Motorized stage

For stereology, testes were embedded in Technovit 7100 resin, cut into 20 µm sections, and stained with Harris’ hematoxylin, as previously described in Ref. [15]. The number of Leydig cells and Sertoli cells was determined by the optical disector method using an Olympus BX50 microscope fitted with a motorized stage (Prior Scientific Instruments, Cambridge, UK) and Stereologer software (Systems Planning Analysis, Alexandria, VA).

Live-cell microscopy was performed using Olympus IX81 motorized inverted microscope, Orca CCD camera (Hamamatsu Photonics, Japan), motorized stage (Prior Scientific, Cambridge, UK), and 37°C incubation chamber (Solent Scientific, Segensworth, UK) fitted with appropriate filter sets and a 40× NA 1.42 oil objective. Images were collected in the 490 nm (Venus, sfGFP), 550 nm (mCherry), and 435 nm (CFP) channels using Micro-Manager software (Edelstein et al., 2014 (link)). Image analysis was performed using a customized plug-in tool in ImageJ (Schindelin et al., 2012 (link)), which calculates mean fluorescence intensity (MFI) by measuring average, background-subtracted gray values over regions of interest (ROIs) of defined diameter around manually selected points in the cell.

A Zeiss Axioplan2 fluorescence microscope connected to a CCD camera and equipped with a motorized stage (Prior Scientific Instruments, Rockland, MA) was used to acquire microscopic images. MetaMorph software (Molecular Devices, Sunnyvale, CA) was used to control the microscope as well as to generate a montage of the entire tumor from the captured image frames. Photoshop CS6 software (Adobe Systems, McLean, VA) was used for post-acquisition processing.

All spectra were recorded on a modified Olympus BX51 microscope coupled to a Raman spectrometer. A Prior Scientific motorized stage was used to move the sample. Dark-field images were recorded using a Lumenera Infinity2 charge-coupled device (CCD) camera. A 632.8-nm single-frequency diode laser was used as the excitation source. Intensities were derived from the laser power on the sample measured using a Thorlabs PM16-121 power meter. For room temperature measurements, the laser was focused on a diffraction-limited spot of diameter ~1 μm. Integration times used for BPT, MBN, and MPy were 0.2, 1, and 1 s, respectively. Excitation and collection were performed using a dichroic beamsplitter and an Olympus MPLFLN100XBD NA (numerical aperture) 0.9 objective. Elastically scattered laser light was removed using two Thorlabs NF633-25 notch filters. Scattered light was imaged onto an Andor Newton EMCCD coupled to a HORIBA Triax 320 spectrometer. For low-temperature measurements, the laser was focused on a ~2-μm-diameter spot, and an integration time of 3 s was used. An Oxford Instruments Microstat HiRes cryostat, an Olympus LMPLFLN100XBD NA 0.8 objective, and two Semrock NF03-633E-25 single-notch filters were used. Scattered light was imaged on an Andor Newton EMCCD coupled to an Andor Shamrock 303i spectrometer.

Cells were seeded onto 8-well plastic-bottom slides (Ibidi GmbH, Martinsried, Germany) at a density of 8 × 10⁴ cm⁻². The imaging medium was Leibowitz L-15 (Thermo Fisher Scientific) supplemented with FBS and antibiotics as described above. Expression of AURKA-Venus was induced 24 h prior to imaging, which was carried out on an Olympus CellR widefield imaging platform comprising an Olympus IX81 motorized inverted microscope, Orca CCD camera (Hamamatsu Photonics, Japan), motorized stage (Prior Scientific, Cambridge, UK) and 37°C incubation chamber (Solent Scientific, Segensworth, UK) fitted with appropriate filter sets and a 60× NA 1.42 oil objective. Images were acquired using the Olympus CellR software as 1 µm stacks and exported as 12 bit TIFF stacks for display as maximum-intensity projections in ImageJ.