Journal: RSC Advances

Article Title: Design, synthesis, and mechanistic evaluation of novel pyrazole/thiazole chalcone hybrids as dual tubulin polymerization and COX-2 inhibitors with potent antiproliferative activity

doi: 10.1039/d6ra03557d

Figure Lengend Snippet: Selected thiazole/chalcone-based tubulin polymerization inhibitors (I–VII) and combretastatin A-4.

Article Snippet: The effects of compounds 9a–o on tubulin polymerization were assessed using a fluorescence-based tubulin polymerization assay (Cytoskeleton, Inc., Cat. No. BK011P), with combretastatin A-4 as the reference inhibitor.

Techniques:

Journal: RSC Advances

Article Title: Design, synthesis, and mechanistic evaluation of novel pyrazole/thiazole chalcone hybrids as dual tubulin polymerization and COX-2 inhibitors with potent antiproliferative activity

doi: 10.1039/d6ra03557d

Figure Lengend Snippet: Tubulin polymerization inhibitory activity of compounds 9a–o and the reference inhibitor CA-4, expressed as IC 50 values (µM), as determined from a fluorescence-based polymerization assay monitored kinetically for 60 min at 37 °C. Data are presented as mean ± SEM.

Article Snippet: The effects of compounds 9a–o on tubulin polymerization were assessed using a fluorescence-based tubulin polymerization assay (Cytoskeleton, Inc., Cat. No. BK011P), with combretastatin A-4 as the reference inhibitor.

Techniques: Activity Assay, Fluorescence, Polymerization Assay

Journal: RSC Advances

Article Title: Design, synthesis, and mechanistic evaluation of novel pyrazole/thiazole chalcone hybrids as dual tubulin polymerization and COX-2 inhibitors with potent antiproliferative activity

doi: 10.1039/d6ra03557d

Figure Lengend Snippet: Superimposition of the co-crystallized (green) and redocked (brown) colchicine poses within the colchicine-binding site of tubulin (PDB ID: 4O2B).

Article Snippet: The effects of compounds 9a–o on tubulin polymerization were assessed using a fluorescence-based tubulin polymerization assay (Cytoskeleton, Inc., Cat. No. BK011P), with combretastatin A-4 as the reference inhibitor.

Techniques: Binding Assay

Journal: RSC Advances

Article Title: Design, synthesis, and mechanistic evaluation of novel pyrazole/thiazole chalcone hybrids as dual tubulin polymerization and COX-2 inhibitors with potent antiproliferative activity

doi: 10.1039/d6ra03557d

Figure Lengend Snippet: Binding interactions of compound 9l within the colchicine-binding site of tubulin (PDB ID: 4O2B): (A) 2D interaction diagram and (B) 3D binding mode.

Article Snippet: The effects of compounds 9a–o on tubulin polymerization were assessed using a fluorescence-based tubulin polymerization assay (Cytoskeleton, Inc., Cat. No. BK011P), with combretastatin A-4 as the reference inhibitor.

Techniques: Binding Assay

Journal: PLoS ONE

Article Title: The Human Minor Histocompatibility Antigen1 Is a RhoGAP

doi: 10.1371/journal.pone.0073962

Figure Lengend Snippet: (A) Sequence alignment of HMHA1 with the typical RhoGAP, p50RhoGAP, and the structurally-related BAR-GAPs, GRAF1 and OPHN1. Green indicates two matching amino acids. Pink indicates three matching amino acids. Purple indicates four matching amino acids. The arginine finger region is indicated with a black bar. (B) 3D model of the protein-protein complex between RhoA and the HMHA1 RhoGAP domain highlighting the catalytic residues (in sticks, colour coding as indicated; P-loop-Switch I-Switch II of RhoA in light green). The homology model for the GAP domain of human HMHA1 is based on the structure of the human p50RhoGAP domain (PDB ID: 1tx4), using Phyre. The position of the HMHA1 GAP domain in the complex with human RhoA (from RhoA⋅GDP⋅AlFx⋅p50RhoGAP; PDB ID: 1tx4) was obtained through its overlay on the p50RhoGAP domain. The RhoGAP domain of GRAF1 from Gallus gallus (PDB ID: 1f7c) was superimposed onto the model of the HMHA1 GAP domain. (C) HMHA1 C1-GAPtail has in vitro GAP activity towards Rac1, Cdc42, and RhoA but not towards Ras (purple bars). p50RhoGAP was used as a positive control (red bars). GTPases or HMHA1 only were used as a control and as a measure for intrinsic nucleotide hydrolysis (yellow bars). Data are mean values of two independent experiments. Error bars indicate SD. (D) HMHA1 GAP activity is inhibited by the N-terminal BAR domain as full-length HMHA1 has no GAP activity while C1-GAPtail, lacking the N-terminal region, shows GAP activity (purple bars). GTPases or HMHA1 only were used as a control and as a measure for intrinsic hydrolysis (yellow bars). Data are mean values of two independent experiments. Error bars indicate SD.

Article Snippet: In vitro GAP activity of HMHA1 was measured using a RhoGAP Assay (BK105; Cytoskeleton) according to the manufacturers' recommendations.

Techniques: Sequencing, In Vitro, Activity Assay, Positive Control, Control

Journal: Cancer gene therapy

Article Title: Pals1 functions in redundancy with SMAP1 to inhibit Arf6 in order to prevent Rac1-dependent colorectal cancer cell migration and invasion.

doi: 10.1038/s41417-022-00570-2

Figure Lengend Snippet: Fig. 1 Loss of Pals1 in a colorectal cancer cell line HCT116 results in TJ defects, enhanced migration and invasion. A Immunostaining of confluent HCT116 and HCT116ΔPals1 cells with indicated antibodies, B Activation of Rac1 in wild type and Pals1-deficient HCT116 was quantified using G-LISA assay. C Representative images and quantification of the FRET signal of a biosensor targeting active Rac1, transfected in HCT116 and HCT116ΔPals1 cells. Results are representative of 4 experiments. D Quantification of Rac1 biosensor FRET signals at the cell body and the cell cortex in HCT116 and HCT116ΔPals1 cells. Scale bars are 20 µm in A, C.

Article Snippet: The Rac1 G-LISA activity assay (#BK128, Cytoskeleton Inc.) was performed according to manufacturer’s instructions, using 0.5 mg/ml protein concentration of cell lysates.

Techniques: Migration, Immunostaining, Activation Assay, Transfection

Journal: Cancer gene therapy

Article Title: Pals1 functions in redundancy with SMAP1 to inhibit Arf6 in order to prevent Rac1-dependent colorectal cancer cell migration and invasion.

doi: 10.1038/s41417-022-00570-2

Figure Lengend Snippet: Fig. 2 Deletion of Pals1 in Caco-2 cells does not result in enhanced migration and invasion or upregulation of active Arf6 or Rac1. A Immunostaining of confluent Caco-2 and Caco-2ΔPals1 cells with the indicated antibodies. B Representative images from wound healing assays of Caco-2 and Caco-2ΔPals1 cells and the corresponding quantification (N = 3). C Representative images and quantification of transwell matrigel invasions assays of Caco-2 and Caco-2ΔPals1 cells (N = 5). D Western blot and CBB-stained gel of pulldown experiments to detect active Arf6 from lysates of Caco-2 and Caco-2ΔPals1 cells (N = 6). E Western blot and CBB-stained gel of pulldown experiments to detect active Rac1 from lysates of Caco-2 and Caco-2ΔPals1 cells (N = 3). F Representative images and quantification of the FRET signal of a biosensor targeting active Rac1, transfected in Caco-2 and Caco-2ΔPals1 cells. Results are representative of 4 experiments. Scale bars are 20 µm in A and F, 100 µm in B.

Article Snippet: The Rac1 G-LISA activity assay (#BK128, Cytoskeleton Inc.) was performed according to manufacturer’s instructions, using 0.5 mg/ml protein concentration of cell lysates.

Techniques: Migration, Immunostaining, Western Blot, Staining, Transfection

Journal: Cancer gene therapy

Article Title: Pals1 functions in redundancy with SMAP1 to inhibit Arf6 in order to prevent Rac1-dependent colorectal cancer cell migration and invasion.

doi: 10.1038/s41417-022-00570-2

Figure Lengend Snippet: Fig. 3 Pals1-deficient DLD1 do not exhibit increased Arf6/Rac1 activity or enhanced cell migration/invasion. A Immunostaining of confluent DLD1 and DLD1ΔPals1 cells with the indicated antibodies. B Representative images from wound healing assays of DLD1 and DLD1ΔPals1 cells and the corresponding quantification (N = 3). C Representative images and quantification of transwell matrigel invasion assays of DLD1 and DLD1ΔPals1 cells (N = 3). D Western blot and CBB-stained gel of pulldown experiments to detect active Arf6 from cell lysates of DLD1 and DLD1ΔPals1 cells (N = 8). E Western blot and CBB-stained gel of pulldown experiments to detect active Rac1 from cell lysates of DLD1 and DLD1ΔPals1 cells (N = 3). Scale bars are 20 µm in A and 100 µm in B.

Article Snippet: The Rac1 G-LISA activity assay (#BK128, Cytoskeleton Inc.) was performed according to manufacturer’s instructions, using 0.5 mg/ml protein concentration of cell lysates.

Techniques: Activity Assay, Migration, Immunostaining, Western Blot, Staining

Journal: Cancer gene therapy

Article Title: Pals1 functions in redundancy with SMAP1 to inhibit Arf6 in order to prevent Rac1-dependent colorectal cancer cell migration and invasion.

doi: 10.1038/s41417-022-00570-2

Figure Lengend Snippet: Fig. 4 Knockout of Pals1 in mesenchymal-like RKO cells does not affect cell motility. A Western blot analysis of the expression of E-Cadherin in different colorectal cancer cell lines. B Representative images from wound healing assays of RKO and RKOΔPals1 cells and the corresponding quantification (N = 6). C Representative images and quantification of transwell matrigel invasion assays of RKO and RKOΔPals1 cells (N = 4). D Western blot and CBB-stained gel of pulldown experiments to detect active Arf6 from cell lysates of RKO and RKOΔPals1 cells (N = 6). E Western blot and CBB-stained gel of pulldown experiments to detect active Rac1 from cell lysates of RKO and RKOΔPals1 cells (N = 3). Scale bars are 100 µm in B.

Article Snippet: The Rac1 G-LISA activity assay (#BK128, Cytoskeleton Inc.) was performed according to manufacturer’s instructions, using 0.5 mg/ml protein concentration of cell lysates.

Techniques: Knock-Out, Western Blot, Expressing, Staining

Journal: Cancer gene therapy

Article Title: Pals1 functions in redundancy with SMAP1 to inhibit Arf6 in order to prevent Rac1-dependent colorectal cancer cell migration and invasion.

doi: 10.1038/s41417-022-00570-2

Figure Lengend Snippet: Fig. 6 SW48ΔPals1 cells display enhanced Arf6/Rac1 activation and increased cell migration, which can be rescued by SMAP1 transfection. A Immunostaining of confluent SW48 and SW48ΔPals1 cells with the indicated antibodies. B Representative images and quantification of the FRET signal of a biosensor targeting active Rac1, transfected in SW48 and SW48ΔPals1 cells. Results are representative of 3 experiments. C Western blot of cell lines with and without SMAP1 overexpression. Empty vector was used as negative control. D, E Quantification of cell migration (scratch assay, D) and invasions assay (E) of the indicated cell lines. F Rac1 activation of the indicated cell lines quantified by G-LISA. G Western blot and CBB-stained gel of pulldown experiments to detect active Arf6 from cell lysates of the indicated cell lines (N = 3). H Survival probability of colorectal cancer patients with only low Pals1 expression, only low SMAP1 expression or low Pals1 and low SMAP1 expression. Scale bars are 20 µm in A and B.

Article Snippet: The Rac1 G-LISA activity assay (#BK128, Cytoskeleton Inc.) was performed according to manufacturer’s instructions, using 0.5 mg/ml protein concentration of cell lysates.

Techniques: Activation Assay, Migration, Transfection, Immunostaining, Western Blot, Over Expression, Plasmid Preparation, Negative Control, Wound Healing Assay, Staining, Expressing

Journal: Bone research

Article Title: IRF1-mediated upregulation of PARP12 promotes cartilage degradation by inhibiting PINK1/Parkin dependent mitophagy through ISG15 attenuating ubiquitylation and SUMOylation of MFN1/2.

doi: 10.1038/s41413-024-00363-3

Figure Lengend Snippet: Fig. 5 ISG15 upregulated by PAPR12, increasing ISGylation of MFN1/2 and then attenuating the ubiquitylation and SUMOylation to inhibit PINK1/Parkin-dependent mitophagy. a Immunoblotting (IB) analysis of ubiquitylation and ISG15 in PHCs following transfection with KD-ISG15 or KD-NC. b Protein quantification of (a) using ImageJ. n = 3 per group. c IB analysis of ubiquitylation of mitochondrial proteins. Mitochondria were isolated from KD-NC and KD-ISG15 PHCs transfected with a HA-ubiquitin construct. d Protein quantification of (d) using ImageJ. n = 3 per group. e IB analysis of SUMO2/3 and ubiquitin with SUMO2/3-conjugated proteins in KD-NC and KD-ISG15 PHCs immunoprecipitated using SUMO2/3 affinity beads. f IB analysis of MFN1/2, MFN1, MFN2, ubiquitin, SUMO2/3 and ISG15 in KD-NC and KD-ISG15 PHCs treated with or without MG-132 after immunoprecipitation of MFN1/2. The molecular marker of IB plots of ubiquitin, SUMO2/3, and ISG15 conjugates with MFN1/2 is 70 kD and above when considering the molecular weight of MFN1/2 at 85 kD. g Quantification of MFN1/2, ubiquitin, SUMO2/3, and ISG15 of (f) using ImageJ after immunoprecipitation of MFN1/2. n = 3 per group. h IB analysis of MFN1/2, MFN1, MFN2, ubiquitin, SUMO2/3, and ISG15 in PHCs following either knockdown or overexpression of PARP12 after immunoprecipitation of MFN1/2. i Quantification of MFN1/2, ubiquitin, SUMO2/3, and ISG15 of (h) using ImageJ. n = 3 per group. j MitoTracker Red and LysoTracker Green staining of PHCs was observed by confocal microscopy. Scale bars: 10 µm. k Western blot analysis of PARP12, ISG15, LC3B, p62, MFN1, MFN2, PINK1, Parkin, COL2A1, aggrecan, MMP13, RUNX2, Bcl2, Bax, and NLRP3 inflammasome activity in PHCs coinfected with PARP12 KD-03 shRNA and ISG15-OE adenovirus. n = 3 per group. l ROS staining with PARP12 knockdown and ISG15 overexpression. Scale bars: 100 µm. m JC-1 staining in PHCs following transfection with PARP12 knockdown and ISG15 overexpression. Scale bars: 100 µm. Data are presented as the mean ± SD. Paired t-test (b, d, i) and one-way analysis of variance followed by Tukey’s multiple comparison test (g) were used for statistical analysis. *P < 0.05, **P < 0.01, and ***P < 0.001

Article Snippet: According to the manufacturer’s instructions of SUMOylation 2/3 affinity beads (BK162, Cytoskeleton, Inc.), SUMO2/3-conjugated proteins in chondrocytes lysates were immunoprecipitated.

Techniques: Western Blot, Transfection, Isolation, Ubiquitin Proteomics, Construct, Immunoprecipitation, Marker, Molecular Weight, Knockdown, Over Expression, Staining, Confocal Microscopy, Activity Assay, shRNA, Comparison

Journal: Bone research

Article Title: IRF1-mediated upregulation of PARP12 promotes cartilage degradation by inhibiting PINK1/Parkin dependent mitophagy through ISG15 attenuating ubiquitylation and SUMOylation of MFN1/2.

doi: 10.1038/s41413-024-00363-3

Figure Lengend Snippet: Fig. 8 PARP12 modulates osteoarthritis (OA) pathogenesis in monosodium iodoacetate (MIA)-treated rats. a Experimental diagram of the MIA OA rat model treated with XAV-939 or PARP12 overexpression (OE) adenovirus. Rats were evaluated at age of 10 weeks. n = 6 per group. b 3D reconstruction images of micro-CT scanning of the knees of rats treated with XAV-939 or PARP12-OE adenovirus. n = 5 per group. c–f Analysis of BV/TV, BS/TV, trabecular thickness, and trabecular numbers. n = 5 per group. g Western blot analysis of PARP12, COL2A1, aggrecan, MMP13, RUNX2, Bcl2/Bax, LC3B, p62, MFN1, MFN2, PINK1, Parkin and NLRP3 inflammasome activity in chondrocytes of rats treated with XAV-939 or PARP12 overexpression (OE) adenovirus. n = 3 per group. h Representative images of Safranin O and IHC staining of PARP12, COL2A1 and MMP13. Scale bars: 250 µm (first row) and 50 µm (second row). i Quantification of macroscopic score based on staining results in (h). n = 3 per group. j–l Quantification of PARP12, COL2A1, and MMP13 positive chondrocytes based on staining results in (h). n = 3 per group. m–o ROS staining, ATP level and JC-1 staining in chondrocytes of rats treated with XAV-939 or PARP12 OE adenovirus. p Schematic representation of the mechanism by which IRF1-mediated upregulation of PARP12 promotes cartilage degradation by inhibiting PINK1/Parkin dependent mitophagy through ISG15 attenuating the ubiquitylation and SUMOylation of MFN1/2. Data are presented as the mean ± SD. Paired t-test (c–f, j–l, n) and non-parametric Mann-Whitney U test (i) were used for statistical analysis. *P < 0.05, **P < 0.01, ***P < 0.001

Article Snippet: According to the manufacturer’s instructions of SUMOylation 2/3 affinity beads (BK162, Cytoskeleton, Inc.), SUMO2/3-conjugated proteins in chondrocytes lysates were immunoprecipitated.

Techniques: Over Expression, Micro-CT, Western Blot, Activity Assay, Immunohistochemistry, Staining, MANN-WHITNEY

Journal: Saudi Pharmaceutical Journal : SPJ

Article Title: Design, synthesis, molecular docking, and in vitro studies of 2-mercaptoquinazolin-4(3 H )-ones as potential anti-breast cancer agents

doi: 10.1016/j.jsps.2024.101971

Figure Lengend Snippet: Inhibition assay of the synthesized quinazolinone compounds (3a, 3b, 3e, 3 g, and 3 h) against KSP (% inhibition at 2 µM and IC 50 ), using Ispinesib as positive control, and against PI3Kδ using Idelalisib as positive control.

Article Snippet: For IC 50 value measurement, a microtubule-activated ATPase kinetic assay kit (Cytoskeleton, Cat. # BK060) was used as per the manufacturer’s instructions ( ).

Techniques: Inhibition, Synthesized, Positive Control, Control, Activity Assay

Journal: Translational oncology

Article Title: NF1-RAC1 axis regulates migration of the melanocytic lineage.

doi: 10.1016/j.tranon.2020.100858

Figure Lengend Snippet: Fig. 1. Loss of NF1 reduces RAC1-driven melanoblast migration. A. Scratch-like migration assay representing the percentage of cell coverage after 6 h, 9 h and 12 h using either WT or NF1+/−melanoblasts (MB) in the presence of a RAC1 activator (CN04). B. RAC1 activity was measured by G-lisa in WT and NF1+/−melanoblasts (MB). C. Scratch-like migration assay after 3 h, 6 h, 9 h and 12 h in NF1+/−melanoblasts 48 h-post transfection with either a scramble siRNA (SCR) or with an NF1-specific siRNA (siNF1). D. Expression status of NF1 and expression of phosphorylated and non-phosphorylated ERK and AKT in NF1+/−melanoblasts by western blot. α-actinin was used as a loading control. GTP-RAC1 pulldown and total lysates were blotted with α-RAC1 antibody. E. Scratch-like migration assay representing the percentage of cell coverage after 9 h and 12 h in NF1+/−melanoblasts 48 h-post transfection with either a scramble siRNA (SCR) or with an NF1-specific siRNA (siNF1) and in the presence or absence of a RAC1 activator (CN04). *: SCR vs. siNF1, #: -CN04 vs. +CN04. F. GTP-RAC1 pulldown and total lysates were blotted with α-RAC1 antibody in the presence or absence of a RAC1 activator (CN04). **P < 0.01, *P < 0.05, ns: not significant (unpaired Student's t-test). All error bars represent the SEM of at least three independent experiments.

Article Snippet: The amount of activated RAC1 was determined by western blot using a RAC1 specific antibody (Cytoskeleton inc. Cat. # BK035).

Techniques: Migration, Activity Assay, Transfection, Expressing, Western Blot, Control

Journal: Translational oncology

Article Title: NF1-RAC1 axis regulates migration of the melanocytic lineage.

doi: 10.1016/j.tranon.2020.100858

Figure Lengend Snippet: Fig. 2. Loss of NF1 increases melanoma migration and is associated with increased PREX1 expression. A. NF1 mRNA expression under NF1 silencing with two siRNAs (NF1.6 and NF1.11) in SK-mel-23, Mel501, and SK-mel-103 melanoma cell lines. B. PREX1 mRNA expression under NF1 silencing with two siRNAs in SK-mel-23, Mel501, and SK- mel-103 cell lines. C. Scratch-like migration assay representing the percentage of cell coverage after 6 h, 12 h and 24 h under NF1 silencing in SK-mel-23, Mel501, and SK-mel- 103 cell lines. D. Scratch-like migration assay as in C, after additional transfection with siRNA control (scramble) or with PREX1 siRNA (siPREX1). E. Scratch-like migration assay as in C. in the absence (control) or presence (RAC1 inhibitor) of a RAC1 inhibitor. ***P < 0.001, **P < 0.01, *P < 0.05 (unpaired Student's t-test). All error bars rep- resent the SEM of at least three independent experiments.

Article Snippet: The amount of activated RAC1 was determined by western blot using a RAC1 specific antibody (Cytoskeleton inc. Cat. # BK035).

Techniques: Migration, Expressing, Transfection, Control

Journal: Translational oncology

Article Title: NF1-RAC1 axis regulates migration of the melanocytic lineage.

doi: 10.1016/j.tranon.2020.100858

Figure Lengend Snippet: Fig. 4. PREX is upregulated in low NF1 expressing melanoma metastases. A. Representative microphotographs of Tissue Microarray (TMA) containing primary and metastatic melanoma samples analysed by immunohistochemistry using a specific antibody against NF1, RAC1 and PREX1. Bar, 100 μm. B. Scoring of the immunohistochemistry staining was performed according to our previously described protocol [24]. Duplicates of valid punch samples are represented for each condition. Significance was tested using two-tailed t-test with *P < 0.05 and ns: not significant.

Article Snippet: The amount of activated RAC1 was determined by western blot using a RAC1 specific antibody (Cytoskeleton inc. Cat. # BK035).

Techniques: Expressing, Microarray, Immunohistochemistry, Staining, Two Tailed Test