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: iScience

Article Title: Pathogenic KIAA0586/TALPID3 variants are associated with defects in primary and motile cilia

doi: 10.1016/j.isci.2024.111670

Figure Lengend Snippet: KIAA0586/TALPID3 -affected respiratory epithelial cells cultured under ALI-conditions showed reduced number of beating cilia not sufficient to maintain mucociliary clearance in vitro (A) After full differentiation, ALI-cultures were stained with antibodies directed against acetylated alpha-tubulin (acet tub; green) to mark the ciliary axoneme. Nuclei were stained with Hoechst (blue). (B) Left graph: CBF was measured and within normal range for all tested individuals (control: 12 Hz ± 1 Hz; family 1 [II.I]: 11 Hz ± 7 Hz; family 1 [II.II]: 10 Hz ± 3 Hz). Right graph: the calculated active area reflecting ciliary beat movement was markedly reduced in ALI-cultures of the affected individuals (control: 47% ± 13%; family 1 [II.I]: 1% ± 1%; family 1 [II.II]: 1% ± 1%). (C) Statistical analyses of three independent particle tracking experiments per individual revealed reduced speed of transported particles for KIAA0586/TALPID3- affected ALI-cultures (control: 86 μm/s; family 1 [II.I]: 5 μm/s; family 1 [II.II]: 4 μm/s). (D) Particle tracking videos given as z stack projections are exemplary shown per individual. Scale bars in (D) represent 100 μm.

Article Snippet: Rabbit polyclonal anti-detyrosinated tubulin , Merck , Cat#AB3201; RRID: AB_177350.

Techniques: Cell Culture, In Vitro, Staining, Control

Journal: iScience

Article Title: Pathogenic KIAA0586/TALPID3 variants are associated with defects in primary and motile cilia

doi: 10.1016/j.isci.2024.111670

Figure Lengend Snippet: Primary cilia length measurement (A) Images of primary cilia from patient-derived fibroblasts in comparison to two unrelated healthy controls. The axonemes are shown in magenta (detyrosinated tubulin), basal bodies in green (γ-tubulin). Primary cilia of the patient (II.I and II.II) of family 1 showed normal basal bodies and shortend axonemes. (B) The length of primary cilia from patient-derived fibroblasts and from unrelated controls were quantified. In total 750 cilia per cell line were measured in three independent experiments (250 cilia per experiment). The median of the box blots showed 2.6 μm in controls and 1.4 μm and 1.5 μm in the patients. Statistical analysis was performed applying Kruskal-Wallis statistical test. Scale bars: 5 μm, 1 μm for inset; ∗∗∗: p value < 0.0001; ns: not significant.

Article Snippet: Rabbit polyclonal anti-detyrosinated tubulin , Merck , Cat#AB3201; RRID: AB_177350.

Techniques: Derivative Assay, Comparison

Journal: iScience

Article Title: Pathogenic KIAA0586/TALPID3 variants are associated with defects in primary and motile cilia

doi: 10.1016/j.isci.2024.111670

Figure Lengend Snippet:

Article Snippet: Rabbit polyclonal anti-detyrosinated tubulin , Merck , Cat#AB3201; RRID: AB_177350.

Techniques: Control, Recombinant, Fluorescence, Sequencing, Isolation, Software, Membrane, Microscopy

Journal: Cells

Article Title: Platelet-Rich Plasma Prevents In Vitro Transforming Growth Factor-β1-Induced Fibroblast to Myofibroblast Transition: Involvement of Vascular Endothelial Growth Factor (VEGF)-A/VEGF Receptor-1-Mediated Signaling †

doi: 10.3390/cells7090142

Figure Lengend Snippet: Evaluation of murine NIH/3T3 fibroblast to myofibroblast transition. The cells were induced to differentiate towards myofibroblasts by culturing for 48 h or 5 days in differentiation medium (DM, low serum medium plus 2 ng/mL TGF-β1). Cells cultured in proliferation medium (PM) served as control, undifferentiated cells. To evaluate the effects of PRP on TGF-β1-induced fibroblast-myofibroblast transition, cells were cultured in DM added with 1:50 diluted PRP (DM + PRP). In addition, the cells were cultured in the presence of 1:50 serum-free medium diluted PRP (PRP). ( a – c ) Representative confocal fluorescence images of the cells ( a ) stained with Alexa Fluor 488-conjugated phalloidin to reveal F-actin and immunostained with antibodies against vinculin, ( b ) immunostained with antibodies against α-sma or ( c ) type-1 collagen. In ( b , c ), nuclei are counterstained with propidium iodide. Scale bar: 50 µm. ( d – f ) Histograms showing the densitometric analyses of the intensity of the fluorescence signals for each marker, performed on digitized images. ( g ) Western blotting analysis of α-sma expression. Histogram shows the densitometric analysis of the bands normalized to α-tubulin. Data shown are mean ± S.E.M. and represent the results of at least three independent experiments performed in triplicate. Significance of difference: * p < 0.05 vs. PM; ° p < 0.05 vs. DM.

Article Snippet: After that, the same membranes were washed and immunodetected for the expression of α-tubulin or β-actin assumed as control invariant proteins, by using rabbit polyclonal anti-α-tubulin (1:1000; Merck, Milan, Italy) or mouse monoclonal anti-β-actin antibodies (1:10,000; Sigma).

Techniques: Cell Culture, Fluorescence, Staining, Marker, Western Blot, Expressing

Journal: Cells

Article Title: Platelet-Rich Plasma Prevents In Vitro Transforming Growth Factor-β1-Induced Fibroblast to Myofibroblast Transition: Involvement of Vascular Endothelial Growth Factor (VEGF)-A/VEGF Receptor-1-Mediated Signaling †

doi: 10.3390/cells7090142

Figure Lengend Snippet: Fibroblast VEGFR-1 and VEGF-A expression. NIH/3T3 fibroblastic cells were cultured in differentiation medium (DM, low serum medium plus 2 ng/mL TGF-β1) in the absence or presence of 1:50 diluted PRP (DM + PRP) or in the presence of 1:50 serum-free medium diluted PRP (PRP). Cells cultured in proliferation medium (PM) served as control undifferentiated cells. ( a ) Western blotting analysis of VEGFR-1 expression. Histogram shows the densitometric analysis of the bands normalized to α-tubulin. ( b ) Representative superimposed differential interference contrast (DIC) and confocal fluorescence images of control cells immunostained with antibodies against VEGFR-1 showing the cellular localization of VEGFR-1; the staining (green) is mainly localized at the cell surface. Scale bar: 20 µm. ( c ) Representative confocal fluorescence images of the cells immunostained with antibodies against VEGF-A. Scale bar: 50 µm. Histogram shows the densitometric analysis of the intensity of VEGF-A fluorescence signal performed on digitized images. Data shown are mean ± S.E.M. and represent the results of at least three independent experiments performed in triplicate. Significance of difference: * p < 0.05 vs. PM; ° p < 0.05 vs. DM.

Article Snippet: After that, the same membranes were washed and immunodetected for the expression of α-tubulin or β-actin assumed as control invariant proteins, by using rabbit polyclonal anti-α-tubulin (1:1000; Merck, Milan, Italy) or mouse monoclonal anti-β-actin antibodies (1:10,000; Sigma).

Techniques: Expressing, Cell Culture, Western Blot, Fluorescence, Staining

Journal: Cells

Article Title: Platelet-Rich Plasma Prevents In Vitro Transforming Growth Factor-β1-Induced Fibroblast to Myofibroblast Transition: Involvement of Vascular Endothelial Growth Factor (VEGF)-A/VEGF Receptor-1-Mediated Signaling †

doi: 10.3390/cells7090142

Figure Lengend Snippet: Effect of VEGFR-1 inhibition and of stimulation with soluble VEGF-A on α-sma expression: western blotting analysis. NIH/3T3 fibroblastic cells were cultured in differentiation medium (DM, low serum medium plus 2 ng/mL TGF-β1) in the absence or presence of 1:50 diluted PRP (DM + PRP) or in the presence of 1:50 serum-free medium diluted PRP (PRP). To evaluate the involvement of VEGF-A/VEGFR-mediated signaling in PRP- induced fibroblast response, the cells were treated with the selective pharmacological VEGFR inhibitor, KRN633 (DM + PRP + KRN633; PRP + KRN633). In parallel experiments the cells were cultured in DM in the presence of two different concentrations of soluble VEGF-A (20 ng/mL, DM + VEGF-A 20; 2 ng/mL, DM + VEGF-A 2). Cells cultured in proliferation medium (PM) served as control undifferentiated cells. ( a ) Representative western blots of α-sma and tubulin expression. ( b ) Histogram showing the densitometric analysis of the bands normalized to α-tubulin. ( b ) Data shown are mean ± S.E.M. and represent the results of at least three independent experiments performed in triplicate. Significance of difference: * p < 0.05 vs. PM; ° p < 0.05 vs. DM; # p < 0.05 vs. DM + PRP; § p < 0.05 vs. PRP.

Article Snippet: After that, the same membranes were washed and immunodetected for the expression of α-tubulin or β-actin assumed as control invariant proteins, by using rabbit polyclonal anti-α-tubulin (1:1000; Merck, Milan, Italy) or mouse monoclonal anti-β-actin antibodies (1:10,000; Sigma).

Techniques: Inhibition, Expressing, Western Blot, Cell Culture

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