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: Frontiers in Chemistry

Article Title: Microscopic distribution of taxanes in freeze-fixed stems of Taxus cuspidata

doi: 10.3389/fchem.2024.1437141

Figure Lengend Snippet: Cryo-TOF-SIMS spectra and chemical structures of (A) 10-DAB, (B) BAC, (C) 10-DAT, (D) EDT, (E) CE, (F) paclitaxel, (G) 7-epi-taxol, and (H) 7-xyl-10-DAT. Cryo-TOF-SIMS spectra obtained from the frozen, hydrated transverse surface of T. cuspidata stems from (I) late summer and (J) spring in the phloem region. Standard chemicals of taxanes were dissolved at ca. 50 mM in 80% EtOH and frozen for measurements.

Article Snippet: Standard chemicals including paclitaxel (FUJIFILM Wako Pure Chemical Corp., Osaka, Japan), 10-DAB (Tokyo Chemical Industry Co., Ltd., Tokyo, Japan), BAC and CE (Selleck Chemicals LLC., TX, United States), 7-xyl-10-DAT, 7-epi-taxol, EDT, and 10-DAT (MedChem Express LLC., NJ, United States) were purchased and used as received.

Techniques:

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