistant cells were treated with DMSO, 60 µM 5-FU, or 50 µM DCT for 24 hr and was assayed for ABCB1 ATPase activity (E). Data are represented as means ± SEM. (F and G) Association of P-gp expression with occurrence of cross-resistance. Drug-resistant cells were treated with 5-FU, DCT, or CIS for 24 hr (F) and time-dependently treated with 5-FU for (G). Cells were treated with 20 µM 5-FU then assayed for qRT-PCR using ABCB1-specific primer. (H) P-gp-specific ATPase activity. Cells were treated with increasing 5-FU concentration for 24 hr and cells were subjected to P-gp luminescent ATPase assay. Data are represented as means ± SEM.

Article Snippet: For ABCB1 silencing, cells were transfected with scramble siRNA (FITC-conjugate) or human-specific ABCB1 (MDR1) siRNA duplexes (Santa Cruz Biotechnology) using Lipofectamine 3000 [80].

Techniques: Activity Assay, MTT Assay, Staining, Flow Cytometry, Fluorescence, Expressing, Quantitative RT-PCR, Concentration Assay, ATPase Assay

Journal: Oncotarget

Article Title: Multiplicity of acquired cross-resistance in paclitaxel-resistant cancer cells is associated with feedback control of TUBB3 via FOXO3a-mediated ABCB1 regulation.

doi: 10.18632/oncotarget.9118

Figure Lengend Snippet: Figure 3: High acquired cross-resistance correlates to regulated TUBB3 and FOXO3a expressions with distinct hyperactive ABCB1 transcription in paclitaxel-resistant cancer cells. (A) Schematic diagram of general strategy used for the generation of transient cross-resistance to 5-FU, DCT, or CIS in PacR phenotype cancer cells or ABCB1-GFP transfected HEK293 cells. (B) Growth rate response of indicated cells (lower panel) to 5-FU treatment in a dose-dependent manner. Schematic schedule of treatment is also displayed (upper panel). Cell viability was determined using MTT assay. Data are represented as means ± SEM. (C) Characterization for maintained ABCB1 and ABCC1 mRNA expressions in developed A549-PacR/5-FU and PC-3-PacR/5-FU cells after indicated subsequent cell cultures. Passages of cells were maintained with 1 µM 5-FU final concentration. (D–F) Characterization for TUBB3 and FOXO3a mRNA expressions in indicated developed transient cross-resistance in PacR phenotype derived from A549 (D), PC-3 (E), and in developed transient 5-FU cross-resistance derived from HEK293 (F) cells. (G and H) Western blot analysis of A549, PC-3-PacR cells (G) and HEK293 cells transfected with either empty vector or ABCB1-GFP (H) cells, all with developed transient cross-resistance to indicated drugs. Cells were assessed for expressions of indicated proteins after 24 hr cell culture. (I) Flow cytometric determination of verapamil-induced apoptosis in indicated cells. Cells were treated with or without 100 µM verapamil for 24 hr. Data are shown as bar graph represented as means ± SEM. (J) Western blot analysis of indicated cells for expressions of apoptotic markers Bax, Bcl-2, and p53. Cells were treated with or without 100 µM verapamil for 24 hr. (K) Intracellular ATP level assessment in indicated cells. Cells were treated with or without 100 µM verapamil for 24 hr and ATP levels were determined in 104 fraction of cells. RLU, relative luciferase units. Data are represented as means ± SEM. (L) Determination of verapamil-induced inhibition of P-gp/ABCB1 in indicated cells assessed through qRT-PCR (left), Western blotting (center) and confocal microscopy (right). Cells were treated with 60 µM verapamil for 24 hr. Confocal images shown were magnified to 80 µm.

Article Snippet: For ABCB1 silencing, cells were transfected with scramble siRNA (FITC-conjugate) or human-specific ABCB1 (MDR1) siRNA duplexes (Santa Cruz Biotechnology) using Lipofectamine 3000 [80].

Techniques: Transfection, MTT Assay, Concentration Assay, Derivative Assay, Western Blot, Plasmid Preparation, Cell Culture, Luciferase, Inhibition, Quantitative RT-PCR, Confocal Microscopy

Journal: Oncotarget

Article Title: Multiplicity of acquired cross-resistance in paclitaxel-resistant cancer cells is associated with feedback control of TUBB3 via FOXO3a-mediated ABCB1 regulation.

doi: 10.18632/oncotarget.9118

Figure Lengend Snippet: Figure 4: FOXO3a activity involves ABCB1 regulation to control TUBB3 response in paclitaxel-resistant cancer cells with transient 5-FU cross-resistance. (A) Characterization of ABCB1 and TUBB3 expressions at both protein (upper panel) and mRNA (lower panel) levels after transient transfections with indicated vectors or siRNA in A549-PacR and PC-3-PacR both with developed 5-FU transient cross-resistance. Cells were transfected with empty vector or FOXO3a-GFP and scrambled siRNA or ABCB1 siRNA for 48 hr. Data are represented as means ± SEM. (B) Protein expressions (upper panel) of ABCB1 and TUBB3 and mRNA levels of TUBB3 (lower panel) in cells same as in A after transient transfection with either scrambled siRNA or FOXO3a siRNA for 48 hr. Data are represented as means ± SEM. (C) Effect of proteasome inhibitor, MG132 (MG), on βIII-tubulin distribution. Cells treated with or without 50 µM MG for 24 h were subjected to immunocytochemistry. Magnified images were zoomed at ×80. The cells were stained with TUBB3 antibody and DAPI. (D) Effect of MG on TUBB3 protein expressions in same cells as in A. Cells were transfected with either empty vector or FOXO3a- GFP for 48 hr and treated with or without 50 µM MG132 for 24 hr. Whole-cell lysates were assayed by Western blotting. (E) Detection of ubiquitinated TUBB3 protein in A549-PacR/5-FU cells. Cells were transfected with either scramble siRNA or FOXO3a-siRNA for 48 hr and cells were further transfected with an empty vector or myc-tagged ubiquitin-encoding vector for 24 hr. Immunoprecipitation was performed by TUBB3 antibody, then ubiquitinated proteins were detected by myc antibody assayed by Western blotting. (F) Methylation- specific PCR of ABCB1 -50GC and -110GC boxes in A549-PacR/5-FU cells. Primer sets are designed to amplify methylated (M) and unmethylated (U) alleles. A primer set encoding the whole GC region (T) was used as loading control. Cells were transiently transfected with either empty vector or FOXO3a-GFP for 48 hr and cells were treated with 10 µM PTX for 12 hr (left lane) or 24 hr (right lane). (G) Combined bisulphite restriction analysis of the Inr ABCB1 promoter region in A549-PacR/5-FU cells treated with or without 10 µM PTX, or 40 µM 5-FU for 24 hr or in combination for 24 or 48 hr as indicated. Figures represent the methylation percentages observed in the indicated drug-treated cells obtained from two separate independent experiments. (H) Effect of doxorubicin (Dox) on FOXO3a-induced regulation of Akt-related signals. Cells were transiently transfected with either empty vector or FOXO3a-GFP for 48 hr and cells were treated with or without 2 µM Dox for 8 hr. Protein expression levels were analyzed by Western blotting. (I) Methylated FOXO3a status in

Article Snippet: For ABCB1 silencing, cells were transfected with scramble siRNA (FITC-conjugate) or human-specific ABCB1 (MDR1) siRNA duplexes (Santa Cruz Biotechnology) using Lipofectamine 3000 [80].

Techniques: Activity Assay, Control, Transfection, Plasmid Preparation, Immunocytochemistry, Staining, Western Blot, Ubiquitin Proteomics, Immunoprecipitation, Methylation, Expressing

Journal: Oncotarget

Article Title: Multiplicity of acquired cross-resistance in paclitaxel-resistant cancer cells is associated with feedback control of TUBB3 via FOXO3a-mediated ABCB1 regulation.

doi: 10.18632/oncotarget.9118

Figure Lengend Snippet: Figure 5: Drug-induced secretome factors influence MDR promotion of FOXO3a-regulated P-gp activity in PTX- resistant A549 cells with multiple cross-resistance. (A) Schematic diagram of the drug conditioned media (CM)-exposure procedure used in various assays (B–J). (B and C) Characterization of FOXO3a-mediated multiple cross-drug resistance in A549-PacR cells (B). Cells were preincubated with indicated 1:1 combination ratio of CM from DMSO-, DCT-, 5-FU-treated cells. After 24 hr, cells were transiently transfected with indicated vectors or siRNA for 48 hr and assessed for colony formation (C). Established drug resistant colonies were stained and visualized by crystal violet or sapphire 700. (D and E) TUBB3 knockdown or FOXO3a overexpression (D) and overexpression of ABCB1 and FOXO3a effects on growth rate of A549-PacR/5-FU cells (E). A549-PacR/DCT cells were treated with indicated 5-FU concentrations for 24 h then CM was collected. Adherent A549 cells were exposed to the collected CM for 24 h followed by transient transfection with indicated vectors or siRNA for 48 hr. Cell viability was assessed by MTT assay. Data are represented as means ± SEM. (F) Confocal microscopic analysis of A549-PacR/5-FU cells. Cells were transiently transfected with indicated vectors or siRNA for 48 hr and stained with P-gp antibody and DAPI. Images shown were magnified to 80 µm. (G) Western blot analysis (left) and agarose gel electrophoresis of qPCR products of A549-PacR/5-FU cells. Cells were transiently transfected with indicated vectors or siRNA for 48 hr and assessed for expression studies. (H) ABCB1 drug efflux activity of A549-PacR/5-FU cells. Cells were transiently transfected with either empty vector or FOXO3a-GFP for 48 hr and cells were treated with or without 100 µM 5-FU or 120 µM DCT for 24 hr. Drug-treated cells were assayed for Rhodamine-123 (Rho-123) uptake (left) and untreated cells were assayed for ABCB1 ATPase activity (right). Data are represented as means ± SEM. (I) ABCB1 drug efflux activity of A549-PacR/5-FU cells. Cells were transiently transfected with either scrambled siRNA or TUBB3 siRNA for 48 hr followed by drug treatment and assayed as in H. Data are represented as means ± SEM. (J) Multidrug resistant colonies. Cells were transiently transfected with either scramble siRNA or TUBB3 siRNA for 48 hr and cells were treated with 30 µM 5-FU for 7 days. Resistant colonies were visualized by sapphire 700. (K) Schematic diagram of drug CM exposure and respective TUBB3 feedback after induced gene expression changes via transient gene transfection or siRNA gene silencing. Indicated gene or siRNA transfection schemes were used in L and M for P-gp functional assays. (L) Growth rate of A549-PacR/5-FU cells. Cells were pre-incubated with indicated drug-CM and cells were transiently transfected with indicated genes or siRNA for 48 hr. Cell viability was assessed by MTT assay. Data are represented as means ± SEM. (M) ABCB1 drug efflux activity A549-PacR/5-FU cells. Cells were pre- incubated with indicated drug-CM and cells were transiently transfected with indicated genes or siRNA for 48 hr. Cells were then treated with indicated drugs and concentration for 36 hr. Data are represented as means ± SEM.

Article Snippet: For ABCB1 silencing, cells were transfected with scramble siRNA (FITC-conjugate) or human-specific ABCB1 (MDR1) siRNA duplexes (Santa Cruz Biotechnology) using Lipofectamine 3000 [80].

Techniques: Activity Assay, Transfection, Staining, Knockdown, Over Expression, MTT Assay, Western Blot, Agarose Gel Electrophoresis, Expressing, Plasmid Preparation, Gene Expression, Functional Assay, Incubation, Concentration Assay

Journal: Oncotarget

Article Title: Multiplicity of acquired cross-resistance in paclitaxel-resistant cancer cells is associated with feedback control of TUBB3 via FOXO3a-mediated ABCB1 regulation.

doi: 10.18632/oncotarget.9118

Figure Lengend Snippet: Figure 7: TUBB3 feedback inactivation reverses impaired microtubule stability in A549-PacR cells with developed transient multiple cross-resistance. (A) Microtubule stability in parental, PacR, and developed PacR phenotype cells with multiple transient cross-resistance. Cells were grown in the presence of indicated PTX concentrations for 18 hr. Following cell lysis, pellet (P) and the supernatant (S) protein fractions were separated by centrifugation and resolved on adjacent lanes by electrophoresis. Transferred filters were probed with tubulin antibody. (B) Acetylated tubulin status of parental, PacR, and developed PacR phenotype cells with multiple transient cross-resistance. Cells were grown in the absence or presence 20 nM PTX for 18 hr, and the amount of acetylated tubulin was measured by immunoblotting using antibody specific to acetylated tubulin. (C) Mixing tubulin experiment. Indicated cells were harvested by adding hypotonic buffer with 50 µg/mL PTX for 15 min. Whole cell lysate from PacR phenotype was added to that of developed PacR/5- FU subline at different ratios as indicated, and incubated for an additional 10 min. The polymerized (Pol) and soluble (Sol) protein fractions were processed as described in Materials and Methods. (D) Schematic diagram of transient gene transfection and/or siRNA silencing with respective TUBB3 feedback results used in E and F in A549-PacR/5-FU cells. (E) Confirmation of TUBB3 feedback gene expressions after transient transfection and/or siRNA silencing of indicated genes with C as control (respective empty vector and/or scramble siRNA) and TS as the transfection scheme shown in D. (F) Microtubule stability of A549-PacR/5-FU cells through tubulin polymerization after indicated transient gene and/or siRNA transfections. Cells were grown in the presence of 60 nM PTX for 18 hr. The polymerized (Pol) and soluble (Sol) protein fractions were processed as in C. (G) Illustration of proposed mechanism behind the feedback response of TUBB3 to silencing of FOXO3a and ABCB1 in regulating microtubule stability in A549-PacR/5-FU cells.

Article Snippet: For ABCB1 silencing, cells were transfected with scramble siRNA (FITC-conjugate) or human-specific ABCB1 (MDR1) siRNA duplexes (Santa Cruz Biotechnology) using Lipofectamine 3000 [80].

Techniques: Lysis, Centrifugation, Electrophoresis, Western Blot, Incubation, Transfection, Control, Plasmid Preparation

Journal: Oncotarget

Article Title: Multiplicity of acquired cross-resistance in paclitaxel-resistant cancer cells is associated with feedback control of TUBB3 via FOXO3a-mediated ABCB1 regulation.

doi: 10.18632/oncotarget.9118

Figure Lengend Snippet: Figure 8: Model representation of the feedback control of TUBB3 via FOXO3a-mediated ABCB1 regulation inducing multiplicity in acquired cross-drug resistance. Multiplicity of acquired cross-resistance to structurally different drugs in cancer cells selected for taxane resistance is associated with feedback control of TUBB3 through FOXO3a-mediated ABCB1 regulation inducing hyperfunctional P-gp-associated drug efflux and escape from potential inhibition.

Article Snippet: For ABCB1 silencing, cells were transfected with scramble siRNA (FITC-conjugate) or human-specific ABCB1 (MDR1) siRNA duplexes (Santa Cruz Biotechnology) using Lipofectamine 3000 [80].

Techniques: Control, Inhibition

Journal: The Journal of Biological Chemistry

Article Title: Tissue transglutaminase activates integrin-linked kinase and β-catenin in ovarian cancer

doi: 10.1016/j.jbc.2022.102242

Figure Lengend Snippet: TG2–FN clusters activate β-catenin in OC cells. A , WB for p-FAK Tyr576/577 , FAK, p-ILK Ser246 , ILK, TG2, and GAPDH in OVCAR-5 cells stably transduced with scrambled- or TG2-targeting shRNA and plated on FN-coated plates for 30 min, 1 h, and 2 h. Densitometry quantifies p-FAK Tyr576/577 /FAK and p-ILK Ser246 /ILK ratios and TG2 expression levels (N = 3; ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.01, and ∗∗∗∗ p < 0.0001). B , WB for non–p-active β-catenin, p-GSKα/β Ser21/9 , GSKα/β, TG2, and GAPDH in OVCAR-5 cells stably transduced with scrambled- or TG2-targeting shRNA and plated on FN-coated plates for 30 min, 1 h, and 2 h. Densitometry quantifies non–p-active β-catenin expression levels and p-GSKα/β Ser21/9 /GSKα/β ratio (N = 3; ∗ p < 0.05, ∗∗ p < 0.01, and ∗∗∗∗ p < 0.0001). All antibodies (Abs) were probed on the same membrane. For consistency, the same TG2 and GAPDH representative WB images and TG2 quantification are shown in A and B . C , WB for p-FAK Tyr576/577 , FAK, p-ILK Ser246 , ILK, TG2, and GAPDH in OVCAR-5 cells stably transduced with TG2-targeting shRNA, transfected with pCMV3- TGM2 -C-His, and plated on FN-coated plates for 2 h. Densitometry quantifies p-FAK Tyr576/577 /FAK and p-ILK Ser246 /ILK ratios and TG2 expression levels (N = 3; ∗∗ p < 0.01, ∗∗∗ p < 0.01, and ∗∗∗∗ p < 0.0001). D , WB for non–p-active β-catenin, p-GSKα/β Ser21/9 , GSKα/β, and GAPDH in OVCAR-5 cells stably transduced with TG2-targeting shRNA, transfected with pCMV3- TGM2 -C-His, and plated on FN-coated plates for 2 h. Densitometry quantifies non–p-active β-catenin expression levels and p-GSKα/β Ser21/9 /GSKα/β ratio (N = 3; ∗ p < 0.05, ∗∗∗∗ p < 0.0001). All Abs were probed on the same membrane. For consistency, the same TG2 and GAPDH representative WB images and TG2 quantification are shown in C and D . E , IF staining for TG2 (Alexa Fluor 488, green ) and non–p-active β-catenin (Alexa Fluor 568, red ) in OVCAR-5 cells sh-Ctr or sh-TG2 KD plated on FN for 2 h. F and G , quantification of Alexa Fluor 488 ( green ) and Alexa Fluor 568 ( red ) proteins was calculated by using Metamorph software (N = 3; ∗∗∗∗ p < 0.0001). H , OVCAR-5 cells sh-Ctr or sh-TG2 KD were cotransfected with TCF/LEF1 luciferase reporter and Renilla control plasmid and plated on FN for 2 h. Luciferase signal relative to Renilla activity is expressed as fold increase (N = 6; ∗∗∗ p < 0.001). I , real-time PCR for c-Myc in OVCAR-5 cells sh-Ctr or sh-TG2 KD plated on FN-coated plates for 2 h (N = 6; ∗∗∗ p < 0.001). FAK, focal adhesion kinase; FN, fibronectin; GSKα/β, glycogen synthase kinase-3α/β; IF, immunofluorescence; ILK, integrin-linked kinase; KD, knockdown; LEF1, lymphoid enhancer–binding factor 1; OC, ovarian cancer; TCF, T-cell factor; TG2, tissue transglutaminase; WB, Western blot.

Article Snippet: Polyclonal ILK (clone 4G9), nonphospho (active) β-catenin at Ser33/37 and Thr41 (clone D13A1), p-FAK Tyr576/577 , FAK (clone D5O7U), GSK-3α/β, and p-GSK-3α/β Ser21/9 (clone 37F11) antibodies were from Cell Signaling Technology; and GAPDH from Biodesign International.

Techniques: Stable Transfection, Transduction, shRNA, Expressing, Membrane, Transfection, Staining, Software, Luciferase, Control, Plasmid Preparation, Activity Assay, Real-time Polymerase Chain Reaction, Immunofluorescence, Knockdown, Binding Assay, Western Blot

Journal: The Journal of Biological Chemistry

Article Title: Tissue transglutaminase activates integrin-linked kinase and β-catenin in ovarian cancer

doi: 10.1016/j.jbc.2022.102242

Figure Lengend Snippet: Functional TG2 and integrin β1 inhibition disrupts β-catenin signaling in OC cells. A , WB for p-FAK Tyr576/577 , FAK, p-ILK Ser246 , ILK, and GAPDH in OVCAR-5 cells treated with inhibitory antibodies (Abs) directed against the FN-binding domain of TG2 (clone 4G3) or integrin β1 (clone P5D2) and/or Wnt-3A and plated on FN-coated plates for 2 h. Densitometry quantifies p-FAK Tyr576/577 /FAK and p-ILK Ser246 /ILK ratios (N = 3; ∗ p < 0.05; ∗∗ p < 0.01). B , WB for non–p-active β-catenin, p-GSKα/β Ser21/9 , GSKα/β, and GAPDH in OVCAR-5 cells treated with 4G3 or P5D2 Abs and/or Wnt-3A and plated on FN-coated plates for 2 h. Densitometry quantifies non–p-active β-catenin expression levels and p-GSKα/β Ser21/9 /GSKα/β ratio (N = 3; ∗∗∗ p < 0.001; ∗∗∗∗ p < 0.0001). C , IF staining for phalloidin (Alexa Fluor 488, green ) and non–p-active β-catenin (Alexa Fluor 568, red ) in OVCAR-5 cells treated with 4G3 or P5D2 Abs and/or Wnt-3A plated on FN for 2 h. D , quantification of Alexa Fluor 568 ( red ) proteins was calculated by using Metamorph software (N = 3; ∗∗∗∗ p < 0.0001). E , OVCAR-5 cells were cotransfected with TCF/LEF1 luciferase reporter and Renilla control plasmid, treated with 4G3 or P5D2 Abs and/or Wnt-3A, and plated on FN for 2 h. Luciferase signal relative to Renilla activity is expressed as fold increase (N = 6; ∗∗∗∗ p < 0.0001). F , real-time PCR for c-Myc in OVCAR-5 cells treated with 4G3 or P5D2 Abs and/or Wnt-3A and plated on FN-coated plates for 2 h (N = 6; ∗∗∗∗ p < 0.0001). FAK, focal adhesion kinase; FN, fibronectin; GSKα/β, glycogen synthase kinase-3α/β; IF, immunofluorescence; ILK, integrin-linked kinase; LEF1, lymphoid enhancer–binding factor 1; OC, ovarian cancer; TCF, T-cell factor; TG2, tissue transglutaminase; WB, Western blot.

Article Snippet: Polyclonal ILK (clone 4G9), nonphospho (active) β-catenin at Ser33/37 and Thr41 (clone D13A1), p-FAK Tyr576/577 , FAK (clone D5O7U), GSK-3α/β, and p-GSK-3α/β Ser21/9 (clone 37F11) antibodies were from Cell Signaling Technology; and GAPDH from Biodesign International.

Techniques: Functional Assay, Inhibition, Binding Assay, Expressing, Staining, Software, Luciferase, Control, Plasmid Preparation, Activity Assay, Real-time Polymerase Chain Reaction, Immunofluorescence, Western Blot

Journal: The Journal of Biological Chemistry

Article Title: Tissue transglutaminase activates integrin-linked kinase and β-catenin in ovarian cancer

doi: 10.1016/j.jbc.2022.102242

Figure Lengend Snippet: ILK inhibition blocks β-catenin signaling in OC cells. A and B , WB for p-FAK Tyr576/577 , FAK, p-ILK Ser246 , ILK, non–p-active β-catenin, p-GSKα/β Ser21/9 , GSKα/β, and GAPDH in OVCAR-5 cells plated on FN-coated plates for 2 h and treated or not with cpd-22 and/or Wnt-3A. Densitometry quantifies non–p-active β-catenin expression levels and p-FAK Tyr576/577 /FAK, p-ILK Ser246 /ILK, and p-GSKα/β Ser21/9 /GSKα/β ratios (N = 3; ∗∗∗ p < 0.001; ∗∗∗∗ p < 0.0001). All antibodies (Abs) were probed on the same membrane. For consistency, the same GAPDH representative WB images are shown in A and B . C and D , WB for p-FAK Tyr576/577 , FAK, p-ILK Ser246 , ILK, non–p-active β-catenin, p-GSKα/β Ser21/9 , GSKα/β, and GAPDH in OVCAR-5 cells sh-Ctr or sh-ILK plated on FN for 2 h and treated or not with Wnt-3A. Densitometry quantifies ILK and non–p-active β-catenin expression levels and p-FAK Tyr576/577 /FAK, p-ILK Ser246 /ILK, and p-GSKα/β Ser21/9 /GSKα/β ratios (N = 3; ∗∗ p < 0.01; ∗∗∗ p < 0.001; and ∗∗∗∗ p < 0.0001). E , IF staining for phalloidin (Alexa Fluor 488, green ) and non–p-active β-catenin (Alexa Fluor 568, red ) in OVCAR-5 cells plated on FN-coated plates for 2 h and treated or not with cpd-22 and/or Wnt-3A. F , quantification of Alexa Fluor 568 ( red ) proteins was calculated by using Metamorph software (N = 3; ∗∗∗∗ p < 0.0001). G , OVCAR-5 cells were cotransfected with TCF/LEF1 luciferase reporter and Renilla control plasmid prior to treatment with cpd-22 or Wnt-3A and plated on FN for 2 h. Luciferase signal relative to Renilla activity is expressed as fold increase (N = 6; ∗∗∗∗ p < 0.0001). H , real-time PCR for c-Myc in OVCAR-5 cells plated on FN-coated plates for 2 h and treated or not with cpd-22 and/or Wnt-3A (N = 6; ∗∗∗∗ p < 0.0001). I , OVCAR-5 cells sh-Ctr or sh-ILK were cotransfected with TCF/LEF1 luciferase reporter and Renilla control plasmid prior to treatment with cpd-22 or Wnt-3A and plated on FN for 2 h (N = 6; ∗∗∗ p < 0.01; ∗∗∗ p < 0.001). J , real-time PCR for c-Myc in OVCAR-5 cells stably transduced with scrambled- or ILK-targeting shRNA plated on FN-coated plates for 2 h and treated or not with Wnt-3A (N = 6; ∗∗∗ p < 0.01; ∗∗∗ p < 0.001). cpd-22, compound 22; FAK, focal adhesion kinase; FN, fibronectin; GSKα/β, glycogen synthase kinase-3α/β; IF, immunofluorescence; ILK, integrin-linked kinase; LEF1, lymphoid enhancer–binding factor 1; OC, ovarian cancer; TCF, T-cell factor; WB, Western blot.

Article Snippet: Polyclonal ILK (clone 4G9), nonphospho (active) β-catenin at Ser33/37 and Thr41 (clone D13A1), p-FAK Tyr576/577 , FAK (clone D5O7U), GSK-3α/β, and p-GSK-3α/β Ser21/9 (clone 37F11) antibodies were from Cell Signaling Technology; and GAPDH from Biodesign International.

Techniques: Inhibition, Expressing, Membrane, Staining, Software, Luciferase, Control, Plasmid Preparation, Activity Assay, Real-time Polymerase Chain Reaction, Stable Transfection, Transduction, shRNA, Immunofluorescence, Binding Assay, Western Blot