Materials and Reagents
The following chemicals and reagents were obtained from the indicated companies:
Paclitaxel: Athenex
Anti-a-Tubulin: Proteintech
488-a-Tubulin: Cytoskeleton
Anti-phospho-histone H3: Millipore
Anti-cleaved/activated caspase 3: Cell signaling
ProLong DAPI reagent: Invitrogen
Anti-Mouse IgG Alexa Fluor 488: Invitrogen
Anti-Rabbit IgG Alexa Flour 546 Invitrogen
HiLyte Fluor 488 labeled tubulin Cytoskeleton, Inc.
Mouse experiments
Mice, 129P3/J 4-week-old, or C57BL/6 3-month-old, purchased from Jackson Lab, were intraperitoneal (I.P.) injected with paclitaxel (Athenex) diluted in saline, 12 mg/kg bodyweight. The mice in each cage were randomly allocated to different treatment groups. Food and water were available ad libitum and experiments were performed during the light cycle (7:00 am to 7:00 pm). At the end of the experiment, animals were euthanized via CO2 asphyxiation, followed by cervical dislocation before tissue biopsy and collection. The animals were maintained and cared in the DVR core facility (University of Miami) according to established NIH protocols and handled in strict agreement with good animal practice as approved by the University of Miami Institutional Animal Care and Use Committee (IACUC).
Ultrasound application
Ultrasound was applied by submerging the mouse from neck down in an ultrasonic water bath (Crest Ultrasonics) with water maintained at 32ºC. The mice were exposed to ultrasound for 5 minutes at a 45 kHZ frequency and adjustable intensity to around 120 mW/cm2. The ultrasound frequency and intensity were monitored with a hydrophone (HCT-0320, from Onda Corporation), pressure meter (Onda), and an oscilloscope (OWON). Mice were sedated with isoflurane during the ultrasonic procedure and recovered from the anesthesia (steady and normal heart rate, respiration, and movements) before returning to the cage for normal activities.
We acquired and tested both the bath and probe type ultrasound devices in our study. The bath device produces 45-150 kHz, 50 to 780 mW/cm2 ultrasound waves with adjustable frequency and energy levels (Crest PowerSonic P1100 Model). The probe device produces adjustable 0.5-3 W/cm2 and 1 or 3 MHz ultrasound waves (New PhysioOriginal Ultrasonic Therapy Machine, etc.). When submerged under water or fully wetted, the presence of fur did not significantly affect the transmission of ultrasound intensity to the surface of the skin.
Histological Analyses
At the end of mouse experiments, following euthanizing the mice, paws and skin (around 0.5 cm2) were collected and preserved immediately by immersing them in 10% formalin overnight at 4°C. Subsequently, the samples underwent embedding in paraffin and were sectioned at 7 μm thickness. These sections underwent deparaffinization and rehydration before being subjected to antigen retrieval in a 0.01 M citric buffer at pH 6. Following a PBS wash, the sections were treated with 0.1% Triton X-100 in PBS for 5 minutes at room temperature, followed by a 30-minute incubation in a blocking solution containing 5% BSA.
For immunostaining, the sections were incubated overnight at 4°C in a humidity chamber with the primary antibodies, which were appropriately diluted in the blocking solution. After several washes with PBS, the sections were exposed to a secondary antibody for 1 hour at room temperature. Mounting was achieved using ProLong DAPI reagent (Invitrogen). The specific antibodies utilized were alpha-tubulin (Proteintech 66031-1-Ig), phospho histone 3 (Millipore 09-797), cleaved caspase 3 (Cell Signaling 9694T), anti-mouse IgG Alexa Fluor 488 (Invitrogen A21202), and anti-rabbit IgG Alexa Fluor 546 (Invitrogen A10040).
Preparation and analyses of fluorescently labeled microtubules in vitro
HiLyte Fluor 488 labeled tubulin was obtained from Cytoskeleton Inc. (Denver, USA), and microtubules were polymerized according to the manufacturer’s instructions. Briefly, one aliquot of 488 tubulin (Cytoskeleton, TL488M) was resuspended to 5 mg/mL (45.5 μM) in 4.0 μl of general tubulin buffer (Cytoskeleton, BST01) supplemented with 0.01 volumes of 100 mM GTP (Cytoskeleton, BST06) and 1 μl of microtubules cushion buffer (Cytoskeleton, BST05) to facilitate polymerization. This solution was mixed with 70 μl of an equivalent solution using unlabeled tubulin (Cytoskeleton, T240) for a final labelling ratio of 1:15. Aliquots were placed in an incubator at 37ºC for 20 minutes to allow the tubulin polymerization to microtubules of appropriate length (average ∼10 μm). Upon polymerization, the microtubules were removed from the incubator and treated with 100 μl of paclitaxel/microtubules buffer solution (20 μM paclitaxel in general tubulin buffer). Control and paclitaxel-treated microtubule mixtures were treated with ultrasound (45-150 kHz, 120 W/cm2), and observations were made and images were acquired using a fluorescent microscope at various time points.
Imaging acquisition
Immunofluorescence staining was observed with widefield microscopy using a Plan-Apochromatix 100Å~ objective lens (oil immersion, numerical aperture [NA] 1.4), 20x, and 10x objective lens, on inverted Zeiss Axio Observer Z1 using AxioVision 4.8 software. Images were acquired using a monochrome Zeiss Axio Cam MRm CCD camera and processed using the NIH Image J software.
Image analysis and microtubule length quantification
Microtubules length was analyzed using the NeuronJ plugin from Image J (imagej.nih.gov). For that purpose the images were transformed to 8-bit, vectorized, and the tubulin filaments were measured on a computer screen for at the least 10 filaments in each image.
Statistics
Statistical analysis was made using Graph Pad (version 9.4.1).Experimental values are expressed as mean ± SD of three independent experiments. Statistical analysis was performed using ANOVA. A p-value of less than 0.05 was considered significant.