Journal: International Journal of Molecular Sciences

Article Title: Methionine Adenosyltransferase 1a (MAT1A) Enhances Cell Survival During Chemotherapy Treatment and is Associated with Drug Resistance in Bladder Cancer PDX Mice

doi: 10.3390/ijms20204983

Figure Lengend Snippet: MAT1A is upregulated in cells that survive 48 h drug treatment and overexpression alters cell response to gemcitabine. ( A ) 5637 cells were dosed at ~80% IC50 gemcitabine concentration (0.3 µM) for 48 h in vitro. RNA was collected from remaining gemcitabine treated (5637 Rx , n = 3) and untreated cells (5637, n = 3) before (day zero), during (day one, two) and after (day three, four, six, nine, and 12) drug treatment. ( B ) MAT1A expression was quantified via qPCR at each timepoint and expressed in FC. Statistical analysis was conducted using Student’s t-test, p < 0.0005 (#). ( C ) Quantitative PCR showing relative MAT1A gene expression in MAT1A plasmid transfected 5637 bladder cancer cells (5637 MAT1A + ) expressed as FC, ( n = 3 biological replicates), p < 0.0005 (#) compared to expression at day four in post drug treated 5637 cells (5637 Rx ) (NS, non-significant). ( D ) Representative gel of MAT1A protein expression relative to control b-tubulin analyzed via Western blotting using the Jess system (ProteinSimple). ( E ) Normalized MAT1A /b-tubulin protein expression calculated using Jess system densitometry expressed as percent of wildtype MAT1A expression in 5637 control cells ( n = 6 biological replicates), p < 0.005 (**). ( F ) IC50 gemcitabine drug toxicity curve between empty vector transfected and MAT1A transfected cells ( n = 5 biological replicates per condition per dose).

Article Snippet: Antibodies were diluted with ProteinSimple antibody diluent as follows: MAT1A primary antibody (undiluted, Abcam, Cambridge, United Kingdom, ab174687), beta-Tubulin (1:100, LI-COR Biosciences, Lincoln, ME, Catalog no. 926-42211).

Techniques: Over Expression, Concentration Assay, In Vitro, Expressing, Real-time Polymerase Chain Reaction, Plasmid Preparation, Transfection, Western Blot

Journal: Journal of Experimental & Clinical Cancer Research : CR

Article Title: Therapeutic efficacy of a novel βIII/βIV-tubulin inhibitor (VERU-111) in pancreatic cancer

doi: 10.1186/s13046-018-1009-7

Figure Lengend Snippet: Effect of VERU-111 on the expression of β-tubulin isotypes in PanCa cells. ( A ) Effect of VERU-111 on mRNA expression of βI, βIIa, βIIb, βIII, βIVa, βIVb, βV and βVI-tubulins in Panc-1 (i) and AsPC-1 (ii) cells. Briefly, cells were treated with the indicated concentrations of VERU-111 for 24 h. RNAs were isolated and transcribed for cDNA preparation. qPCR was performed to determine the mRNA expression of indicated tubulin isotypes. GAPDH was used as an internal control. Bar graphs represent relative fold expression of various tubulins mRNA. (Values mean ± SEM; n = 3). Asterisk (*) denotes the significant value p < 0.05. ( B ) Effect of VERU-111 on protein levels of various β-tubulin isotypes in Panc-1 (i) and AsPC-1(ii) cells. Cells were treated with vehicle or indicated concentrations of VERU-111 for 24 h and cell lysates were subjected for Western blot analysis. Equal loading of protein in each well was confirmed by stripping and re-probing of the blots with GAPD for all isoform were provided in Additional file : Figure S2

Article Snippet: Antibody βIIa - tubulin (cat. # TA345669) and βIVa-tubulin (cat. # TA-340088) were obtained from OriGene.

Techniques: Expressing, Isolation, Western Blot, Stripping Membranes

Journal: Journal of Experimental & Clinical Cancer Research : CR

Article Title: Therapeutic efficacy of a novel βIII/βIV-tubulin inhibitor (VERU-111) in pancreatic cancer

doi: 10.1186/s13046-018-1009-7

Figure Lengend Snippet: VERU-111 repressed the expression of βIII-tubulin and restored miR-200c expression in PanCa cells. ( A ) Effect of VERU-111 (i), colchicine (ii), vinorelbine (iii) and paclitaxel (iv) treatment on the mRNA expression of βIII-tubulin in PanCa cells as determined by qPCR analysis. Bar graphs represent fold change mRNA expression of βIII-tubulin compared to control group. (Values means ±SEM; n = 3). p < 0.05. ( B ) Western blot analysis results indicating the effect of VERU-111, colchicine and vinorelbine on β-tubulin III in Panc-1 cells at 24 h post-treatment. (C) PanCa cells (Panc-1) were treated with control (vehicle) or VERU-111, colchicine, vinorelbine and paclitaxel at 5–10 nM for 18 h. These cells were processed for immunofluorescence analysis using anti- βIII-tubulin antibody (green) and DAPI (blue). The images were captured with a Zeiss 710 Confocal microscope and Zen imaging software (Zeiss) at × 63 magnifications. (D) Effect of VERU-111 on the expression of miR-200c in Panc-1 (i), AsPC-1 (ii) and HPAF-II (iii) cells as determined by qPCR analysis. RNU6B was used as an internal control. ( E ) Effect of VERU-111 on the expression of βIII-tubulin in miR-200c mimic or inhibitor transfected Panc-1 cells as determined by qPCR (i) and WB analysis (ii). Cells were transfected with 100 nM of miR-200c mimic (pre-200c) or miR-200c inhibitor or scrambled miRNA (negative control) for 48 h followed by VERU-111 (20 nM) treatment for 24 h. RNA was isolated and transcribed for cDNA and mRNA expression of βIII-tubulin was determined by qPCR (i). Data in bar graph indicate fold change mRNA expression of βIII-tubulin. (Values mean ± SEM; n = 3). Asterisk (*) denote the significant value p < 0.05. In a same parallel experiment, protein lysates were prepared and subjected for Western blot analysis to determine the protein levels of βIII-tubulin. Equal loading of protein was determined by stripping and probing the blot with GAPDH antibody. (F) Comparative effect of VERU-111, colchicine and vinorelbine, on cell viability of Panc-1 (i), AsPC-1 (ii), and HPAF-II (iii) cells as determined by MTT assay. Line bar graphs indicate percent cell viability compared to control group in response to VERU-111, colchicine, vinorelbine, and paclitaxel treatment after 48 h treatment. Values in graph represent mean ± SEM of three independent experiments. Asterisk (*) denotes the significant value p < 0.05

Article Snippet: Antibody βIIa - tubulin (cat. # TA345669) and βIVa-tubulin (cat. # TA-340088) were obtained from OriGene.

Techniques: Expressing, Western Blot, Immunofluorescence, Microscopy, Imaging, Software, Transfection, Negative Control, Isolation, Stripping Membranes, MTT Assay

Journal: Journal of Experimental & Clinical Cancer Research : CR

Article Title: Therapeutic efficacy of a novel βIII/βIV-tubulin inhibitor (VERU-111) in pancreatic cancer

doi: 10.1186/s13046-018-1009-7

Figure Lengend Snippet: VERU-111 inhibits pancreatic tumor growth. (A) Effect of VERU-111 on AsPC-1 cells derived xenograft tumors in athymic nude mice. Representative images of AsPC-1 cells derived xenograft tumor bearing mice of control and VERU-111 treated group. ( B ) Line graph showing average tumor volume of control and VERU-111 treatment group different time points. Data shown in the graph represent mean ± SEM of six tumors of each group. ( C ) Average tumor weight of control and VERU-111 treatment group. ( D ) Net tumor growth of control and VERU-111 treatment group. Data in bar graph represent mean ± SEM of six tumors in each group. Asterisk (*) denotes the significant value p < 0.05. ( E ) Representative images of H&E staining of excised xenograft tumors of control (i) and VERU-111 treatment (ii) group. Effect of VERU-111 on the expression of PCNA, βI, βIII, βIVb and βIVb in excised tumor tissues of control (i) and VERU-111 treated (ii) mice as determined by Immunohistochemistry. ( F) Effect of VERU-111 on mRNA expression of βI, βIII, βIVb and βIVb in excised xenograft tumors of control and VERU-111 treated mice as determined by qPCR. Bar graph represents fold mRNA expression of βI, βIII, βIVb and βIVb (mean ± SEM; n = 4). Asterisk (*) denotes the significant value p < 0.05. ( G ) Effect of VERU-111 on the expression of miR-200c in excised xenograft tumors of control and VERU-111 treated mice as determined by qPCR ( H ) and representative images of in situ hybridization. ( I ) Proposed model illustrating possible molecular mechanisms of VERU-111 for the inhibition of pancreatic tumor growth. VERU-111 destabilizes microtubule fiber integrality (de-polymerization) via inhibitions of βIII/βIV isotypes, cell cycle arrest and induction of apoptosis. Moreover, VERU-111 also induces miR-200c expression, which negatively regulates β-tubulin III, leading to apoptosis induction and inhibition of invasion/migration of PanCa cells

Article Snippet: Antibody βIIa - tubulin (cat. # TA345669) and βIVa-tubulin (cat. # TA-340088) were obtained from OriGene.

Techniques: Derivative Assay, Staining, Expressing, Immunohistochemistry, In Situ Hybridization, Inhibition, Migration

Journal: Journal of Cancer

Article Title: Dihydroorotate dehydrogenase Inhibitors Target c-Myc and Arrest Melanoma, Myeloma and Lymphoma cells at S-phase

doi: 10.7150/jca.14835

Figure Lengend Snippet: DHODH expression in melanocytes, PBMCs (normal cells) and A375, H929, Ramos (cancer cells). Equal amount of protein lysate was used for Western blot analysis with human primary DHODH antibody. Tubulinβ served as protein loading control. Data represents mean ± SE of 3 independent experiments. *, P<0.05 and ***, P < 0.001.

Article Snippet: Loading controls - β actin, GAPDH and tubulin β antibodies were procured from SignalChem, Canada.

Techniques: Expressing, Western Blot, Control

Journal: Journal of Cancer

Article Title: Dihydroorotate dehydrogenase Inhibitors Target c-Myc and Arrest Melanoma, Myeloma and Lymphoma cells at S-phase

doi: 10.7150/jca.14835

Figure Lengend Snippet: DHODH inhibitors affect DHODH enzyme activity but not the DHODH protein expression in A375 cells. A, DHODH enzymatic assay with A771726 and BQR using pure human DHODH enzyme. B, DHODH enzymatic assay with A771726 and BQR using A375, H929 and Ramos cells lysates. C, A375 cells were incubated with 10, 30, 100 and 200 µM A771726 and 0.016, 0.05, 0.15 and 0.45 µM of BQR for 24 and 48 hours and cell viability was determined by Trypan blue stain. D, Western blot of A375 cell lysate after treatment with A771726 and BQR. Cells were harvested at 48 hours after treatment for Western blot analysis. Tubulinβ served as protein loading control. Values shown are mean ± SE of 3 independent experiments. *, P < 0.05, **, P < 0.01 and ***, P <0.001.

Article Snippet: Loading controls - β actin, GAPDH and tubulin β antibodies were procured from SignalChem, Canada.

Techniques: Activity Assay, Expressing, Enzymatic Assay, Incubation, Staining, Western Blot, Control

Journal: Journal of Cancer

Article Title: Dihydroorotate dehydrogenase Inhibitors Target c-Myc and Arrest Melanoma, Myeloma and Lymphoma cells at S-phase

doi: 10.7150/jca.14835

Figure Lengend Snippet: DHODH inhibitors target c-Myc and p21 signaling proteins in cancer cells. Equal amounts of protein lysates were used for Western blot analysis with the antibodies indicated. Tubulin-β and GAPDH served as protein loading control. A, A375 cells were treated with DMSO or DHODH inhibitors and cells were harvested at the indicated time point. B, H929 cells were treated with DMSO or DHODH inhibitors and cells were harvested at 48 hours after treatment. C, Ramos cells were treated with DMSO or DHODH inhibitors and cells were harvested at 48 hours after treatment. D, A375 cells plated in 6-well plates were transfected with DHODH shRNA (2 μg) or the control shRNA (2 μg) for 48 hours before immunoblotted with antibodies against c-Myc and p21. E, A375 cells plated in 6-well plates were transfected with DHODH siRNA (4 nM) or the control siRNA (4 nM) for 48 hours before harvesting for Western blot assays as described above. Data represents mean ± SE of 3 independent experiments. *, P <0.05, **, P <0.01, ***, P <0.001 and ****, P<0.0001.

Article Snippet: Loading controls - β actin, GAPDH and tubulin β antibodies were procured from SignalChem, Canada.

Techniques: Western Blot, Control, Transfection, shRNA