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: The Journal of Neuroscience

Article Title: Overexpression of HGF Retards Disease Progression and Prolongs Life Span in a Transgenic Mouse Model of ALS

doi: 10.1523/JNEUROSCI.22-15-06537.2002

Figure Lengend Snippet: Attenuation of caspase-1 induction in double transgenic mice. A, Immunostaining of tubulin III (green) and caspase-1 (red) in the lumbar spinal cord at 6 months of age (n = 3 in each group). Caspase-1 was specifically induced in large tubulin III-positive neurons of G93A transgenic mice, whereas double transgenic mice showed much lower levels of caspase-1. Inset is magnified view.B, Comparison of the fluorescence intensity of caspase-1 in motoneurons. Fluorescence intensity of caspase-1-IR in each motoneuron was determined by image analysis using Photoshop after collecting the confocal image; average ± SE of 40 motoneurons was expressed. C, Western blot analysis of caspase-1 in the spinal cords of wild-type, HGF, G93A, and G93A/HGF mice.

Article Snippet: For immunohistochemistry, antibodies specific for c-Met (polyclonal) ( Sun et al., 1999 ; Funakoshi and Nakamura, 2001 ), HGF (polyclonal) (Tokushu Meneki), human SOD (monoclonal) (Sigma), GFAP (monoclonal) (Sigma), β-tubulin (βIII) (monoclonal) (Babco, Richmond, CA), and caspase-1 (polyclonal) (Santa Cruz), were applied to sections for 1–4 hr at room temperature or overnight at 4°C after blocking with 5% goat serum and mouse IgG blocking reagents (M.O.M Kit, Vector Laboratories, Burlingame, CA).

Techniques: Transgenic Assay, Immunostaining, Fluorescence, Western Blot

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