Ultraview vox

In order to investigate relative levels of IL-2Rβ within the Jurkat cell population, cells were incubated with antibody against IL-2Rβ. 100,000 cells were resuspended in fresh medium and incubated with primary antibody against IL-2Rβ (Novus Biologicals) at 1:10 dilution for one hour at room temperature. The cells were then washed three times and incubated for one hour with Alexa 647-labeled secondary antibody (Life Technologies) at 1:200 dilution for one hour at room temperature, followed by three additional washes. Cells were analyzed in two ways: by flow cytometry using a BD LSR Fortessa and by fluorescent microscopy using a PerkinElmer UltraVIEW VoX spinning disk confocal microscope with a Nikon Ti-E camera. The relative intensity of the Alexa 647 signal was quantified for each cell and used to assess the heterogeneity of available IL-2Rβ within the population. In order to investigate relative levels of IL-2Rα and IL-2Rβ on the same cell, cells were incubated with FITC-labeled antibody against IL-2Rα (BioLegend) and APC-labeled antibody against IL-2Rβ. Cells were analyzed using flow cytometry, as above.

Cells were seeded in 1 glass bottom well chamber one day before analysis and pre-sensitized with 10 μM bromodeoxyuridine (BrdU, GE Healthcare) for 24 h. Before laser irradiation, cells were incubated with 1 µM THZ1 (1604810-83-4) for 1 h, at 37 °C and 5% CO2. The cells were then maintained under the same conditions using the Temperature Control Chamber (PerkinElmer UltraView VoX), and images were taken with a Nikon Eclipse Ti microscope equipped with a 63x oil objective and equipped with a Perkin Elmer spinning disk. Images were collected every 4 s for 10 min after irradiation. Cellular nuclei were irradiated with a 355 nm UV ablation laser at a power setting of 0.15, a repetition rate of 200 Hz, a pulse energy >60 μJ, pulse length< 4 ns (Rapp OptoElectronic). The intensity of the GFP signal was measured using ImageJ software for at least 20 cells per condition from three biological replicates.

HUVECs transduced with a lentiviral vector containing VEC-EGFP (HUVECs- VEC-EGFP) and without any lentiviral treatment (HUVECs-NT, non-treated) were maintained separately in 3.5 dishes. HUVECs-NT were transfected with Cy3 Labeled GAPDH siRNA according to Lipofectamine RNAiMax procedure. 24 hours after siRNA transfection, HUVECs- VEC-EGFP and HUVECs-NT were washed with PBS, trypsinized and mixed into 3.5 cm glass bottom dish. 4 hours after mix, the digital fluorescent images were captured by a PerkinElmer UltraView VoX spinning disk confocal system with a Nikon eclipse Ti inverted microscope.

Fluorescence microscopy experiments were performed on mid-log yeast cultures in synthetic media at the indicated temperatures. Live yeast cell imaging data in all Figures were acquired with a 100× CFI Plan Apochromat VC oil-immersion objective lens (1.4 NA), using a PerkinElmer Ultraview Vox spinning disk confocal microscope that consists of a Nikon TiE inverted stand attached to a Yokogawa CSU-X1 spinning disk scan head, a Hamamatsu C9100-13 EMCCD camera, Prior NanoscanZ piezo focus, and a Nikon Perfect Focus System (PFS). All images were collected as square images with 512 × 512 pixels. The number of cells observed in experiments is reported in the figures and figure legends. The brightness and contrast of images were linearly adjusted and cropped in Photoshop (Adobe) for presentation.
For vacuole staining in Fig S3, 5 μM FM4-64 (Invitrogen) was added to mid-log cell cultures in YPD media for 15 min at 26°C. Cultures were then resuspended in fresh YND media and incubated for 1 h in at 26°C. 0.1 mM CellTracker Blue CMAC (Invitrogen) was then added 15 min before heat stress and imaging. For the myriocin treatment in Fig S6, 2 μM myriocin was added to mid-log cell cultures in YND media for 1 h at 26°C before heat stress and imaging.