Varian cary eclipse fluorescence spectrophotometer

ChT-L (β5) activity of CPs was monitored by fluorescence spectroscopy using the model substrate Suc-LLVY-AMC. Purified yCPs (66 nM in 100 mM Tris-HCl, pH 7.5) were incubated with 300 μM substrate for 1 h at room temperature or 37 °C. The reactions were stopped by diluting samples 1:10 in 20 mM Tris-HCl, pH 7.5. AMC fluorophores released by proteasomal activity were measured in triplicate with a Varian Cary Eclipse Fluorescence Spectrophotometer (Agilent Technologies) at λexc=360 nm and λem=460 nm.

Thermal-denaturations were monitored by the intrinsic fluorescence of tryptophan and tyrosine residues on a Varian-Cary Eclipse Fluorescence Spectrophotometer (Agilent, USA) with excitation and emission slits of 5 nm. Excitation filter was fixed in automatic position and the emission filter was left open. Sample concentrations were 0.013 mg·ml⁻¹ (2 µM) in 0.1 M buffer (sodium acetate pH 5.0, MES pH 6.0 and Hepes pH 7.0). All the measurements were performed using a 10-mm path length cell and changes at 315, 330 and 350 nm were monitored after excitation at 280 or 295 nm. The scan rate was 60°C·h⁻¹, with an average time of 1 s, and data were collected every 0.2°C. The unfolding curves were analyzed using a two-state model as described previously [52] (link). The reported thermal denaturation midpoints for the proteins are the average of the three denaturations over the different wavelengths explored (Table 2).

Cell viability was measured by a Resazurin Cell Viability Assay Kit (Immunological Science), following the instructions of the manufacturer’s protocol. In summary, parental cells and CSCs were plated in 96-well plates on top of the extracellular matrix gel prepared as described above or directly on the top of the well (2D cultures). Viable cells were counted by Trypan Blue dye exclusion, and 7 × 10³ cells were seeded in each well.
After 24 h, both cell lines were treated as follows: GEM, C12GEM, and C18GEM for 72 h (2D experiments) or 7 days (3D experiments). To determine their mechanism of intracellular transport in 2D cultures, cells were also treated with different membrane entry inhibitors: dipyridamole (Dip, Sigma–Aldrich, St. Louis, MO, USA), methyl-β-cyclodextrin (MβCD, Sigma–Aldrich, St. Louis, MO, USA), chlorpromazine (CPM, Sigma–Aldrich, St. Louis, MO, USA), sulfo-N-succinimidyl oleate (SSO, Cayman).
After treatments, 15 μL of resazurin were added directly in the medium of each 96-well. After about 3 h of incubation at 37 °C, the fluorescent signal was obtained using a Varian Cary Eclipse Fluorescence Spectrophotometer (Agilent Technologies, Santa Clara, CA, EUA), at 535 nm of excitation wavelength and 590 nm of emission wavelength. The data obtained in each treatment were normalized with the respective control group.

Emission spectra were obtained using a Varian Cary Eclipse Fluorescence Spectrophotometer (Agilent Technologies, Santa Clara, CA, USA) and 100‐µL Quartz cuvette. For fluorescence resonance energy transfer (FRET) measurements samples were excited at 435 nm (CyPet excitation) and emission was monitored from 450 to 600 nm with both slits set to 1 nm. Affinity experiments were carried out using 1 µm of FRET fusion protein, in which the CyPet and YPet FRET pair were separated by both Myo1 IQ motifs, with varying concentrations of Cam1 in a final volume of 100 µL in analysis buffer of 140 mm of KCl, 2 mm of MgCl₂, 20 mm of MOPS, pH 7.0 with 2 mm of EGTA, CaCl₂ or Ca²⁺‐EGTA as required.

An assay was performed to check for the toxic effects of the procedure on the cells. Cell viability was evaluated using the LIVE/DEAD Viability/Cytotoxicity Kit (Thermo Fisher Scientific). In this assay, the intracellular esterase activity determines the conversion of non-fluorescent cell-permeant calcein-AM to intense fluorescent calcein. The dye is retained within the live cells, producing intense green fluorescence (Ex/Em wavelengths: 495/515 nm). Ethidium homodimer-1 (EthD-1) enters the cells with damaged membranes and undergoes a 40-fold enhancement upon binding to nucleic acids, thereby producing a bright red fluorescence in dead cells (Ex/Em wavelengths: 560/635 nm).
EthD-1 is excluded by the intact plasma membrane of live cells. Non-encapsulated cells were used as positive controls (viable cells), and 0.1% Triton X-100 treated cells were used as negative controls (dead cells). The fluorescence emissions were measured separately for calcein-AM at 530 nm and ethidium homodimer-1 at 645 nm (Varian Cary Eclipse Fluorescence Spectrophotometer, Agilent). Cell viability was analysed at 0, 24, 48 and 72 hrs after shielding in culture. Each experiment was conducted in triplicates (n = 3), and the data were represented as mean ± SD. $$\mathrm{Percentage}\mathrm{viability}(\%)=\frac{\mathrm{Absorbance}\mathrm{of}\mathrm{treated}\mathrm{cells}}{\mathrm{Absorbance}\mathrm{of}\mathrm{control}\mathrm{cells}}\times 100$$

Trp fluorescence variation was used to determine the protein-ligand dissociation constant Kd at 23 °C in 50 mM Tris HCl, pH 8.0 containing 200 mM NaCl, 10% glycerol, 10 mM DTT. The fluorescence intensity emitted by XIAP-BIR1 Trp73 or cIAP2-BIR1 Trp76 was measured by varying the concentration of the different ligands. The tests were performed with a spectral fluorimeter (Varian Cary Eclipse Fluorescence Spectrophotometer, Agilent Technologies), recording the fluorescence emission spectra between 300 and 400 nm (excitation 280 nm). XIAP-/ cIAP2-BIR1 protein samples were concentrated at 5 μM.
Two-fold dilutions of FC2 and derivatives prepared to reach final concentrations ranging from 106 to 0.2 μM. 8 μL of each FC2 dilution were mixed with 180 μL of the protein solutions and fluorescence was measured in a 200 μL quartz cuvette. The Kd values were obtained through the GraFit5 program (Erithacus Software Limited, 2010), fitting the fluorescence values (F) with the following equation dependent on three parameters (M, m, K_d): $$\begin{gathered} F=M-\frac{\left(M-m\right)}{[P_T]}\left[P_I\right]; \\ \mathrm{with} \left[P_I\right]=\frac{\left[P_T\right]+\left[I_T\right]+K_d-\sqrt{{\left(\left[P_T\right]+\left[I_T\right]+K_d\right)}^2-4\left[P_T\right]{\left[I_T\right]}^2}}{2} \end{gathered}$$
where F is the fluorescence intensity, [P_T]/[I_T] are the total protein/inhibitor concentrations, M/m is the max/min of fluorescence and [P_I] is the concentration of the protein bound to the inhibitor. All the analyses were performed with GraFit version 5 (Erithacus Software Ltd., Horley, UK).

One millimolar laurdan (Sigma-Aldrich) solution in dimethyl sulfoxide (DMSO) was added to the vesicle suspension in order to have a lipid/probe ratio of 20:1. This mixture was incubated in the dark for 30 min and further diluted in HEPES-buffered saline to a final concentration of 5 μM laurdan and 100 μM phospholipids. Flagella were purified as previously described by mechanical shearing (17 (link)). Increasing quantities of H7 flagella were progressively added to the laurdan/liposome mixture and incubated each time in the dark for 20 min in a circulating water bath at 37°C. Generalized polarization (GP) (23 (link)) was calculated from the emission intensities at 440 nm and 490 nm after excitation at 390 nm, according to the following equation (1): $$\text{GP}=\frac{I_{440}-I_{490}}{I_{440}+I_{490}}$$ using a Varian Cary Eclipse fluorescence spectrophotometer (Agilent Technologies). The relative ΔGP was obtained by subtracting the GP value in the absence of flagella from all GP values, for each size of liposome.