Journal: Brain

Article Title: PRUNE is crucial for normal brain development and mutated in microcephaly with neurodevelopmental impairment

doi: 10.1093/brain/awx014

Figure Lengend Snippet: PRUNE and tubulin. ( A ) Top : Cell-based microtubules co-sedimentation assay and SDS/PAGE analysis showing the binding of FLAG-tagged PRUNE to microtubule polymers (MT) using in vitro whole protein extracts from SHSY5Y clones overexpressing wild-type FLAG-tagged PRUNE or empty vector (as negative control), with immunoblotting with antibodies against anti-FLAG, anti-β-tubulin and anti-kinesin V (as a positive control due to its known binding to microtubule polymers). Wild-type FLAG-tagged PRUNE was found in the pellet (P) fraction in the presence of microtubule polymers, while it was found only in the supernatant (S) fraction in the absence of microtubule polymers, indicating microtubule binding. Bottom : Co-immunoprecipitation assay using Flag-tagged wild-type, D30N and R297W PRUNE protein expression in SHSY5Y inducible cell clones. The whole protein extract from empty vector (EV, as negative control), wild-type, D30N and R297W PRUNE-overexpressing cells incubated with antibodies against β-tubulin or α-tubulin to immunoprecipitate (IP), endogenous β-tubulin ( left ) or α-tubulin ( right ). A band of the expected size (60 kDa) was detected by western blotting using an anti-Flag antibody in the immunoprecipitate fraction from wild-type and D30N-overexpressing clones, indicating binding of PRUNE wild-type and D30N with both β- and α-tubulin. Flag-tagged R297W PRUNE was detected with a long exposure ( and ). ( B ) Microtubule nucleation assay. SHSY5Y-inducible cells overexpressing wild-type, D30N, R297W PRUNE proteins were treated with doxycycline followed by immunofluorescence staining with β-tubulin antibody (red), and DAPI for DNA staining (blue). Cells containing microtubule asters with a diameter longer than 5 µm were scored and the results from a representative experiment in triplicate are shown. Left : The immunofluorescence analysis performed on the inducible clones after 2 min at 37°C showing some representative asters (in red) for each clone (Scale bars = 5 µm). The chart on the right indicates the percentage of cells with aster diameters longer than 5 µm. Wild-type PRUNE expressing clones show a higher percentage of cells containing asters longer than 5 µm, compared to those expressing D30N and R297W PRUNE (∼160 nuclei per clone were counted; ). ( C ) In vitro microtubule polymerization assay performed using wild-type (black), D30N (orange) and R297W (green) PRUNE purified from E. coli. The standard polymerization reaction, alone or in presence of the purified wild-type or mutated (D30N and R297W) PRUNE protein, incubated with tubulin and followed by absorbance readings at 360 nm (excitation at 360 nm, and emission at 420 nm; EnSpire manager software) to evaluate the maximum absolute curve slope. Polymerization curves are shown for the three phases of polymerization; I (nucleation), II (growth), III (steady state). The polymerization rate is enhanced (∼2-fold) in presence of wild-type PRUNE (black) in comparison with microtubules alone (blue). Polymerization in the presence of D30N PRUNE (orange) is unaffected, while it is unregulated by R297W PRUNE (green). Both mutations result in a notable delay of microtubule polymerization rate, which is particularly evident during the nucleation phase (phase I). The curves shown represent the average of n = 3 independent experiments, expressed as mean ± SD of samples assayed in triplicate. See for standard polymerization alone, and in the presence of 3 µM paclitaxel or 3 µM nocodazole, used as positive and negative controls, respectively.

Article Snippet: Twenty microlitres of porcine brain tubulin (10 mg/ml; #T240-DX; Cytoskeleton) was supplemented with 1 mM GTP, 1 mM EGTA, 1 mM DTT and 80 μl of BRB80 buffer (#BP01; Cytoskeleton) consisting of 80 mM PIPES, 2 mM MgCl 2 , 0.5 mM EGTA, pH 6.9 and allow to polymerize at 37°C for 30 min by adding paclitaxel (#TXD01; Cytoskeleton) drop wise (starting from 0.02 mM up to 2 mM).

Techniques: Sedimentation, SDS Page, Binding Assay, In Vitro, Clone Assay, Plasmid Preparation, Negative Control, Western Blot, Positive Control, Co-Immunoprecipitation Assay, Expressing, Incubation, Immunofluorescence, Staining, Polymerization Assay, Purification, Software, Comparison

Journal: JCI insight

Article Title: Biallelic MAD2L1BP (p31comet) mutation is associated with mosaic aneuploidy and juvenile granulosa cell tumors.

doi: 10.1172/jci.insight.170079

Figure Lengend Snippet: Figure 5. Mutated p31comet is sensitive to proteosomal degradation causing slowed proliferation and decreased sensitivity to chemotherapeutic drugs in patients’ fibroblasts. (A) Analysis of p31comet protein half-life using cycloheximide. Fibroblasts were treated with cycloheximide at indicated time points, and p31comet was analyzed by immunoblotting (β-tubulin as internal control). The blots are representative of 3 experiments. (B) Densitometric analysis of the immunoblots. Intensities of the p31comet bands in relation to β-tubulin standard. Expression levels were normalized to untreated cells. The dashed line indicates t1/2. The blots are representative for 3 repeated experiments with consistent results. (C) Inhibition of proteasomal degradation by MG132 increas- es p31comet levels. Fibroblasts were treated with 10 μM MG132 for 2 and 4 hours, and p31comet protein accumulation was analyzed by Western blot (β-actin as internal control). The blots are representative for 3 experiments. (D) Growth curves of fibroblasts from 2 controls and both patients. Data were analyzed by a 2-tailed, unpaired Student’s t test at each time point. Error bars represent the SD (n = 4). (****P < 0.0001; ***P < 0.001; **P < 0.01; *P < 0.05.) Each experiment was repeated 3 times. (E) Dose response to paclitaxel. Cell viability of fibroblasts from controls and patients were treated for 72 hours with the indicated concentrations of paclitaxel normalized to DMSO vehicle. Error bars represent the SD (n = 4). Data were analyzed by 2-tailed, unpaired Student’s t test (****P < 0.0001; ***P < 0.001; **P < 0.01). Each experiment was repeated 3 times.

Article Snippet: For SAC override, Taxol-arrested HeLaK cells were harvested by shake-off and released for the indicated times by replating into medium supplemented with aurora B inhibitor ZM447439 (Tocris Biosciences, Bio-Techne, 5 μM), Taxol (Tocris Biosciences, Bio-Techne, 0.2 μg/mL), and cycloheximide (MilliporeSigma, 30 μg/mL).

Techniques: Western Blot, Control, Expressing, Inhibition

Journal: Current biology : CB

Article Title: Synaptic-like Vesicles Facilitate Pioneer Axon Invasion.

doi: 10.1016/j.cub.2019.06.078

Figure Lengend Snippet: Figure 7. Modulation of Actin Structures Alters Vesicles at the DREZ (A, C, and E) Confocal z-projections of pioneer growth cone actin organization in Tg(sox10:syn-gfp) in animals treated with DMSO (A), SU6656 (C), and paclitaxel (E) from a 24-h time lapse starting at 48 hpf. White arrows denote actin concentrations at DREZ. White box denotes insets on right. Dashed blue lines denote DRG tracings. Note: fluorescence outside of white box in (E) is a spinal oligodendrocyte. (B, D, and F) Growth cone Syn-GFP intensity tracings throughout navigation in DMSO (B; n = 6), SU6656 (D; n = 7), and paclitaxel (F; n = 7) DRG. Inhibition of actin- based invasion abrogates Syn-GFP accumulation, and promotion of actin-based invasion generates robust Syn-GFP accumulation. Green line denotes representative graph. Dashed black line denotes mean ± SEM. Blue box denotes DREZ. (G) Percent of axons that demonstrate vesicle accumulations at DREZ (n = 6 DMSO; n = 7 SU6656; n = 7 paclitaxel). (H and I) Duration (H) and time of initiation (I) of vesicle concentrates at the DREZ showing changes in vesicles when actin invasion is altered (n = 6 DMSO; n = 7 SU6656; n = 7 paclitaxel). (J) Model of actin and synaptic-like vesicle coordinated invasion of the spinal cord by DRG pioneer axons. SEM is shown. Tukey’s HSD was used for (H) and (I). Scale bars, 10 mm. See also Video S6.

Article Snippet: REAGENT or RESOURCE SOURCE IDENTIFIER Antibodies Rabbit anti-Synapsin 1/2 Synaptic Systems Cat #: 106 002; RRID: AB_2622240 Anti-Rab8a mAb produced in mouse Abnova Cat #: H00004218-M02; RRID: AB_519014 Anti-MMP14 antibody produced in rabbit Sigma Cat #: SAB4501901; RRID: AB_10744760 Znp-1 (anti-synaptotagmin 2) DHSB RRID: AB_10013783 SV2 (synaptic vesicle glycoprotein 2A) DHSB RRID: AB_2315387 Alexa Fluor 594 goat anti-mouse Thermo Fisher Cat #: A-11032; RRID: AB_2535767 Alexa Fluor 594 goat anti-rabbit Thermo Fisher Cat #: R37117; RRID: AB_2534116 Alexa Flour 647 goat anti-mouse Thermo Fisher Cat #: A-21235; RRID: AB_2535806 Alexa Flour 647 goat anti-rabbit Thermo Fisher Cat #: A-20991; RRID: AB_2535814 Alexa Flour 488 goat anti-rabbit Thermo Fisher Cat #: A-11034; RRID: AB_2576217 Bacterial Strains Mix & Go Competent Cells Zymo Research Cat #: T3007 Chemicals, Peptides, and Recombinant Proteins Paclitaxel Acros Cat #: 328420050; CAS: 33069-62-4 GM6001 Santa Cruz Biotechnology Cat #: sc-203979; CAS: 142880-36-2 SU6656 Santa Cruz Biotechnology Cat #: sc-203286; CAS: 330161-87-0 LR Clonease II Plus Thermo Fisher Cat #: 12538120 TRIzol Thermo Fisher Cat #: 15596026 Chloroform Sigma-Aldrich Cat #: C2432; CAS: 67-66-2 Isopropanol Sigma-Aldrich Cat #: W292907; CAS: 67-63-0 ProtoScript II First Strand cDNA Synthesis Kit New England Biolabs Cat # E6300S BseRI New England Biolabs Cat #: R5081S Critical Commercial Assays RNAscope Multiplex Fluorescent Reagent Kit v2 Advanced Cell Diagnostics Cat #: 323100 RNAscope Probe-Dr-mmp14a Advanced Cell Diagnostics Cat #: 575081 RNAscope Probe-Dr-polr2 Advanced Cell Diagnostics Cat #: 559921 RNAscope 3-plex Negative Control Probe Advanced Cell Diagnostics Cat #: 320871 Opal 650 Fluorophore Akoya Biosciences Cat #: FP1496001KT Experimental Models: Organisms/Strains

Techniques: Inhibition

Journal: International journal of molecular sciences

Article Title: Flubendazole Enhances the Inhibitory Effect of Paclitaxel via HIF1α/PI3K/AKT Signaling Pathways in Breast Cancer.

doi: 10.3390/ijms242015121

Figure Lengend Snippet: Figure 1. Flubendazole enhances the proliferation inhibitory effect of PTX in MDA-MB-231 and MCF-7 cells. (A,B) heat map of mean IC50 values of flubendazole and paclitaxel in MDA-MB-231 and MCF-7 cell lines at different concentrations. Cell viabilities were measured by MTT assay. Combination indexes

Article Snippet: Reagents used in this study were as follows: MTT (M2128), DAPI (D9542), Flubendazole (HY-B0294), Paclitaxel (HY-B0015), MMP-2 (87809, CST), E-cadherin (14472, CST), β-actin (66009-1-Ig, Proteintech, Rosemont, IL, USA), N-Cadherin (13116, CST), PI3K (4255, CST), p-PI3K (17366, CST), AKT (9272, CST), p-AKT (4060S, CST), mTOR (2972, CST), p-mTOR (5536, CST), HIF1α (36169, CST) p62 (88588, CST), LC3 (4108, CST), ATG5 (12994, CST).

Techniques: MTT Assay

Journal: International journal of molecular sciences

Article Title: Flubendazole Enhances the Inhibitory Effect of Paclitaxel via HIF1α/PI3K/AKT Signaling Pathways in Breast Cancer.

doi: 10.3390/ijms242015121

Figure Lengend Snippet: Figure 2. Flubendazole enhances the metastasis inhibitory effect of PTX in MDA-MB-231 and MCF-7 cells. (A–D) scratch assay of MDA-MB-231 and MCF-7 cells treated with vehicle, flubendazole, paclitaxel, flubendazole combined with paclitaxel for 24 h. Representative images and quantitative analysis of scratch cells are shown. Scale bar,100 µm. (E–H) transwell assay of MDA-MB-231 and MCF-7 cells treated with vehicle, flubendazole, paclitaxel, flubendazole combined with paclitaxel for 24 h. Repre- sentative images and quantitative analysis of percentage of positive ratios are shown. Scale bar, 40 µm. (I–P) immunofluorescence analysis of the E-cadherin and MMP-2 antibody in MDA-MB-231 and MCF-7

Article Snippet: Reagents used in this study were as follows: MTT (M2128), DAPI (D9542), Flubendazole (HY-B0294), Paclitaxel (HY-B0015), MMP-2 (87809, CST), E-cadherin (14472, CST), β-actin (66009-1-Ig, Proteintech, Rosemont, IL, USA), N-Cadherin (13116, CST), PI3K (4255, CST), p-PI3K (17366, CST), AKT (9272, CST), p-AKT (4060S, CST), mTOR (2972, CST), p-mTOR (5536, CST), HIF1α (36169, CST) p62 (88588, CST), LC3 (4108, CST), ATG5 (12994, CST).

Techniques: Wound Healing Assay, Transwell Assay, Immunofluorescence

Journal: International journal of molecular sciences

Article Title: Flubendazole Enhances the Inhibitory Effect of Paclitaxel via HIF1α/PI3K/AKT Signaling Pathways in Breast Cancer.

doi: 10.3390/ijms242015121

Figure Lengend Snippet: Figure 3. Combination of flubendazole and paclitaxel leads to aberrant mitosis and induces apoptosis in MDA-MB-231 and MCF-7 cells. (A–D) immunofluorescence image of α-Tubulin and β-Tubulin antibodies in MDA-MB-231 and MCF-7 cells after treatment by vehicle, flubendazole, paclitaxel, fluben- dazole combined with paclitaxel. Scale bar, 10 µm. (E–H) cell cycle distribution assay of MDA-MB-231 and MCF-7 cells treated with vehicle, flubendazole, paclitaxel, flubendazole combined with paclitaxel.

Article Snippet: Reagents used in this study were as follows: MTT (M2128), DAPI (D9542), Flubendazole (HY-B0294), Paclitaxel (HY-B0015), MMP-2 (87809, CST), E-cadherin (14472, CST), β-actin (66009-1-Ig, Proteintech, Rosemont, IL, USA), N-Cadherin (13116, CST), PI3K (4255, CST), p-PI3K (17366, CST), AKT (9272, CST), p-AKT (4060S, CST), mTOR (2972, CST), p-mTOR (5536, CST), HIF1α (36169, CST) p62 (88588, CST), LC3 (4108, CST), ATG5 (12994, CST).

Techniques: Immunofluorescence

Journal: International journal of molecular sciences

Article Title: Flubendazole Enhances the Inhibitory Effect of Paclitaxel via HIF1α/PI3K/AKT Signaling Pathways in Breast Cancer.

doi: 10.3390/ijms242015121

Figure Lengend Snippet: Figure 4. Transcriptome analysis of MDA-MB-231 cells treated with vehicle, paclitaxel, flubendazole combined with paclitaxel for 24 h. (A) volcanic map of significantly differentially expressed genes in paclitaxel group and flubendazole combined with paclitaxel group. (Red dots indicate genes with FDR < 0.05 and log2FoldChange > 1; Blue dots indicate genes with FDR < 0.05 and log2FoldChange < −1. (B) pathway bubble diagram of differentially expressed gene enrichment in paclitaxel group and fluben- dazole combined with paclitaxel group. (C) PI3K/AKT signaling pathway-related genes differentially expressed heat map. (D) HIF1α and AKT genes interact with some genes of PI3K/AKT signaling pathway. (E,F) Western blotting analysis of HIF1α, PI3K, p-PI3K, AKT, p-AKT, mTOR, and p-mTOR expression in MDA-MB-231 and MCF-7 cells treated with vehicle, flubendazole, paclitaxel, flubendazole combined with paclitaxel for 24 h. β-actin was used as a loading control. Quantification of Western blotting analysis is shown. Data are expressed as mean ± SEM. All data are representative of at least three independent experiments. * p < 0.05, ** p < 0.01, *** p < 0.001, compared with control; # p < 0.05, ## p < 0.01, ### p < 0.001 compared with the flubendazole and paclitaxel combinational group; ns, no significance.

Article Snippet: Reagents used in this study were as follows: MTT (M2128), DAPI (D9542), Flubendazole (HY-B0294), Paclitaxel (HY-B0015), MMP-2 (87809, CST), E-cadherin (14472, CST), β-actin (66009-1-Ig, Proteintech, Rosemont, IL, USA), N-Cadherin (13116, CST), PI3K (4255, CST), p-PI3K (17366, CST), AKT (9272, CST), p-AKT (4060S, CST), mTOR (2972, CST), p-mTOR (5536, CST), HIF1α (36169, CST) p62 (88588, CST), LC3 (4108, CST), ATG5 (12994, CST).

Techniques: Western Blot, Expressing, Control

Journal: International journal of molecular sciences

Article Title: Flubendazole Enhances the Inhibitory Effect of Paclitaxel via HIF1α/PI3K/AKT Signaling Pathways in Breast Cancer.

doi: 10.3390/ijms242015121

Figure Lengend Snippet: Figure 5. Flubendazole combined with paclitaxel suppresses tumor growth in vivo. (A,B) whole- body optical imaging system of subcutaneous breast cancer animal model of nude mice formed by injecting MDA-MB-231-luc cells after vehicle, flubendazole, paclitaxel, flubendazole combined with paclitaxel treatment for 24 h. Color scale, min = 50, max = 9691. (C) the images of isolated tumors derived from nude mice after vehicle, flubendazole, paclitaxel, flubendazole combined with paclitaxel treatment. (D,E) line graph of volume and weight of isolated tumors from nude mice with time. (F,G) the expression of Ki67 in MDA-MB-231 tumor sections of nude mice after vehicle, flubendazole, paclitaxel, flubendazole combined with paclitaxel treatment. Representative images and quantitative analysis of the percentage of positive ratios are shown. Scale bar, 40 µm. (H,I) TUNEL assay in tumor sections of nude mice after vehicle, flubendazole, paclitaxel, flubendazole combined with paclitaxel treatment. Representative images and quantification of TUNEL-positive cells are shown. Scale bar, 40 µm. (J) line graph of weight of nude mice with time. (K) H&E staining images representing the heart, liver, spleen, lung, and kidney of nude mice in each group are shown. Scale bar, 40 µm. Data are expressed as mean ± SEM. All data are representative of at least three independent experiments. * p < 0.05, ** p < 0.01, *** p < 0.001, compared with control; ## p < 0.01, ### p < 0.001 compared with the flubendazole and paclitaxel combinational group.

Article Snippet: Reagents used in this study were as follows: MTT (M2128), DAPI (D9542), Flubendazole (HY-B0294), Paclitaxel (HY-B0015), MMP-2 (87809, CST), E-cadherin (14472, CST), β-actin (66009-1-Ig, Proteintech, Rosemont, IL, USA), N-Cadherin (13116, CST), PI3K (4255, CST), p-PI3K (17366, CST), AKT (9272, CST), p-AKT (4060S, CST), mTOR (2972, CST), p-mTOR (5536, CST), HIF1α (36169, CST) p62 (88588, CST), LC3 (4108, CST), ATG5 (12994, CST).

Techniques: In Vivo, Optical Imaging, Animal Model, Isolation, Derivative Assay, Expressing, TUNEL Assay, Staining, Control

Journal: International journal of molecular sciences

Article Title: Flubendazole Enhances the Inhibitory Effect of Paclitaxel via HIF1α/PI3K/AKT Signaling Pathways in Breast Cancer.

doi: 10.3390/ijms242015121

Figure Lengend Snippet: Figure 6. Flubendazole combined with paclitaxel suppresses tumor metastasis in vivo. (A,B) optical imaging system of heart, liver, spleen, lung, and kidney isolated from breast cancer animal model formed by injecting MDA-MB-231-luc cells into the vein of nude mice. Color scale, min = 41, max = 4861. Representative images and quantitative analysis of bioluminescent in lung sections are shown. (C–H) the expression of E-cadherin, MMP-2, and N-cadherin in lung sections of nude mice. Representative images and quantitative analysis of the percentage of positive ratios are shown. Scale bar, 40 µm. Data are expressed as mean ± SEM. All data are representative of at least three independent experiments. * p < 0.05, ** p < 0.01, *** p < 0.001, compared with control; ## p < 0.01, ### p < 0.001 compared with the flubendazole and paclitaxel combinational group.

Article Snippet: Reagents used in this study were as follows: MTT (M2128), DAPI (D9542), Flubendazole (HY-B0294), Paclitaxel (HY-B0015), MMP-2 (87809, CST), E-cadherin (14472, CST), β-actin (66009-1-Ig, Proteintech, Rosemont, IL, USA), N-Cadherin (13116, CST), PI3K (4255, CST), p-PI3K (17366, CST), AKT (9272, CST), p-AKT (4060S, CST), mTOR (2972, CST), p-mTOR (5536, CST), HIF1α (36169, CST) p62 (88588, CST), LC3 (4108, CST), ATG5 (12994, CST).

Techniques: In Vivo, Optical Imaging, Isolation, Animal Model, Expressing, Control

Journal: International journal of molecular sciences

Article Title: Flubendazole Enhances the Inhibitory Effect of Paclitaxel via HIF1α/PI3K/AKT Signaling Pathways in Breast Cancer.

doi: 10.3390/ijms242015121

Figure Lengend Snippet: Figure 7. Flubendazole inhibits the proliferation and migration of paclitaxel-resistant MCF-7 cells. (A,B) 3D cell culture assay of MCF-7/PTX cells treated with vehicle and flubendazole. Representative images and quantitative analysis of 3D cells are shown. (C,D) colony formation assay of MCF-7/PTX cells treated with vehicle and flubendazole. Representative images and quantitative analysis of colonies

Article Snippet: Reagents used in this study were as follows: MTT (M2128), DAPI (D9542), Flubendazole (HY-B0294), Paclitaxel (HY-B0015), MMP-2 (87809, CST), E-cadherin (14472, CST), β-actin (66009-1-Ig, Proteintech, Rosemont, IL, USA), N-Cadherin (13116, CST), PI3K (4255, CST), p-PI3K (17366, CST), AKT (9272, CST), p-AKT (4060S, CST), mTOR (2972, CST), p-mTOR (5536, CST), HIF1α (36169, CST) p62 (88588, CST), LC3 (4108, CST), ATG5 (12994, CST).

Techniques: Migration, Cell Culture, Colony Assay

Journal: Cancers

Article Title: Preclinical Efficacy and Involvement of AKT, mTOR, and ERK Kinases in the Mechanism of Sulforaphane against Endometrial Cancer

doi: 10.3390/cancers12051273

Figure Lengend Snippet: Sulforaphane, paclitaxel and the combination of sulforaphane and paclitaxel reduced tumor volume when compared to control. Severe combined immunodeficiency (SCID) mice bearing an average tumor volume of 50 mm 3 Ishikawa xenografts were randomized into treatment groups which received daily 0.9% saline (control), daily 50 mg/kg sulforaphane, weekly 10 mg/kg paclitaxel, or daily 50 mg/kg sulforaphane in combination with weekly 10 mg/kg paclitaxel. Each drug was administered via intraperitoneal injection. The arrow denotes the inititation of treatment. n = 10 mice per group. ( A ) average tumor volume over time in groups treated with sulforaphane, paclitaxel, or sulforaphane and paclitaxel. ( B ) average body-weight of mice receiving each treatment regimen over the course of administration. n = 10 mice per group. ( C ) tumor tissues were analyzed by tunnel assay for apoptosis. Red color indicates positive signal. Representative images taken at 10× magnification are shown. ( D ) Schematic model for sulforaphane mediated endometrial cancer tumor inhibition. SFN; sulforaphane.

Article Snippet: Sulforaphane (#S8044; Cas No. 4478-93-7) and paclitaxel (#P0093; Cas No. 33069-62-4) were obtained from LKT laboratories (St. Paul, MN, USA).

Techniques: Control, Saline, Injection, Inhibition