Journal: Osteoarthritis and cartilage

Article Title: SOX9 protein is stabilized by TGF-β and regulates PAPSS2 mRNA expression in chondrocytes

doi: 10.1016/j.joca.2016.10.007

Figure Lengend Snippet: ATDC5 cells were transfected with Sox9 siRNA, Smad2/3 siRNA, or control non-specific (NS) siRNA. Sox9 mRNA expression was significantly reduced in cells expressing Sox9 siRNA (A). Acan mRNA was significantly reduced in the presence of Sox9 siRNA (B). Cells containing NS siRNA or Sox9 siRNA were treated with TGF-β1 and expression of Papss2 mRNA was measured by qPCR (C). In A–C * = p < 0.05 REST, n = 5. Western blots showed Smad2/3 protein levels were reduced in the presence of Smad2/3 siRNA, α-Tubulin was used as a loading control (n = 6) (D). Acan mRNA was significantly down-regulated in the Smad2/3 siRNA-transfected cells compared to cells containing NS siRNA (REST, * = p < 0.05, n = 2) (E). Cells containing NS or Smad2/3 siRNA siRNA were treated with TGF-β1 and Papss2 mRNA was, measured by qPCR (REST, * = p <0.05, n.s = not-significant n = 4) (F).

Article Snippet: To assess whether equivalent amounts of protein were loaded in all wells, either an anti-GAPDH primary antibody (1:1000, Santa Cruz Biotechnology sc-25778) or anti-α-Tubulin primary antibody (1:2500, Rockland Immunochemicals #200-301-880) were used.

Techniques: Transfection, Expressing, Western Blot

Journal: Osteoarthritis and cartilage

Article Title: SOX9 protein is stabilized by TGF-β and regulates PAPSS2 mRNA expression in chondrocytes

doi: 10.1016/j.joca.2016.10.007

Figure Lengend Snippet: In A, mRNA was collected from bovine chondrocytes treated with vehicle or TGF-β1 for the indicated amounts of time. SOX9 mRNA levels were determined by qPCR (REST, no statistically significant differences, n = 5). In B, protein was collected from bovine chondrocytes that were treated with either vehicle or TGF-β1 for 4 or 6 hours. SOX9, pSMAD3, and SMAD2/3 protein levels were determined by Western blot (n = 3). α-Tubulin was used as a loading control. In C, new protein synthesis was inhibited with cycloheximide. Cells were subsequently treated with either vehicle or TGF-β1 for up to 8 hours. Protein was isolated at specified time points, and the levels of SOX9 protein were determined by Western blot (n = 3). pSMAD3 = phosphorylated SMAD3, CHX = cycloheximide.

Article Snippet: To assess whether equivalent amounts of protein were loaded in all wells, either an anti-GAPDH primary antibody (1:1000, Santa Cruz Biotechnology sc-25778) or anti-α-Tubulin primary antibody (1:2500, Rockland Immunochemicals #200-301-880) were used.

Techniques: Western Blot, Isolation

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: Nuclear miR-150 enhances hepatic lipid accumulation by targeting RNA transcripts overlapping the PLIN2 promoter

doi: 10.1016/j.isci.2023.107837

Figure Lengend Snippet:

Article Snippet: Anti-Beta Tubulin Antibody , Boster , Cat# M01857-3.

Techniques: Recombinant, Magnetic Beads, SYBR Green Assay, Lysis, Luciferase, Reporter Assay, cDNA Synthesis, Plasmid Preparation, Software