Topro 3

Following the same experimental protocol of treatment as described above, LCLs and DLBCLs cells were collected and washed with DPBS. After washing, they were fixed with 4% paraformaldehyde for 10 min at RT and washed with DPBS. Actin fibers were revealed using 405-Phalloidin-iFluor reagent (Abcam) as per the manufacturer’s instructions.
Finally, after 30 min of phalloidin incubation, nuclei were stained with TOPRO-3 (1:1000—Fisher scientific) for 15 min at RT. Cells were visualized using a ZEISS LSM 900 confocal microscope (40 × oil lens). Images were constructed using the Fiji software. For actin quantification by flow cytometry, the same experimental protocol was followed. Intracellular actin fibers labeling (45 min of phalloidin staining) was performed after permeabilization (IntraPrep Permeabilization Reagent kit–Beckman Coulter) according to the protocol recommended by the supplier. Acquisitions were performed on the Cytoflex cytometer (Beckman Coulter). Results were analyzed with Kaluza Analysis 2.1 Software. Fold change was calculated based on the mean fluorescence intensity ratio of phalloidin of the test normalized to the control.

Yeast cell samples grown under the three conditions described above were fixed in 4% paraformaldehyde (PFA) in PBS for 15 min at 4 °C. These cells were gently pelleted by centrifugation for 5 min at 3500 rpm, and cells were rinsed 3× with sterile PBS. Subsequent processing steps were carried out in the dark, where samples were covered with aluminum foil and protected from the light. Cell pellets were resuspended in 15 µM Calcofluor White and incubated for 1 h at 37 °C. Samples were then rinsed and pelleted as previously mentioned but with distilled H₂O. After the third rinse, samples were pelleted again, and the pellet was resuspended with 1 µM TO-PRO^®-3 (Fisher Scientific, Los Angeles, CA, USA) and incubated for 30 min at room temperature. Samples were further rinsed and pelleted, as previously mentioned, using distilled H₂O. The resuspended samples were filtered through a 0.2 µM 25 mm Isopore Membrane Filter (GTBP) using a filter manifold. This membrane was removed, placed on a microscope slide, and a drop of antifade was added to the top. A coverslip was placed, and then, the edges were sealed. The samples were viewed using a Zeiss LSM 880 upright laser scanning confocal microscope and an Alpha Plan-APOCHROMAT 63× lens (Zeiss, Oberkochen, Germany). Yeast cell size and cell wall thickness were analyzed using ImageJ [28 (link)].

The following antibodies were used: rabbit anti-GFP (Invitrogen cat. no. A-6455, 1:500). In addition, TO-PRO-3 (Fisher cat. #T3605) and phalloidin conjugated to Alexa Fluor 647 (Invitrogen cat. no. A22287) were used to visualize nuclei of cells and actin filaments. The antibody labeling was done under the standard conditions, which included 20 min of fixation in 4% paraformaldehyde solution, and subsequent incubation with antibodies in PBS that contained 0.2% Triton X-100.

ICC was performed on undifferentiated GBM CSCs, plated at 3.5 × 10⁵ cells/cm² on Matrigel (Becton and Dickinson, San Jose, CA)-coated glass coverslips for 24 h, and on their differentiated progeny.
For intracellular epitopes detection, the cells were permeabilized for 10 min with 0.1% Triton X-100 in PBS. Cells were then incubated with primary antibodies diluted at the appropriate concentration in PBS-10% NGS over night at 4 °C. Secondary antibodies were then added for 1 h at room temperature. Nuclei were counterstained with TOPROIII (Invitrogen), 1:2000 in PBS or DAPI (Fluka, Buchs, Switzerland).