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: Pharmaceuticals

Article Title: The Role of TRPA1 Channels in the Central Processing of Odours Contributing to the Behavioural Responses of Mice

doi: 10.3390/ph14121336

Figure Lengend Snippet: Expression of Trpa1 mRNA in the investigated regions of the olfactory system of C57BL/6 mice ( n = 4). Trpa1 (red) mRNA signal did not colocalise with β-tubulin III (green) immunorective cells in the OE ( a ). Trpa1 (red) mRNA signal colocalised exclusively with NeuN (white) positive neurons in the OB ( b ). Trpa1 (red), Gad1 (white) and Vglut1 (green) mRNA expression in the OB (Bregma 3 mm), ( c ) and in the PC ( d ) (Bregma −1.46 mm). Trpa1 mRNA signal colocalised both with Gad1 and Vglut1 positive neurons in the OB ( c ), but it colocalised only with Vglut1 positive neurons in the PC ( d ). Cell nuclei were counterstained with DAPI (blue) in all areas. Abbreviations: NeuN: neuronal nuclear protein, Gad1: glutamate decarboxylase 1, Vglut1: vesicular glutamate transporter 1, DAPI: 4′,6-diamidino-2-phenylindole. In order to highlight the cell borders, differential interference contrast (DIC) images were merged with the virtual color images. Bars: 10 µm.

Article Snippet: Briefly, after channel development of the RNAscope assay, sections were washed for 2 × 15 min in PBS, incubated overnight at RT with polyclonal rabbit anti-β-tubulin III (Merck, Sigma Aldrich GmbH; Cat. No.: T2200, Schnelldorf, Germany), diluted 1:200 with 2% normal donkey serum for blocking aspecific binding.

Techniques: Expressing

Journal: PLoS ONE

Article Title: Use of Bacterially Expressed dsRNA to Downregulate Entamoeba histolytica Gene Expression

doi: 10.1371/journal.pone.0008424

Figure Lengend Snippet: A. General scheme for dsRNA production. The 5′-terminal portions of the genes were amplified by PCR and cloned into the bacterial vector L4440 for dsRNA transcription. Primers used for quantification of mRNA by qRT-PCR match with the 3′-terminal part of the gene, which was not cloned into the plasmid (marked “Q-PCR template” in the figure), allowing the exclusive quantification of genome-encoded, sense-orientated transcripts. The multicloning site of L4440 vector is bidirectionally flanked by T7 promoters driving the synthesis of RNA complementary strands ( i.e . dsRNA). The recombinant plasmid was transfected into the bacterium HT115, allowing dsRNA purification in order to conduct the parasite soaking experiments or direct feeding of parasites with the expressing bacteria. B. Constructs performed in this work. Three independent DNA fragments were cloned and used for RNAi experiments including β-tubulin-, KERP1- and GFP-encoding genes (upper panel). Upon production of dsRNA, a nuclease-resistant dsRNA was detected in lysates of the recombinant bacterium (bottom panel).

Article Snippet: A rabbit anti-beta-tubulin polyclonal antibody was obtained from Eurogentec (Belgium) by immunization with two synthetic peptides with sequences derived from the amino acid sequence of E. histolytica beta-tubulin (amino acids 50 to 64: FSESSTKRYVPRSIN; and amino acids 324 to 338: DVEEQLYKIREKNPD).

Techniques: Amplification, Clone Assay, Plasmid Preparation, Quantitative RT-PCR, Recombinant, Transfection, Purification, Expressing, Construct

Journal: PLoS ONE

Article Title: Use of Bacterially Expressed dsRNA to Downregulate Entamoeba histolytica Gene Expression

doi: 10.1371/journal.pone.0008424

Figure Lengend Snippet: Following 3, 5 and 7 days of culture of 3×10 5 E. histolytica trophozoites in the presence of bacteria expressing dsRNA from genes encoding GFP (control) or β-tubulin (test), the parasites were counted under the microscope(A). At day 3 post-inoculation, endogenous mRNA was quantified by qRT-PCR (B) and protein levels were observed by western blot (C) according to procedures described in the materials and methods. Each graph represents the mean of 3 independent experiments.

Article Snippet: A rabbit anti-beta-tubulin polyclonal antibody was obtained from Eurogentec (Belgium) by immunization with two synthetic peptides with sequences derived from the amino acid sequence of E. histolytica beta-tubulin (amino acids 50 to 64: FSESSTKRYVPRSIN; and amino acids 324 to 338: DVEEQLYKIREKNPD).

Techniques: Expressing, Microscopy, Quantitative RT-PCR, Western Blot